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UNIVERSIDADE FEDERAL DE SERGIPE
PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA
MESTRADO EM CIÊNCIAS FARMACÊUTICAS
INFLUÊNCIA DE NOVOS MARCADORES
IMUNOFENOTÍPICOS NO PROGNÓSTICO E SOBREVIDA
DE LEUCEMIAS MIELOIDES AGUDAS: UMA REVISÃO
SISTEMÁTICA E META-ANÁLISE
AMANDA FERNANDES DE OLIVEIRA COSTA
SÃO CRISTÓVÃO
FEVEREIRO/2017
UNIVERSIDADE FEDERAL DE SERGIPE
PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA
MESTRADO EM CIÊNCIAS FARMACÊUTICAS
INFLUÊNCIA DE NOVOS MARCADORES
IMUNOFENOTÍPICOS NO PROGNÓSTICO E SOBREVIDA
DE LEUCEMIAS MIELOIDES AGUDAS: UMA REVISÃO
SISTEMÁTICA E META-ANÁLISE
Amanda Fernandes De Oliveira Costa
Dissertação apresentada ao Núcleo de Pós-
Graduação em Ciências Farmacêuticas da
Universidade Federal de Sergipe como requisito
parcial à obtenção do grau de Mestre em
Ciências Farmacêuticas.
Orientadora: Profª Drª Dulce Marta Schimieguel Mascarenhas Lima.
SÃO CRISTÓVÃO
FEVEREIRO/2017
AMANDA FERNANDES DE OLIVEIRA COSTA
INFLUÊNCIA DE NOVOS MARCADORES
IMUNOFENOTÍPICOS NO PROGNÓSTICO E SOBREVIDA
DE LEUCEMIAS MIELOIDES AGUDAS: UMA REVISÃO
SISTEMÁTICA E META-ANÁLISE
Dissertação apresentada ao Núcleo de Pós-
Graduação em Ciências Farmacêuticas da
Universidade Federal de Sergipe como requisito
parcial à obtenção do grau de Mestre em
Ciências Farmacêuticas.
Aprovada em 13/02/2017
______________________________________________________
Orientador: Profª. Drª. Dulce Marta Schimieguel Mascarenhas Lima
_____________________________________________________
1º Examinador: Profª. Drª. Rosana Cipoloti
______________________________________________________
2º Examinador: Prof. Dr. Ricardo Ambrósio Fock
PARECER
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AGRADECIMENTOS
A gratidão é o único tesouro dos humildes, já dizia Shakespeare. E nada mais
digno aos que me acompanharam nessa trajetória do que a minha profunda gratidão.
Com o passar dos anos, aprendi a enxergar a presença de Deus no meu dia a dia, em
detalhes que muitas vezes passavam despercebidos e sem dúvida alguma, apenas
pela sua existência e persistência nessa mera cristã, ele merece a gratidão de toda a
minha vida. Não só por ter me acompanhado, mas por ter me abençoado de tantas
diversas formas e através de tantas pessoas.
Agradeço sempre, eternamente e incansavelmente as duas pessoas que mais
me apoiaram, suportaram minhas dores e compartilharam minhas alegrias, não só por
esses dois anos, mas por toda a minha vida: Minha mãe, Ana Emília e minha avó,
Maria José. Não existe explicação, se não Deus, por serem vocês a fazerem parte de
mim.
Agradeço ao meu companheiro inquestionável. Aquele que lidou com todas as
minhas frustrações durante esse período, que me incentivou a encarar a vida
acadêmica, me ensinou, me ajudou e sempre está ao meu lado. No sossego e no
sufoco, para toda a vida. Meu amado marido, Adonis.
Agradeço imensamente a essa mulher que me mostrou a beleza da
Hematologia e me inspira a ser como ela. Uma grande profissional, uma mulher com
princípios, de Deus, que muitas vezes me mostrou sua amizade, não só na
compreensão, mas na crítica construtiva, quando fiz corpo mole. Professora Doutora
Dulce Marta Schimieguel Mascarenhas Lima, a você serei eternamente grata. Tudo
que eu me tornar daqui para frente também pertencerá a senhora. Muito obrigada.
Ao Professor Doutor Divaldo Pereira de Lyra Junior, o maior incentivador que
conheci nesse caminho. Obrigada por ter se tornado um amigo, por sempre ter tido fé
em mim e por ter me ensinado a buscar sempre ser melhor. O senhor com certeza,
fez e faz parte da minha história. Sempre que converso com o senhor sinto que algo
me é acrescentado e que aprendo algo novo. Essa vitória também é sua. Obrigada.
Aos mestres que caminharam junto comigo, compartilhando seus
conhecimentos valiosos e contribuindo para o meu crescimento acadêmico. O
Professor Doutor Marco Prado Nunes e o Doutor Alex Sandes. Meus sinceros
agradecimentos.
A Daniel Lima, com quem trabalhei nesse projeto. Obrigada por ter me
presenteado sua amizade. Por ter acompanhado todo esse processo, cheio de
primeiras vezes, cheio de incertezas e inseguranças, mas que graças a Deus, foi
finalizado com êxito. E me sinto muito orgulhosa por ele.
Por último e não menos importante, devo muita gratidão aos que
compartilharam o dia a dia comigo, escutaram todas as minhas choradeiras, me
distraíram, me colocaram para cima e acreditaram em mim quando eu fraquejei. Meus
queridos amigos de laboratório, espero levar vocês para a vida. Muito obrigada por
tudo. E que venham novos desafios.
Finalmente, gostaria de agradecer à CAPES, pelo apoio financeiro, sem o qual,
dificilmente poderia ter concluído o curso de Mestrado do Programa de Pós-
Graduação em Ciências Farmacêuticas da UFS e a todos mais que eu não tenha
citado nesta lista de agradecimentos, mas que de uma forma ou de outra contribuíram
para a conclusão dessa jornada.
Muito obrigada. Esse é só o início.
RESUMO
INFLUÊNCIA DE NOVOS MARCADORES IMUNOFENOTÍPICOS NO PROGNÓSTICO E SOBREVIDA DE LEUCEMIAS MIELOIDES AGUDAS: UMA REVISÃO SISTEMÁTICA E META-ANÁLISE. AMANDA FERNANDES DE OLIVEIRA COSTA. SÃO CRISTÓVÃO, 2016.
Apesar dos avanços tecnológicos, o prognóstico e a sobrevida dos pacientes adultos
com leucemia mieloide aguda (LMA) permanecem baixos quando comparados com
outras neoplasias hematológicas. Alguns antígenos identificados pela técnica de
imunofenotipagem por citometria de fluxo podem desempenhar um papel significativo
na compreensão da fisiopatologia, no prognóstico e na sobrevida global dos pacientes
com LMA. Sendo assim, foi realizada uma revisão sistemática e metanálise nas bases
de dados PubMed, Scopus, Science Direct, Web of Science e Cochrane Library
(utilizando as diretrizes do PRISMA). Em onze estudos realizados em um total de 639
pacientes, foram detectados treze antígenos, analisados pela metodologia de
imunofenotipagem por citometria de fluxo. Destes marcadores, doze exibiram um
impacto negativo no prognóstico da LMA. A metanálise demonstrou que a alta
expressão dos marcadores de LMA tem sido associada a uma diminuição nas taxas
de sobrevida em 10 meses (RR 2,55; IC 95%; 1,49-4,37) e 20 meses (RR 2,46; IC
95%; 1,75- 3.45). O conhecimento de que a expressão de novos marcadores
imunofenotípicos pode ser capaz de influenciar o comportamento da doença, parece
ser uma informação promissora, pois demonstra influência no prognóstico e
diminuição da sobrevida dos pacientes com LMA. Isto pode servir de base para a
investigação de diferentes protocolos quimioterápicos, incluindo o estudo prospectivo
de novos alvos terapêuticos.
Palavras-chave: imunofenotipagem; leucemia mieloide aguda; prognóstico; sobrevida.
ABSTRACT
ROLE OF NEW IMMUNOPHENOTYPIC MARKERS ON PROGNOSTIC AND OVERALL SURVIVAL OF ACUTE MYELOID LEUKEMIA: A SYSTEMATIC REVIEW AND META-ANALYSIS. AMANDA FERNANDES DE OLIVEIRA COSTA. SÃO CRISTÓVÃO, 2016. Despite technological advances, the prognosis and survival of acute myeloid leukemia
(AML) adult patients remain low, compared with other hematologic malignancies.
Some antigens detected by immunophenotyping may soon play a significant role in the
pathophysiologic, prognostic, and overall survival (OS) rate of AML patients. Therefore,
we conducted a systematic review and meta-analysis of PubMed, Scopus, Science
Direct, Web of Science, and the Cochrane Library (using PRISMA guidelines). We
analyzed 11 studies and 13 antigens, detected through the immunophenotyping of 639
patients. From them, 12 exhibited a negative impact with AML prognosis. The meta-
analysis demonstrated a high expression of AML markers, which have been associated
with a decrease in survival over 10 months (RR 2.55; IC 95%; 1.49-4.37) and over 20
months (RR 2.46; IC 95%; 1.75-3.45). Knowing that the expression of
immunophenotypic markers, which are not used on a routine basis, might be able to
influence disease behavior, looks promising. Since they have been associated with a
poor prognosis as well as a decrease in survival. This may allow for different
chemotherapeutical protocols, including future studies for new therapeutic targets.
Key word: immunophenotyping; acute myeloid leukemia; prognosis; survival.
SUMÁRIO 1 INTRODUÇÃO ...................................................................................................................... 1
2 REVISÃO DA LITERATURA .............................................................................................. 2
2.1 Neoplasias hematológicas - Leucemias ........................................................................ 2
2.2 Leucemias mieloides agudas (LMAs) ............................................................................ 5
2.3 Imunofenotipagem e marcadores imunofenotípicos em leucemias mieloides
agudas ..................................................................................................................................... 10
2.4. Novos marcadores imunofenotípicos ......................................................................... 15
REFERÊNCIAS ..................................................................................................................... 19
CAPÍTULO 1 .......................................................................................................................... 24
ROLE OF NEW IMMUNOPHENOTYPIC MARKERS ON PROGNOSTIC AND
OVERALL SURVIVAL OF ACUTE MYELOID LEUKEMIA: A SYSTEMATIC REVIEW
AND META-ANALYSIS ......................................................................................................... 25
ANEXOS ................................................................................................................................. 55
ANEXO A - Supplementary Information ............................................................................. 55
ANEXO B - Carta de aceite do artigo ................................................................................. 59
ANEXO C - Normas da Revista ........................................................................................... 61
ÍNDICE DE FIGURAS
INTRODUÇÃO
Figura 1: Representação espacial das taxas brutas de incidência de leucemia por 100
mil homens e mulheres no estado de Sergipe. ................................................................... 4
Figura 2: Detector de dispersão de luz frontal: Tamanho celular - FSC (Forward
Scatter) e detector de dispersão de luz em ângulo reto: Complexidade celular - SSC
(Side Scatter). ......................................................................................................................... 11
Figura 3: Marcadores mieloides e suas intensidades de expressão de acordo com o
subtipo de Leucemia mieloide aguda. ................................................................................ 13
Figura 4: Via de transdução de sinal do receptor FLT3. ................................................ 16
Figura 5: Esquema funcional de células CD133+. .......................................................... 17
CAPITULO 1
Figure 1: Flow diagram for study identification. .............................................................. 32
Figure 2: Forest Plot of relative risks and confidence intervals of 10-month
survival.....................................................................................................................................39
Figure 3: Forest Plot of relative risks and confidence intervals of 20-month survival.
.................................................................................................................................................. 40
SUPPLEMENTARY INFORMATION
Supplementary figure S 1: Forest plot with relative risks and confidence intervals of
survival in 10 month. ............................................................................................................. 55
Supplementary figure S 2: Funnel plot of studies on survival in 10 months. ............ 56
Supplementary figure S 3: Funnel plot of studies on survival in 10 months. ............ 57
Supplementary figure S 4: Funnel plot of studies on survival in 10 months. ............ 58
ÍNDICE DE QUADROS E TABELAS
INTRODUÇÃO
Quadro 1: Características clínicas e laboratoriais das leucemias mieloides agudas. . 7
Quadro 2: Classificação do grupo Franco-Americano-Britânico para as leucemias
mieloides agudas. .................................................................................................................... 8
Quadro 3: Classificação da Organização Mundial da Saúde para as leucemias
mieloides agudas. .................................................................................................................... 9
Quadro 4: Painel de triagem para leucemias mieloides agudas. ................................ 14
Quadro 5: Descrição dos marcadores que compõem o painel mandatório do Grupo
Brasileiro de Citometria de Fluxo. ....................................................................................... 14
CAPITULO 1
Table 1: Contingency table (2x2). ...................................................................................... 51
Table 2: Main characteristics of the individual studies analyzed on the systematic
review and meta-analysis. .................................................................................................... 52
Table 3: Main disease and treatment features of the individual studies included on the
systematic review and meta-analysis. ................................................................................ 53
Table 4: Basic features of each antigen analyzed on this systematic review and meta-
analysis .................................................................................................................................... 54
1
1 INTRODUÇÃO
As neoplasias hematológicas são doenças malignas que afetam as células do
sistema hematopoético, caracterizadas por alterações no sangue e/ou em seus
tecidos formadores (KLATSKY et al., 2009). Neste grupo estão descritas as
leucemias, neoplasias caracterizadas pela proliferação exacerbada de células
anormais na medula óssea, provocando um prejuízo na produção de eritrócitos,
leucócitos e plaquetas normais, desencadeando consequentemente anemia, maior
suscetibilidade à infecções e quadros hemorrágicos (PUTZU et al., 2014; WINTERS
et al., 2015). De acordo com o tecido em que se origina o clone leucêmico e com a
evolução da doença, as leucemias podem ser categorizadas em quatro subtipos:
leucemias linfoides crônicas, leucemias linfoides agudas, leucemias mieloides
crônicas e leucemias mieloides agudas (BETZ & HESS, 2010).
As leucemias mieloides agudas (LMAs) consistem em um grupo de doenças
biologicamente heterogêneo, onde há acúmulo clonal de células mieloides imaturas,
exibindo diferentes comportamentos clínicos e fisiopatológicos (OUYANG et al., 2015).
São neoplasias com prognóstico sombrio, que afetam predominantemente adultos
com idade média de 70 anos, com taxas de sobrevida global de 10% em 2 anos e 2%
em 5 anos (STRICKLAND et al., 2016).
A heterogeneidade genética deste grupo de neoplasias hematológicas torna
impraticável executar análises iniciais que possam abranger os diferentes genes
envolvidos e todas as alterações compreendidas nestas doenças, o que pode gerar
dificuldades no diagnóstico, implicando diretamente no direcionamento da terapêutica.
Sendo assim, apesar da crescente importância das características genéticas e
moleculares na subclassificação das leucemias mieloides agudas, as análises
morfológicas e imunofenotípicas são fundamentais para diagnóstico inicial destas
doenças (PETERS & ANSARI, 2011; HASAN et al., 2015).
As análises imunofenotípicas por citometria de fluxo compõem uma ferramenta
de útil para diagnóstico e seguimento da doença, por serem capazes de avaliar
diferentes características celulares simultaneamente, célula por célula. Esta técnica
emprega anticorpos monoclonais, utilizados como marcadores imunofenotípicos na
análise do padrão de expressão de antígenos (Clusters Designations - CDs) das
populações celulares, combinados em painéis de triagem e classificação (DOGEN &
2
ORFAO 2012; FINAK et al., 2016).
Os marcadores imunofenotípicos associados à leucemia (leukemia-associated
phenotypic markers - LAIPS) são úteis para distinguir os precursores mieloides
imaturos normais/reativos das células leucêmicas e são amplamente utilizados em
estudos de doença residual mínima (DRM), além de permitirem a possibilidade de
avaliação do protocolo terapêutico e o desenvolvimento de novos alvos terapêuticos.
(GRIMWADE & FREEMAN, 2014; OMMEN, 2016).
Atualmente, na literatura, não são descritas recomendações claras para a
utilização de novos marcadores imunofenotípicos nos painéis de imunofenotipagem
para LMAs, tampouco sua influência no prognóstico e sobrevida. Assim sendo, foi
realizada uma revisão sistemática da literatura e uma metanálise dos dados, com o
objetivo de identificar as publicações relevantes sobre a influência destes novos
marcadores imunofenotípicos no prognóstico e sobrevida dos pacientes com
leucemias mieloides agudas.
2 REVISÃO DA LITERATURA
2.1 Neoplasias hematológicas - Leucemias
As neoplasias hematológicas compõem um grupo de doenças malignas com
características distintas, classificadas de acordo com a linhagem das células das quais
se originam e representam cerca de 8% dos casos de neoplasias nos países
desenvolvidos. Essas doenças afetam o sistema hematopoético, apresentando
diversas alterações morfológicas, mutações genéticas e perda de função celular
(BETZ & HESS, 2010).
São classificadas de acordo com a morfologia, imunofenotipagem, citogenética
e perfil molecular. Dentro da linhagem linfoide são categorizados os linfomas, o
mieloma múltiplo e as leucemias linfoides crônicas e agudas. As neoplasias originadas
da linhagem mieloide são categorizadas em desordens mieloproliferativas, síndromes
mielodisplásicas e leucemias mieloides crônicas e agudas (BETZ & HESS, 2010;
ARBER et al., 2016).
As leucemias são caracterizadas pela proliferação neoplásica maligna e
generalizada ou acúmulo de células hematopoéticas malignas na medula óssea com
3
ou sem envolvimento do sangue periférico, linfonodos e baço (WINTERS et al., 2015).
De acordo com sua incidência, foram relatadas como sendo o 9º tipo de câncer mais
comum em homens e o 10º mais comum em mulheres no mundo (JEMAL et al., 2011).
De acordo com o Instituto Nacional do Câncer (INCA), em 2014 foram relatados, no
Brasil, 5.050 casos novos de leucemia em homens e 4.320 em mulheres e a estimativa
para 2016 é de 5.540 novos casos em homens e de 4.530 em mulheres. Na figura 1
é possível observar a incidência de leucemias no estado de Sergipe em homens e
mulheres.
As leucemias são neoplasias que se apresentam clinicamente de forma
inespecífica. Características laboratoriais como anemia, trombocitopenia e
leucocitose ou leucopenia são identificados e associados a sintomas como fadiga,
dispneia, dor de cabeça, dor no peito e aparecimento de hematomas ou hemorragias,
particularmente do nariz e gengivas. Mesmo quando a contagem de leucócitos está
normal, os pacientes podem apresentar disfunção do sistema imunológico
evidenciada pela cicatrização lenta de feridas da pele, febre, infecções de repetição
e, em casos raros, sepse. As manifestações gastrintestinais são comuns
principalmente em casos de recidiva e podem ocorrer úlceras não só na boca e região
anorretal, mas também no trato gastrintestinal (ROSE-INMAN; KUEHL, 2014).
4
Figura 1: Representação espacial das taxas brutas de incidência de leucemia por 100 mil homens e mulheres no estado de Sergipe. Fonte: http://www.inca.gov.br/estimativa/2014/mapa.asp?ID=8
As leucemias podem afetar pessoas de todas as idades e, embora as causas
para o desenvolvimento de leucemias ainda não sejam bem conhecidas, alguns
fatores de risco que predispõem a doença são a quimioterapia prévia, doenças
genéticas causadas por cromossomos anormais, síndrome mielodisplásica prévia,
exposição à radiação ionizante e exposição ocupacional ao benzeno (RHOMBERG et
al., 2011). São categorizadas de acordo com o desenvolvimento e evolução da doença
em dois grandes grupos: leucemias agudas e leucemias crônicas (WINTERS et al.,
2015).
As leucemias agudas apresentam proliferação clonal acompanhada de
bloqueio maturativo variável, originando diferentes subtipos de leucemias. A célula em
que ocorre a transformação leucêmica é um precursor que perde a capacidade de
acompanhar o processo de maturação normal. A origem tecidual desse precursor irá
5
definir qual subtipo de leucemia aguda irá se desenvolver. Quando este precursor é
de origem linfoide, desenvolvem-se as leucemias linfoides agudas e quando o
precursor advém de linhagem mieloide, são formadas as leucemias mieloides agudas
(MERINO, 2010).
2.2 Leucemias mieloides agudas (LMAs)
As leucemias mieloides agudas são doenças clonais do tecido hematopoético
onde há uma proliferação exacerbada de células jovens da linhagem mieloide,
denominadas “blastos mieloides” ou “mieloblastos”, ocasionando produção
insuficiente de células sanguíneas maduras normais. O processo neoplásico que
origina o clone leucêmico pode surgir em qualquer estágio do desenvolvimento
celular, ou seja, em qualquer fase da hematopoese (FERRARA; SCHIFFER, 2013).
A proliferação exacerbada seguida de comprometimento na diferenciação
celular ocorre devido a mutações moleculares através da ativação de algumas classes
de genes, chamados oncogenes, que começam a produzir proteínas quiméricas
responsáveis pelo descontrole da divisão, diferenciação, amadurecimento e apoptose
celular. Os genes responsáveis pela supressão de tumores ou anti-oncogenes
também podem apresentar alterações, perdendo suas funções de transdução e
produção de proteínas relacionadas à inibição do crescimento celular e diminuição da
proliferação, aumentando o crescimento desregulado dessas células anormais
(LOGAN et al., 2015).
De acordo com dados do Instituto Nacional do Câncer (INCA, 2016), é uma
neoplasia de mau prognóstico, sendo o tipo mais comum de leucemia em adultos,
diagnosticada em idade média de 65 anos e com uma incidência ligeiramente maior
em homens de descendência europeia. Apresenta uma taxa de sobrevida em 5 anos
de 50% para pacientes de até 45 anos e de 2% para pacientes com mais de 75 anos
no momento do diagnóstico.
As taxas de remissão para LMAs apresentam um decréscimo com o aumento
da idade do paciente, chegando a atingir 90% em crianças, 70% em adultos jovens,
60% em adultos de meia idade, e 40% em pacientes mais velhos (ROSE-INMAN;
KUEHL, 2014). Quanto mais jovem o paciente, maior as taxas de sobrevida global e
melhor é o seu prognóstico. A taxa de sobrevida relativa de 5 anos para pacientes
entre 0 e 19 anos é de 62,8%, 48,8% em pacientes com idade entre 20 e 49 anos, de
6
28,0% para os pacientes em torno de 50 a 64 anos e de 5,4%, em pacientes com
idade superior 65 anos (DESANTIS, et al., 2014).
As leucemias mieloides agudas, assim como todas as leucemias, são
idiopáticas, ou seja, acontecem sem uma causa definida. Porém, alguns fatores de
risco são conhecidos e associados a doença, como a exposição à radiação ionizante,
a exposição ocupacional ao benzeno e alguns agentes alquilantes (WANG; BAILEY,
2015).
Algumas alterações moleculares também contribuem para a expansão clonal
leucêmica das células hematopoéticas jovens, fazendo com que elas percam a sua
capacidade de diferenciação e gerando anormalidades morfológicas. Além disso,
alterações cromossômicas como inserções, deleções e translocações são
relacionadas às LMAs, sendo responsáveis pela supressão de genes que regulam o
ciclo celular, induzindo a perda dos mecanismos normais de proliferação,
diferenciação maturação e/ou da morte celular programada (FARAONI et al., 2015).
As manifestações clínicas das LMAs estão descritas no Quadro 1, onde se
relaciona os principais sinais clínicos e achados laboratoriais apresentados por
portadores dessas doenças (MILLER; PILISHOWSKA, 2014).
Inicialmente as leucemias foram classificadas com base somente em
investigações citomorfológicas e citoquímicas. A morfologia ainda é importante, mas
a imunofenotipagem e a citogenética foram incorporadas em sistemas de classificação
atuais para que haja um delineamento mais preciso da linhagem hematopoética, do
estágio de diferenciação celular e prognóstico da doença (MILLER; PILISHOWSKA,
2014). As LMAs possuem subtipos distintos que são definidos pela morfologia
específica, citogenética e expressão molecular (FARAONI et al., 2014).
Nos últimos anos foram sugeridos diversos sistemas de classificação para as
LMAs e, dentre estes, os de maior relevância são os propostos pelo sistema Franco-
Americano-Britânico (FAB), e pela Organização Mundial da Saúde (ROSE-INMAN;
KUEHL, 2014).
7
Quadro 1: Características clínicas e laboratoriais das leucemias mieloides agudas.
EXAME SINAIS
CLÍNICO
-Palidez
-Hepatomegalia/esplenomegalia
-Linfadenopatia
-Febre em consequência de infecções
-Petéquias e outras manifestações hemorrágicas
-Dor óssea
-Hipertrofia gengival
-Infiltrações cutâneas
HEMOGRAMA
-Dosagem hemoglobina baixa
-Contagem de leucócitos < 1.000/μL a 200.000/μL
-Neutropenia e presença de blastos
-Anemia normocrômica e normocítica;
-Trombocitopenia pode ser severa
MIELOGRAMA >20% de blastos
Fonte: Adaptada de MILLER; PILISHOWSKA, 2014.
A classificação publicada pelo grupo Franco-Americano-Britânico entre 1976 e
1994 para as leucemias agudas foi a primeira mundialmente aceita e avalia critérios
morfológicos, citoquímicos e a presença de mais de 30% de blastos na medula óssea.
Primeiramente ela classificava as LMAs em seis subtipos (M1 até M6). Em 1985 foram
adotados critérios imunofenotípicos, incluindo o subtipo LMA M7, através da
confirmação de blastos plaquetários e o subtipo M0, através de marcadores
monoclonais (Quadro 2) (BAIN; ESTCOURT, 2013).
A classificação FAB está aos poucos sendo substituída pela classificação da
OMS, porém mesmo com a classificação da OMS em vigor há mais de dez anos, a
classificação FAB ainda é muito utilizada para os subtipos das LMAs na maioria dos
países em desenvolvimento devido à limitação de análises citogenéticas (MAHMOOD
et al., 2014).
A classificação da Organização Mundial da Saúde, publicada inicialmente em
2001 e com última atualização em 2016, expandiu o uso da imunofenotipagem e
incluiu a avaliação de alterações genéticas e moleculares específicas na classificação
8
das LMAs e outras neoplasias hematológicas, abordando esses critérios na
classificação dos seus subtipos, como é mostrado no quadro 3 (ARBER et al., 2016).
Quadro 2: Classificação do grupo Franco-Americano-Britânico para as leucemias
mieloides agudas.
CLASSIFICAÇÃO FAB PARA LEUCEMIA MIELOIDE AGUDA
M0 MPO (mieloperoxidase) positiva por método imunológico ou ultraestrutural CD13+
ou CD33+ ou CD11b+
M1 Blastos indiferenciados em alta%. Bastonete de Auer presentes ou ausentes.
Mieloperoxidase ou Sudan Black positivos em > 3% dos blastos.
M2 Blastos indiferenciados e diferenciação até promielócito que contém granulações primárias abundantes. Bastonetes de Auer são frequentes. Mieloperoxidase ou
Sudan Black positivos em > 3% dos blastos.
M3 Grande quantidade de promielócitos hipergranulares, com ou sem bastonetes de
Auer. Sudan Black e mieloperoxidase fortemente positivos.
M4 Diferencia-se de M2 por ter > 20% de células monocíticas na medula óssea e/ou
sangue. Diferencia-se de M5 por ter > 20% de promielócitos e mieloblastos na MO e/ou sangue periférico. Alfa naftil esterase positiva nas células monocíticas
M5a Blastos grandes, com citoplasma abundante, levemente basófilo e com projeções
citoplasmáticas. Diferenciação monocítica. Sudan Black e mieloperoxidase - Alfanaftil esterase + nas células monocíticas. >80% das células são monoblastos.
M5b >80% das células são monócitos ou promonócitos. Alfa naftil esterase positiva nas
células monocíticas.
M6
30% de blastos mieloides (mieloblastos ou promielócitos) e > 50% de blastos da série vermelha (eritroblastos). Muitas vezes há > 30% de megaloblastos, formas bizarras. PAS fortemente positivo nas células eritroblásticas e megaloblásticas.
Componente monocítico >80% das células não eritroides.
M7 Megacariócitos pequenos ou megacarioblastos >30% das células nucleadas da
MO. FAB: classificação franco-americana-britânica; MO: medula óssea; CD: grupo de diferenciação; Mb: mieloblasto; PAS: ácido periódico de Schiff. Fonte: Adaptado de BAIN; ESTCOURT, 2013
A OMS classifica as LMAs de acordo com anormalidades genéticas recorrentes
e mutações nos oncogenes Nucleophosmin 1 (NPM1) e potencializador de ligação de
proteína alfa (CEBPA) em três grandes categorias: favorável, intermediaria e não
favorável (ILYAS et al., 2015; STRICKLAND et al., 2016). Diferente da classificação
FAB, que adotava o valor de 30% de blastos para caracterizar o diagnóstico de
leucemias mieloides agudas, a OMS adotou, desde sua primeira publicação o valor
de 20% de blastos encontrados na contagem diferencial de 200 células no sangue
periférico ou 500 células na medula óssea (ESTCOURT; BAIN, 2013).
9
Todas as atualizações incorporadas pela Organização Mundial da Saúde foram
importantes não só para o diagnóstico, mas também para a identificação de doença
residual mínima e para a definição do prognóstico das LMAs. Além disso, evidenciou
a importância de se conhecer o histórico de citotoxicidade induzida por terapia prévia
e de ocorrência de síndrome mielodisplásica e mostrou o valor da análise das
amostras de sangue periférico e medula óssea na identificação de características
especificas de células blásticas ou não blásticas das LMAs (MAHMOOD et al., 2014).
Quadro 3: Classificação da Organização Mundial da Saúde para as leucemias
mieloides agudas.
TIPO DE LMA SUBTIPO DE LMA
LMAs com anormalidades genéticas recorrentes
- LMA com {t(8;21) (q22;q22.1)}, {LMA 1 fator de ligação núcleo alfa/ fator de transcrição 1 relacionado
com o nanico ; translocado para 1 (relacionado a ciclina-D) (CBF-ALFA/ETO)}
- Leucemia promielocítica aguda {LMA com t(15;17) (q22; q11-12} e com variantes {Leucemia
promielocítica/ Receptor alpha do ácido retinóico (PML/RAR-ALFA).
- LMA com eosinófilos anormais na medula óssea {inv(16)(p13q22} {t(16;16)(p13;q11)}, {fator de ligação núcleo beta/cadeia de miosina pesada 11, musculo
liso (CBFb/MYH11)} - LMA com anormalidades em 11q23-Lysine(k)-
specific methyltransferase 2A (LML)
LMAs com alterações relacionadas à mielodisplasia
- Com síndrome mielodisplásica pregressa - Sem síndrome mielodisplásica pregressa
Neoplasias mieloides relacionados à terapia (NM-T)
- Relacionados a agentes alquilantes - Relacionados ao inibidor da topoisomerase II
- Outros tipos
LMAs sem outra especificação (LMA, SOE)
-LMA pouco diferenciada -LMA sem maturação -LMA com maturação
-Leucemia mielomonocítica aguda -Leucemia monoblástica e monocítica aguda
-Leucemia eritróide pura -Leucemia megacariocítica aguda
-Leucemia basofílica aguda -Pan-mielose com mielofibrose aguda
Sarcoma mieloide
Proliferações mieloides relacionadas a síndrome de
Down
Mielopoese anormal transitória (MAT) Leucemia mieloide associada com síndrome de Down
Fonte: Adaptado de ARBER, 2016.
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A citogenética e os estudos moleculares são capazes de detectar
anormalidades dentro do clone leucêmico, sendo de muita utilidade na definição do
diagnóstico e/ou prognóstico. Alterações cromossomais e moleculares recorrentes
quando identificadas ao diagnóstico, fornecem informações de prognóstico valiosas
tais como: Resposta a indução da quimioterapia, risco de recidiva e sobrevida global
do paciente. Sendo assim, pode-se observar que a citogenética seguida da genética
molecular são fundamentais para desvendar a heterogeneidade das LMAs (ILYAS et
al., 2015).
Além da morfologia e da citogenética, a imunofenotipagem por citometria de
fluxo é uma técnica que possibilita a análise de múltiplos parâmetros (MILLER;
PILISHOWSKA, 2014) para avaliação qualitativa e quantitativamente de padrões de
expressão de antígenos (Clusters designations – CDs) em populações celulares, por
meio da ligação com anticorpos monoclonais específicos, e que auxilia tanto na
diferenciação de tecido, mieloide ou linfoide, quanto na caracterização da etapa
maturativa da célula afetada (PETERS & ANSARI, 2011).
2.3 Imunofenotipagem e marcadores imunofenotípicos em leucemias mieloides agudas
A imunofenotipagem por citometria de fluxo é uma técnica que começou a
surgir com o desenvolvimento dos corantes citológicos. Porém, somente em 1950, foi
provada a detecção de antígenos através de anticorpos fluorescente por Coons e
Kaplan. Com isso, o uso de fluorescência se tornou habitual e a citometria de fluxo
usual nas áreas de hematologia e imunologia (KHAN; BOLTON, 2014). Mack foi o
inventor do precursor dos citômetros de fluxo de hoje, especialmente quando se diz
respeito à separação de células em 1965. O primeiro dispositivo de citometria de fluxo
baseado em fluorescência foi desenvolvido em 1968 por Göhde (NASSAR et al.,
2015).
Nos dias atuais, os citômetros de fluxo modernos são empregados em pesquisa
biológica e diagnósticos clínicos para a determinação de número e concentração de
um ou mais tipos de células sanguíneas. Essa análise é realizada pela passagem de
célula por célula por uma corrente estreita onde incide um feixe de laser. Sinais ópticos
como dispersão frontal da luz (FSC- Forward scatter), dispersão lateral da luz (SSC -
Side scatter) (Figura 2) e emissão de luz fluorescente (FL – Fluorescência relativa)
11
são simultaneamente mensurados para obter informações como o tamanho das
células, granulosidade ou complexidade interna (HUANG et al., 2014).
A imunofenotipagem por citometria de fluxo é uma técnica de análise e
quantificação de proteínas em suspensões celulares. Hoje, é um passo crítico quando
se trata de investigação e tomadas de decisões clínicas para leucemias, HIV e uma
série de outras doenças e serve como uma ferramenta para a triagem de testes
genéticos e moleculares recorrentes (O'NEILL et al., 2015). Além disso, as análises
são realizadas em apenas algumas horas, tornando-a uma ferramenta veloz, ao
contrário dos ensaios moleculares, que além de serem mais lentos necessitam de um
alto nível de conhecimento técnico (PETERS & ANSARI, 2011).
Os dados gerados pela citometria de fluxo são analisados pela aplicação de
regiões ou “gates” que permitem que sejam selecionados os dados de interesse e
descartados os dados que não são interessantes. Essas regiões podem formar
combinações que, ao serem aplicadas, torna viável a imunofenotipagem detalhadas
de toda a população de células. Citômetros modernos estão disponíveis com oito
cores ou mais, permitindo a avaliação de dez ou mais parâmetros simultaneamente
(TUTE, 2011).
Figura 2: Detector de dispersão de luz frontal: Tamanho celular - FSC (Forward Scatter) e detector de dispersão de luz em ângulo reto: Complexidade celular - SSC (Side Scatter). Fonte: http://regmed.musc.edu/flowcytometry/flowcytometry.html
12
A imunofenotipagem por citometria de fluxo é uma das principais técnicas de
relevância para a classificação e o diagnóstico de neoplasias hematológicas e, nos
últimos 20 anos, ganhou espaço como principal forma de analisar características
celulares em amostras de sangue periférico, medula óssea, linfonodos, biopsias,
liquido cefalorraquidiano e ainda outras amostras suspeitas de neoplasias
hematológicas (KALINA et al., 2012).
No estudo das características imunofenotípicas das LMAs, os pacientes
apresentam duas características distintas: Uma população de células que representa
uma fase inicial da hematopoese e uma população de células que representa uma
fase de maturação mais tardia. As duas são geneticamente relacionadas. Estas
populações de células de sinalização têm também perfis distintos, que parecem
representar LMAs de diferentes estágios de diferenciação. Indicadores internos
funcionais, marcadores de superfície celular, e conjuntos centrais de marcadores
podem ser examinados simultaneamente para elaborar um quadro mais completo de
sinalização celular e identificar essas populações. Além disso, existe uma série de
painéis que permitem analisar a ocorrência de apoptose, vias de sinalização, ciclo
celular e danos no DNA (NOLAN, 2011).
Os marcadores imunofenotípicos de linhagem mieloide mais frequentes são:
Mieloperoxidase (MPO), CD13, CD33, CD117, CD15, sendo os principais o CD13, o
CD33 e a MPO (MILLER; PILISHOWSKA, 2014). Porém, devido a evidente
heterogeneidade imunofenotípica apresentada pelas LMAs, existem marcadores bem
característicos de acordo com seus diversos subtipos. Na figura 3 é a expressão de
cada marcador imunofenotípico está representada de acordo com a cor, quanto mais
escura, mais forte a expressão do marcador. Como pode ser observado, o CD34, que
é um marcador de blastos não está presente em alguns subtipos, por serem
compostos de células mais maduras. O CD64 e o CD36 são fortemente expressos em
subtipos de linhagem monocítica e já perdem um pouco a expressão na LMA
mielomonocítica, que também é caracterizada por um componente mieloide
(mieloblastos). Já o CD71 e o 235a são fortemente expressos na Eritroleucemia,
sendo o CD71 exclusivo desse subtipo de LMA (IKOMA et al., 2016).
No Brasil, o Grupo Brasileiro de Citometria de Fluxo (GBCFLUX), padronizou
em 2015, painéis de triagem para leucemias agudas, incluindo as leucemias mieloides
agudas, como é mostrado no quadro 4.
13
Todos os painéis foram concebidos para acomodar diferentes níveis de
recomendações para a precisão do diagnóstico e classificação, para permitir algum
grau de flexibilidade de acordo com os recursos laboratoriais locais disponíveis. A
proposta dos painéis para LMAs teve como objetivo a detecção e classificação das
LMAs com foco no perfil de avaliação de linhagem e maturação de células blásticas
(Quadro 5). As recomendações mandatórias contêm os requisitos mínimos para
identificação, quantificação e classificação de LMAs (IKOMA et al., 2015).
Apesar de já existirem esses marcadores imunofenotípicos predefinidos nos
painéis de diagnóstico para LMAs, ainda há uma grande necessidade do estudo de
novos marcadores para serem incluídos na rotina com o objetivo de cada vez mais
aumentar a precisão no diagnóstico devido à heterogeneidade desse grupo de
neoplasias, para que sejam definidas as medidas de tratamento mais adequadas e,
consequentemente haja uma melhoria na sobrevida global e uma diminuição na taxa
de mortalidade associada a tratamento das LMAs (LARSEN et al, 2011).
Figura 3: Marcadores mieloides e suas intensidades de expressão de acordo com o subtipo de Leucemia mieloide aguda. Fonte: Adaptado de ARBER et al., 2016.
14
Quadro 4: Painel de triagem para leucemias mieloides agudas.
PAINEL MANDATÓRIO
TUBO 1 HLA-DRFITC/CD117PE/CD45PercP/CD34APC
TUBO 2 CD16FITC/CD13PE/CD45PercP/CD11bAPC
TUBO 3 CD36FITC/CD64PE/CD45PercP/CD14APC
TUBO 4 CD71FITC/CD235aPE/CD45PercP/CD33APC
TUBO 5 CD15FITC/CD61PE/CD45PercP/CD13APC
TUBO 6 CD2FITC/CD56PE/CD45PercP/CD4APC
Fonte: Adaptado de IKOMA et al., 2015.
Quadro 5: Descrição dos marcadores que compõem o painel mandatório do Grupo
Brasileiro de Citometria de Fluxo.
Marcador Imunofenotípico Descrição
HLA-DR; CD45; CD34 Marcadores de blastos
CD117 Marcador de blastos/linhagem mieloide
CD13 Marcador de linhagem mieloide
CD33 Marcador de linhagem mieloide
CD11b Marcador de expressão assincrônica
CD16 Marcador de expressão assincrônica
CD15 Marcador de linhagem neutrofílica/assincrônica
CD14 Marcador de linhagem monocítica/assincrônica
CD64 Marcador de linhagem monocítica
CD36 Marcador de linhagem
monocítica/eritróide/megacariocítica
CD71 Marcador de linhagem eritróide
CD235a Marcador de linhagem eritróide
CD56 Marcador de células Natural Killer
CD4 Marcador de linhagem monocítica
CD2 Marcador para identificação de expressão linfoide
aberrante
Fonte: Adaptado de IKOMA et al., 2015
15
2.4. Novos marcadores imunofenotípicos
O estudo de novos marcadores imunofenotípicos é realizado com o intuito de
ampliar a pesquisa na diferenciação das células leucêmicas e seu impacto no
prognóstico e sobrevida dos pacientes com LMA. Alguns marcadores podem ser
destacados por auxiliarem na determinação de prognóstico e por estarem associados
a uma baixa sobrevida, como pode ser observado nos seguintes antígenos: O CD87,
o CD135, o CXCR4 (CD184), o CD133, o TRAILR2 (CD262), o TRAILR3 (CD263) e o
TNFR1.
CD87: É um receptor glicosilado que, ligado a uma serina protease específica
ativadora de plasminogênio (uPA), inicia a conversão de plasminogênio em plasmina.
Além disso, o CD87 exerce vários efeitos regulatórios sobre a migração celular,
adesão de leucócitos, quimiotaxia e na transdução de sinais citoplasmáticos ao
citoesqueleto (ATFY et al., 2011).
A uPA promove a migração celular devido a possibilidade de iniciar a proteólise
pericelular; o complexo-uPA agrupa e polariza sítios focais da célula-substrato,
favorecendo a atividade da plasmina na degradação das membranas basais,
facilitando o movimento celular através das barreiras do tecido. Já a quimiotaxia e a
adesão acontecem pela ligação de proteínas aos receptores dos 3 domínios ligados
por dissulfureto (D1, D2 e D3). Uma mudança na conformação de uPA dependente
de uPAR revela esses domínios quimiotáticos, promovendo a atração de leucócitos
polimorfonucleares. A adesão à matriz celular ocorre com a participação da
fibronectina e vitronectina, ligantes que irão se unir a uPAR e favorecer a adesão
celular (SHEN et al., 2015).
CD135: Denominado FLT3 (fms related tyrosine kinase 3) é um receptor de tirosina-
quinase (RTK) ligado à membrana, com domínios extracelulares: transmembranar,
justamembranar e domínio tirosina-quinase. Pertence à subfamília da classe III das
RTK e é expresso em células mieloides e progenitoras linfoides, com expressão
variável em linhagem monocítica madura. A interação do receptor com o ligante FLT
(FL) leva a uma alteração conformacional com exposição de um de seus domínios,
dimerizando o receptor e ativando a enzima tirosina-quinase. Essa reação conduz a
uma fosforilação dos sítios intracelulares e a ligação com proteínas, formando
16
complexos proteicos, iniciando uma cascata de reações de fosforilação que ativam
uma série de mediadores secundários e promovem a transdução de sinal no núcleo.
Essa série de eventos regula a diferenciação celular, proliferação e apoptose, como
mostra a figura 4 (MESHINCHI; APPELBAUM, 2009).
O CD135 desempenha um papel importante na regulação da hematopoese
normal e crescimento celular. Junto com fatores de crescimento como CSF e IL-3
promovem a proliferação de células progenitoras hematopoéticas primitivas, bem
como as células comprometidas mieloides e precursores linfoides. Sua expressão foi
avaliada em células leucêmicas e dados sugerem que níveis muito elevados de
receptores de FLT3 podem promover a ativação típica do receptor do tipo selvagem
em células malignas (SHARAWAT et al., 2013).
Fonte adaptada: MESHINCHI; APPELBAUM, 2009.
CXCR4: é um receptor de quimiocina CXC 4 para fator derivado do estroma 1 (SDF1),
que desempenha um papel no desenvolvimento da hematopoese e organização do
sistema imunológico. Esses receptores são de uma família de sete domínios
transmembranares, que estão na superfície celular acoplados a G-proteína-CXCR1
(MANNELLI et al., 2015).
Figura 0-4: Via de transdução de sinal do receptor FLT3.
17
As quimiocinas são proteínas pequenas que são secretadas e podem ser
agrupadas em duas subfamílias principais com base em dois resíduos de cisteína
conservados, separados por um aminoácido interveniente, representando por CXC ou
quimiocinas CC. Os receptores das quimiocinas estão presentes em muitos tipos
celulares. Inicialmente, foram identificados em leucócitos, desenvolvendo um papel
importante no “homing” dessas células para os locais de inflamação. No entanto,
durante os últimos anos, as células hematopoéticas e não-hematopoéticas foram
encontradas expressando receptores para várias quimiocinas, em tecidos de
microambientes diferentes. As interações entre esses receptores e suas respectivas
quimiocinas ajudam a coordenar o curso e organização de células dentro de vários
compartimentos dos tecidos (BAE, et al. 2015).
CD133: Este antígeno (prominin-1) é uma molécula com cinco domínios
transmembranares em células progenitoras hematopoéticas normais primitivas. Sua
expressão está associada com as funções de crescimento celular, desenvolvimento e
origem de tumores sólidos, bem como a infiltração e resistência a quimioterapia, como
mostra a figura 5 (LI, 2013).
Fonte adaptada: LI, 2013. Figura 0-5: Esquema funcional de células CD133+.
18
TRAILR2 (CD262), TRAILR3 (CD263) e TNFR1: O antígeno CD262 é um marcador
imunofenotípico expresso em células T e Natural Killer (NK), responsável pela indução
de apoptose em células de origem linfoide. Eles agem sobre a regulação do sistema
imune e são responsáveis pela indução de morte celular em células de leucemia
linfoide crônica e linfoma não-Hodgkin. O CD263 é expresso em neutrófilos e
granulócitos e atua sobre a regulação da apoptose por meio da atividade de ligação
competitiva. O TNFR1 é um mediador de citotoxicidade, expresso em granulócitos,
monócitos e linfócitos que também está envolvido em outas funções como ativação
endotelial e adesão, proliferação de células T, dentre outros (SCHMOHL et al., 2015).
19
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24
CAPÍTULO 1
25
ROLE OF NEW IMMUNOPHENOTYPIC MARKERS ON
PROGNOSTIC AND OVERALL SURVIVAL OF ACUTE
MYELOID LEUKEMIA: A SYSTEMATIC REVIEW AND META-
ANALYSIS
Costa AFO¹, Menezes DL¹, Pinheiro LHS¹, Sandes AF², Nunes MAP³, Lyra Junior DP¹,
Schimieguel DM¹
¹Department of Pharmacy, Laboratory of Hematology, Federal University of Sergipe,
Aracaju, Sergipe, Brazil.
²Fleury Group, Hematology Division, São Paulo, São Paulo, Brazil.
³Department of Medicine, Federal University of Sergipe, Aracaju, Sergipe, Brazil.
*Corresponding author:
[email protected] (AF)
26
Abstract
Despite technological advances, the prognosis and survival of acute myeloid leukemia
(AML) adult patients remain low, compared with other hematologic malignancies.
Some antigens detected by immunophenotyping may soon play a significant role in the
pathophysiologic, prognostic, and overall survival (OS) rate of AML patients. Therefore,
we conducted a systematic review and meta-analysis of PubMed, Scopus, Science
Direct, Web of Science, and the Cochrane Library (using PRISMA guidelines). We
analyzed 11 studies and 13 antigens, detected through the immunophenotyping of 639
patients. From them, twelve exhibited a negative impact with AML prognosis. The
meta-analysis demonstrated a high expression of AML markers, which have been
associated with a decrease in survival over 10 months (RR 2.55; IC 95%; 1.49-4.37)
and over 20 months (RR 2.46; IC 95%; 1.75-3.45). Knowing that the expression of
immunophenotypic markers, which are not used on a routine basis, might be able to
influence disease behavior, looks promising. However, they have been associated with
a poor prognosis as well as a decrease in survival. This may allow for different
chemotherapeutical protocols, including future studies for new therapeutic targets.
Introduction
Acute myeloid leukemia (AML) is an aggressive hematological malignancy
characterized by clonal proliferation of immature myeloid cells at various stages of
maturation 1, 2. The genetic heterogeneity of this group of hematological malignancies
makes it impractical to perform initial analyses that can encompass different genes
involved in AML. This makes diagnoses difficult, which can negatively influence
therapeutic strategy 3, 4. Even with major improvements in the understanding and
treatment of AML over the past several years, few advances have been made in the
27
outcomes and survival of AML patients 5, 6. Complete remission is expected for
approximately 60%‒70% of adults with AML after the induction phase of treatment, but
only about 25% survive three or more years with the possibility of being cured 7. The
5-year relative survival rate for patients from birth to 19 years has been reported to be
62.8%, but declines to 5.4%, for patients older than 65 years 8.
Multiparameter flow cytometry (MFC) immunophenotyping provides relevant
information for AML diagnosis, classification, and monitoring. MFC allows
identification, quantification, and lineage assessment of abnormal blast cells and
disease classification according to the maturation stage to be made 9, 10. AML presents
highly heterogeneous immunophenotypic profiles, which is probably due to genetic
diversity. Leukemia-associated phenotypic markers (LAIPS) are useful to discriminate
between normal/reactive immature myeloid precursors from leukemic cells and is
commonly used in minimal residual disease (MRD) studies. In addition, LAIPS are also
associated with molecular alterations with well-recognized prognostic implications
(such as CD19 expression in AML with RUNX1-RUNX1T1) 11, 12.
Previous studies show that a great number of distinct antigens affect AML
prognosis and prediction. Nevertheless, difficulties are still found in performing
accurate risk stratification for diagnoses based on immunophenotypic features 13.
Improving the accuracy of prognostic assessment of AML may allow the treatment to
be more specific and risk-adapted, increase the probability of cure, and minimize
treatment-related morbidity and mortality 14.
Since there are no clear recommendations and no consistent approaches to the
use of new markers in the immunophenotypic panels for AML evaluations as well as
for the markers' influence on determining prognosis and survival in clinical practice, a
systemic review was done. In this review, we aimed to identify relevant publications
28
about the influence of these new monoclonal antibodies used as immunophenotypic
markers in the prognosis and survival of AML patients.
Material and methods
A systematic review was performed based on a scientific research protocol
describing the aims and methods used. Within the limitations of the research in this
area, this synthesis was performed according to the Preferred Reporting Items for
Systematic Reviews and Meta-Analyses (PRISMA) statement 15.
The question of this systematic literature review was: “Are there new
immunophenotypic markers that may aid in the prognosis and survival of acute myeloid
leukemia?”
Search Strategy
The literature search was conducted using Pubmed, Science Direct, Web of
Science, Scopus and Cochrane Library databases for articles published from 2012 to
2015. In addition, the reference lists of relevant papers were searched for additional
AML studies.
The following search terms consisted of a range of pertinent terms: 1) antigens
CD (MeSH); 2) antigens, differentiation (MeSH); 3) biological Markers (MeSH); 4)
tumor markers, 5) biological (MeSH) and prognosis (MeSH); 6) survival rate (MeSH);
7) survival analysis (MeSH) and leukemia myeloid, acute (MeSH); or 8) acute
leukemia.
Study selection
The articles found in the search were compared with the previously defined
inclusion criteria to determine the relevance of the study: (1) papers published from
2012 to 2015; (2) articles published in English, Spanish, and Portuguese; (3) articles
29
that used immunophenotyping in their methodologies; (4) articles assessing
monoclonal antibodies not included in the consensus diagnostic panels for AML
(Euroflow consortium 16); (5) articles assessing AML cases; and (6) articles with
available abstract and full text.
Systematic and literature reviews, meta-analysis, editorials, conference
proceedings and books were excluded from the study.
Two reviewers independently evaluated the titles and abstracts of the identified
publications by applying the inclusion criteria. Potentially relevant articles were
retrieved in full. The final inclusion of articles into our systematic review was based on
agreement between both reviewers. In case of any disagreements between the two
reviewers, a third reviewer inspected the full text article and finalized the decision
whether or not to use the article.
Rating quality of individual studies
The methodological quality of each individual study was evaluated using the
Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)
assessment scale, which consisted of 22 items 17. High scores meant that there was
sufficient information and good design. STROBE was a highly feasible and applicable
method to use for evaluating systematic reviews of observational studies.
Data extraction and management
From the included studies, information regarding several parameters was
obtained: (1) journal of publication; (2) The Journal Citation Reports (JCR) impact
factor; (3) location; (4) study design; (5) aim of the study; (6) number of samples
analyzed; (7) AML classification; (8) most incident subtype; (9) immunophenotypic
marker; (10) prognostic value; (11) first induction treatment protocol (12) follow up; (13)
survival; (14) limitations; and (15) STROBE scores. For inaccessible or incomplete full
30
texts, authors were contacted for additional information.
Statistical analysis
A meta-analysis of the relative risks related to the probability of survival at 10 to
20 months was performed. For analysis purposes, low expression of the
immunophenotypic marker was considered as absent, and high expression was
considered as present.
Survival analysis or time for the event were analyzed by dichotomous data using
the knowledge of the situation of all patients in the study at 10 and 20 months 18. A
contingency table was constructed (Table 1) to analyze every connection and then the
relative risks were calculated using this equation:
𝑅𝑖𝑠𝑘 𝑜𝑓 𝑡ℎ𝑒 𝑒𝑣𝑒𝑛𝑡 𝑜𝑛 𝑡ℎ𝑒 𝑔𝑟𝑜𝑢𝑝 𝑤𝑖𝑡ℎ 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛
𝑅𝑖𝑠𝑘 𝑜𝑓 𝑡ℎ𝑒 𝑒𝑣𝑒𝑛𝑡 𝑜𝑛 𝑡ℎ𝑒 𝑔𝑟𝑜𝑢𝑝 𝑤𝑖𝑡ℎ 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛=𝑎 𝑎 + 𝑏
𝑐 𝑐 + 𝑑 1
A correction value of 0.5 was used in order to enable the statistical analysis
using the absence of death in the absence of one of the immunophenotypic markers
in a 10 month follow up of survival and no patient survival in the presence of the
markers in a 20 month follow up of survival as effect measures 5, 29
The heterogeneity of the meta-analysis was assessed using the Cochran Q and
Higgins I² tests. We used the relative risk as an effect measure after taking into
consideration the number of people who would be alive in the absence and presence
of the immunophenotypic marker expression at 10 and 20 months. Meta-effect
estimates were reported and relative risks summarized with their 95% confidence
intervals. The funnel graphs and regression testing of asymmetry were used to assess
potential publication bias related to survival at both 10 and 20 months. The bias was
considered significant at p = 0.05. All analyses were performed with the program R
version 3.3.1 19 and the "Metafor" package 20.
31
Results
The literature search
The literature search retrieved 9,950 articles. After screening titles and
reviewing abstracts, we identified 30 potentially relevant articles that focused on
immunophenotypic markers and MFC (Fig. 1). In the final analysis, a total of 11 studies
were included in the qualitative synthesis of this review .One of them was found after
a hand search of reference lists 5, 21‒30. Three of them were used on the meta-analysis
for 10-month survival (21,29,30) and four were used on the 20-month survival
analysis(5,28,29,30).
32
Figure 1: Flow diagram for study identification.
Study Characteristics
An overview of the characteristics of the 11 studies included in the final analysis
is summarized in Tables 2 and 3. The sample size ranged from 12–142 patients with a
33
total of 639 hematological samples analyzed in all studies 22, 23, 25, 28. Two studies used
the World Health Organization (WHO) classification for AML, six used the French-
American-British (FAB) classification, and three used both FAB and WHO to classify
the AML subtypes 5, 21‒30. The most reported subtype in the articles was FAB M2
followed by FAB M4.
Most studies were performed in countries with high scientific and technologic
development, including the United States (US), Germany, and Japan 5, 21‒24, 26, 29. The
Journal Citation Reports (JCR) impact factor found in the search ranged from 1.826 to
22.268 24, 29. There were only two pediatric studies, and the others focused on adults
25, 30. The expression of 13 different antigens was evaluated in all articles (Table 4). All
11 studies were based on proving the impact of novel antigens on AML prognosis while
five of them were cohort studies that also proved the influence of these markers on
patient survival 5, 21, 28‒30. The most common chemotherapy regimen was
anthracycline-based induction therapy (3+7), although other alternative treatments
were chosen by some groups.
The methodological quality of observational studies conducted with the
STROBE tool showed that eight articles scored > 90% revealing a high methodological
quality of the included studies. One article scored 19 points, which was equivalent to
86.4%, four scored 20 points, which was equivalent to 91%, and four scored 21 points,
which was equivalent to 95.4% 5, 21, 24, 25‒30.
Prognostic value and survival
Most articles showed that the expression of the evaluated antigens has a
negative impact on prognosis of AML. Only one article showed that CD263 has a
positive value on prognosis, and two articles showed that the values of CD90 and ILT3
were not determined 25, 26, 29. All five cohort articles showed a decreased survival
34
related to the high expression of the immunophenotypic markers.
Two articles by the same authors discussed CD82, a member of the tetraspanin
superfamily that was originally identified as an accessory molecule in T-cell activation
and in nonimmune cells in integrin-mediated cell adhesion to the extracellular matrix.
Both articles evaluated the expression of CD82 in the self-renewing leukemia stem cell
(LSC) compartments (CD34+/CD38- cells) and the CD34+/CD38+ compartments of
AML cells. They showed that LSC expressed a higher amount of CD82 than
CD34+/CD38+ AML cells; these findings suggested that overexpression of CD82 may
render LSC able to adhere to the bone marrow (BM) niche where it appears to regulate
maintenance of leukemia stem cells within the BM niche. In addition, they showed that
down-regulation of CD82 in LSC may stimulate mobilization of these cells from the BM
niche to PB and sensitize them to chemotherapeutic agents 22, 23.
CD87 is a urokinase plasminogen activator receptor that initiates the conversion
of plasminogen to the protease plasmin. CD87 is involved in signal transduction of
cytoplasmic signals to the cytoskeleton. Atfy et al. showed that high expression of this
antigen is associated with a decrease in AML patients' overall survival. Regarding
prognostic values, the authors presented an association of CD87+ with clinical
features; this association predicted a more aggressive course of the disease with a
negative prognostic impact. They suggested that CD87 expression should be included
in the initial diagnostic AML work-up 5.
CD93 is a C-type lectin transmembrane receptor that is involved in the
modulation of phagocytosis, inflammation, and cell adhesion. Using flow cytometry,
Iwasaki et al. evaluated the CD93 expression profile on CD34+CD38- cells of various
AML subtypes and normal cord blood. They observed that CD93 was expressed on a
significant percentage of cells in the LSC fraction of MLL-rearranged leukemias, while
35
this marker was negative on LSC subpopulations within non-MLL leukemias and
normal cord blood cells. Since MLL rearrangement is associated with a dismal
prognosis in acute leukemia, expression of CD93 in the LSC compartment of AML
cases may be a useful surrogate marker to identify this AML subgroup 24.
CD135 (FLT3) is a tyrosine kinase receptor that has a significant role in
leukemogenesis. Sharawat et al. evaluated CD135 and CD117 expressions in a cohort
of 115 AML patients (64 pediatric and 51 adults) and showed that CD135 was
expressed in 82% of all cases. There was no association of CD135 expression with
the FLT3 internal tandem duplication mutation, a molecular abnormality associated
with unfavorable AML prognosis.. Nevertheless, co-expression of CD135 and CD177
was associated with a decrease in event free survival (EFS) and overall survival (OS)
in multivariable analysis in both age groups 30.
Mannelli et al. investigated the expression of CXCR4 (CD184) in AML. CXCR4
is a receptor for stromal-derived factor 1 (SDF1) that plays a very important role in
hematopoiesis development and organization of immune system. After analyzing
whole blasts and CD34+ cells in 142 adult non-M3 AML cases, the authors used mean
fluorescence intensity (MFI) and showed a correlation between high CXCR4
expression and decrease in EFS and OS in addition to an unfavorable prognosis. In
addition, CXCR4 expression was associated with high leukemic burden, as estimated
by DHL level and white blood cell and peripheral blast cell counts 28.
CD133 is a novel five transmembrane molecule expressed on primitive normal
hematopoietic progenitors. Tolba et el. evaluated the expression of this antigen in 30
AML cases and 30 acute lymphoblastic leukemia patients and observed that CD133
was expressed in 56.6% of AML (n=17). The authors observed an important correlation
between the expression of CD133 and the survival of AML patients and demonstrated
36
a decrease in OS with an increase in CD133 expression. As for the prognostic value,
it was concluded that CD133 expression was highly associated with poor prognosis in
AML patients 21.
In 46 AML patients, Schmohl et al. assessed the expression of the death
receptors, including TRAILR1, 2, and 3 (CD261, 262, and 263, respectively), TNFR1
(CD120a), and FAS (CD95). CD262 is involved in induction of apoptosis in lymphoid
cells. CD263 is a marker of neutrophilic granulocytes that participates in apoptosis
regulation and inhibition of cell death through competitive binding activity. TNFR1 is
expressed on monocytes, lymphocytes, and granulocytes, and is involved in
cytotoxicity mediation. The authors concluded that CD262 and TNFR1 expressions
showed significantly shorter OS, earlier disease onset, and higher probability of
relapse in AML patients. Conversely, CD263 expression exhibited an enhanced OS.
As for prognostic value, high expressions of CD262 and TNFR1 were found to be
associated with unfavorable prognostic groups, and CD263 was found in cases with
favorable risk 29.
Dobrowolska et al. evaluated one member of the immunoglobulin-like
transcripts (ILT3 expression) in normal and leukemic myeloid precursors in 20 healthy
individuals and 37 AML cases. ILT3 is a member of the large family of ILT molecules,
leukocyte Ig-like receptors (LIRs), and monocyte/macrophage Ig-like receptors (MIRs).
The authors showed that ILT3 was expressed in all cases of AML displaying monocytic
differentiation but not in the AML subtypes M1/M2 and M3. It was shown that
expression of ILT3 by leukemic cells contribute to the inhibition of tumor specific T cell
responses. As for the prognostic impact, frequent cytogenetic abnormalities observed
in AML patients with ILT3+ were those associated with intermediate prognosis, but it’s
potential value as a prognostic marker, particularly in cytogenetically normal AML,
37
remains to be determined 26.
Larsen et al. evaluated hMICL expression in one article in 93 AML patients.
hMICL is a heavily glycosylated transmembranal C-type lectin with an unknown
function.. Cryopreserved mononuclear cells and bone marrow samples were used in
the analysis. They showed that hMICL was found to be restricted to the
CD45low/SSClow population of AML cells. The authors suggest that no loss of hMICL
expression in AML patients may be an early indicator of relapse. In addition, hMICL
preserved fluorescence intensity at relapse, suggesting that this antigen could be a
tool for minimal residual disease quantification by flow cytometry in AML 27.
CD90, a cell-surface glycoprotein, seems to be involved in proliferation and
expansion processes, and CD96 is a member of the immunoglobulin superfamily. In
one article, Chaves-Gonzales et al. evaluated the expression of these two antigens in
two distinct primitive cell population compartments from bone marrow. CD34+CD38-
cells (enriched for HSC) and CD34+ CD38+ cells (enriched for HPC) obtained from 12
pediatric AML patients were used for the analysis. The results showed that CD90
showed slight incremental increases in patients who reached remission and did not
relapse. As for CD96, the authors describe greater expression in relapse and higher
levels in AML cells than in normal bone marrow cells before and after chemotherapy
25.
Meta-analysis
Five cohorts were analysed at first, in which the results of quantitation of six
immunophenotypic markers were evaluated (5, 21,28, 29,30). The expression of these
markers are considered independent events, since their pathogenic significance are
not necessarily interconnected within certain pathways. The survival analysis over 10
38
months showed a great heterogeneity with a significant Cochran Q test (Q [df = 5]; =
11.3330; p = 0.0452) and the Higgins I² test showed a result of 83.33%, indicating high
heterogeneity and suggesting an impediment to achieving the meta-analysis. Despite
that impediment, the tests were still performed showing a meta-analytical estimate of
no significant risk (1.49). The funnel plot and regression testing for evaluation of
publication bias showed a large asymmetry (t = 5.0896; df = 4; p = 0.007).
(Supplementary fig. S1‒2).
Therefore, two studies were considered responsible for the asymmetry in the
analysis and withdrawn of the analysis in 10 months, remaining four events in the three
articles included (5, 29, 30). On the 20-month analysis, one article was removed for not
having information regarding 10-month survival, remaining five events in the four
articles included (5, 28-30).
The Q Cochran test for 10 months survival showed that the studies included in
the meta-analysis would be homogeneous (Q (df = 3) = 1.0512; p = 0.7889), and the
Higgins I² test showed a result of 0% indicating no heterogeneity. The same results
were found in relation to the markers collected at 20 months, both in relation to Q
Cochran test (Q (df = 4) = 2.6083; p = 0.6254) and the Higgins I² test (0%). It was
decided then, to perform the two meta-analyses using the fixed effects model.
The meta-analytical estimate represented by the relative risks of survival rate at
10 months (Fig. 2) was 2.55 (95% CI: 1:49‒4:37) when analyzed for the risk of survival
in the absence of the immunophenotypic marker expression and its relationship with
the risk of survival in the presence the immunophenotypic marker expression. The
estimate at 20 months (Fig. 3) was 2:46 (95% CI: 1.75‒3.45). Both sets of values were
significant.
39
Figure 2: Forest Plot of relative risks and confidence intervals of 10-month
survival.
40
Figure 3: Forest Plot of relative risks and confidence intervals of 20-month
survival.
Publication bias potential
After resetting the final models, especially the 10-month survival, the funnel plot
and the regression test for analysis of the asymmetry were used to assess the potential
for publication bias. Both tests showed that publication bias was not significantly
associated with both the 10 and 20 month survival (t = 0.9055, df = 2; p = 0.4608 and
t = 0.2818; df = 3; p = 0.7964, respectively) (Supplementary fig. S3 – 4).
Discussion
MFC has an important role in AML diagnosis, classification, and evaluation of
treatment effectiveness. Over the last several years, important achievements were
obtained in the field, including improvements in flow cytometry instrumentation (> 8
41
colors) and new analytical strategies. However, the discovery of new
immunophenotypic markers for AML diagnosis was limited, and immunophenotypic
panels have remained similar over the last twenty years. Therefore, there is still a need
for new markers for leukemic myeloid cells to be included in clinical routines to increase
the value of MFC not only as a diagnostic but also prognostic tool for monitoring of
MRD and for development of drugs for targeted therapy 31.
In this systematic review, we observed that some markers could be used for
AML diagnosis and during the follow up. For example, the high expression of CD87 is
an indicator of morphologically and antigenically poorly differentiated disease,
especially in the M4 and M5 subtypes 5. The inhibitory receptor ILT3 is a highly
sensitive and specific marker for both diagnosis and monitoring of AML with monocytic
differentiation; ILT3 as a marker is particularly useful in the differential diagnosis of AML
with monocytic differentiation and microgranular acute promyelocitic leukemia, two
leukemia subtypes that require different treatment strategies 26. Another marker,
hMICL, expressed significantly higher fluorescence intensity compared to normal bone
marrow, suggesting suitability of this antigen as a pan-AML marker 27. Regarding cell
adhesion, CD82 has been shown to have a high expression in AML cells suggesting
an increase of adhesion to the BM niche. On the other hand, down-regulation of CD82
in AML cells may stimulate circulation of these cells into peripheral blood 22.
Many prognostic factors have been established in the past few decades in AML,
including age, cytogenetic abnormalities, white blood cell count, serum lactate
dehydrogenase, and the presence of antecedent hematologic disorder. This
systematic review has shown that the high expression of CD87 on peripheral blood
blasts was associated with relapse and poor prognosis and could be incorporated into
the initial diagnostic work-up of AML patients 5. CXCR4 could be also included due to
42
its high expression on the surface of the entire leukemic population and demonstration
of its influence on the overall CR rate and as poor prognostic factor for DFS and OS
28. Another interesting study described the association between high expression of
CD93 and loss of CDKN2B (p15Ink4b) expression; a cell-cycle inhibitor gene that has
been shown to play a prominent role in leukemia pathogenesis and correlate with poor
prognosis 24.
The demonstration that the high expression of these immunophenotypic
markers influence not only the survival of AML patients but also the prognosis, brings
up some questions. Since immunomarkers are signaling those important prognostic
data and it is known that molecular mutation can also have influence on the disease
pathways regarding both survival and prognostic features, how these two information
can be combined on determine better accurate prognostic factors, and what
association could be found between them? We could observe that this data are rarely
combined on the articles included on this systematic review and meta-analysis. It was
evidenced that majority of patients that expressed high CXCR4 also presented the
NPM1 mutated gene, what gives it a worst prognosis 28. Another study showed that
FLT3 receptor (CD135) was associated with the FLT3 Internal tandem duplication (ITD)
mutation, which confers a poor prognosis 30. Since gene mutations are of utmost
importance in deciding the patients' risk-stratification, the knowledge of this association
can bring advancement on treatment decision and give even more precision to it,
raising the chances of cure. From this preliminary information, it is possible to envision
future studies involving the connection of these two types of markers to predict
prognosis and survival of AML.
Despite the advancement of therapeutic progress, the overall survival of AML
patients remains low 5. One method to attempt to increase these patients' survival
43
could be a more specific chemotherapeutic protocol for this heterogeneous group of
diseases. It is also necessary to identify in advance those patients who have greater
resistance to treatment, higher relapse rates, and lower survival. We performed a
meta-analysis using the fixed effects model. Four new immunophenotypic markers,
CD133, CD135, TRAIL2 (CD262), and TNFR1 (CD120a) were demonstrated and
showed correlation with lower survival at 10 and 20 months. Thus, these antigens can
be used as early markers in combination with other prognostic factors for risk
stratification of relapse. CD133 antigen appears to be expressed restrictively in the
more immature cell population, and examination of the articles has made it possible to
observe that expression of the CD133 in acute leukemia could be correlated with an
immature phenotype of the myeloid blasts and highly associated with poor prognosis
5. Proliferation regulators (such as tyrosine kinase receptors) play an important role in
the pathogenesis of acute myeloid leukemia. The high expression of CD135 was
associated with lower EFS and OS 30. As verified by this study, the expression of death
receptors is typically associated with the apoptotic regulation of leukemic blasts that
demonstrated a significant association of TRAILR2 expression on blasts from patients
in adverse risk groups and showed a negative impact on overall survival. Results
concerning TNFR1 showed that this receptor is for the immune-modulating cytokine
TNF. TNFR1 may play a role in initiation and proliferation of AML blasts due to its high
expression, which appears to be related to a lower survival in AML patients 29.
Technically, this meta-analysis demonstrated that meta-analytical estimates,
represented by the relative survival risk at 10 and 20 months, were both significant
when the risk of survival in the absence and presence of the markers was analyzed.
Notably, in our analysis, there was no heterogeneity among the cohort studies, which
implied that the present method to combine the results from these studies was
44
reasonable. Thus, the conclusions from this analysis should be credible. However, due
to the relatively limited number of cases included in these studies, further analyses of
larger series of patients are needed to confirm these preliminary observations.
Minimal residual disease is a term used to describe detection of subclinical
levels of leukemia using multiparameter flow cytometry or molecular-based
approaches. Employing the MFC for minimal residual disease detection appears to be
a reliable method for obtaining rapid and objective patient remission status, provide
early end points in clinical trials, and to inform patient management of a patient's status.
Emerging evidence indicates that MRD detection in patients with AML is also
associated with poor prognosis, and early therapeutic interventions may be of clinical
benefit. In addition, other studies have reported on the progression of new drug
development that target specific areas of the leukemia cells; this is important since the
current treatment can help, but does not always cure, AML patients. In this review, we
observed that some antigens can be useful both as a marker for MRD and potential
therapeutic target. ILT3 is an antigen expressed in AML displaying monocytic
differentiation that supports differentiation and subsequent AML diagnosis of AML. It
may also be a candidate marker for MRD detection in AML patients due to its high
sensitivity, specificity, and stable expression. This antigen also can be a target for
therapy in AML with monocytic differentiation as a result of inhibition of ILT3 signaling
with specific antibodies or antagonists, which may render ILT3+ AML cells more
susceptible to differentiation agents and anti-tumor T cell responses 26. CD93, an AML
with MLL rearrangements marker expressed on leukemia stem cells (LSC), may also
be a useful therapeutic target candidate for anti-LSC therapy and prognostic marker
for quantitation of minimal residual disease 24. hMICL is a pan-AML marker uniformly
present on cells. It can be poorly immunophenotypically lineage-characterized and
45
difficult to monitor for residual disease and development of drugs for targeted therapy
27. The glycoprotein CD82 could be an attractive target for LSC eradication, due to its
important role in regulation of the AML cell survival and their adhesion to bone marrow
microenvironment 22.
There are some limitations that should be noted in our systematic review and
meta-analysis. One of the most important is the lack of actual patient data included in
the analysis. Despite that, it is known that AML is a disease with low incidence. In the
US, the estimate for 2024 predicts that leukemia, including AML, will represent 3% of
all cancers in male and females 8. It was notable that some data discrepancies were
found as in the case with hMICL, in which the author associated its high hMICL
expression with relapse, but its role in the prognosis was not clear. More data are
necessary to establish the extent that hMICL-based immunophenotyping can detect
treatment failure, which is common in most AML patients. The same results were
observed for CD90 and CD96, in which the author also shows an association of its
high expression with relapse, but does not associate either CD90 or 96 with prognosis.
CD96, especially, does not demonstrate high fluorescence in the first diagnosed
sample, only in the normal bone marrow sample.
Regarding the limitations of our systematic review and meta-analysis, survival
information was extracted from survival curves and not from a mortality table.
Secondly, positive and negative expression were considering basing on the MFI of
each study Third, the criteria to determine the positive or negative antigen expression
varied across the included studies. Fourth, from the six immunophenotypic markers
included in the meta-analysis, two were removed as they caused heterogeneity
between the studies, which may have been due to some methodological differences.
Fifth, we only searched for articles published in English, Spanish, and Portuguese and
46
may have missed relevant publications in other languages. Finally, some studies
included in the qualitative synthesis also evaluated M3 AML patients, a subgroup
recognized for its excellent prognosis. However, only two studies included in the meta-
analysis contained M3 AML cases, both with a low number of patients (< 10%, table 3)
and did not cause any heterogeneity on the analysis.
Despite the limitations listed above, the present analysis revealed the prognostic
value of new antigen expression in AML. Although the high expression of CD133,
CD135, TRAIL2 and TNFR1 was not significant in most of the individual studies
regarding the occurrence of the outcome, when they were collected in the meta-
analysis a high correlation was observed with poor prognosis and low DFS and OS.
However, further prospective studies with larger sample sizes are required to include
these new immunomarkers in MFC routine of acute myeloid leukemia.
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50
Author Contributions Statement
Costa, A.F.O performed the systematic research, selected the studies, extracted
an analysed data and wrote the article; Menezes D.L performed the systematic
research, selected the studies and wrote the article; Pinheiro, L.H.S organized the
figures and tables and wrote the article; Sandes, A.F. analysed data, wrote and
reviewed the article; Nunes, M.A.P. did the meta-analytical analysis and wrote the
article; Lyra Junior, D.P. analysed data and reviewed the article and Schmieguel, D.M.
Selected the studies, analysed data, wrote and reviewed the article.
Additional Information
Competing financial interests
The authors declare no competing financial interests.
Figure Legends
Figure 1. Flow diagram for study identification. Flow chart of how the research was
systematically conducted for study identification.
Figure 2. Forest Plot of relative risks and confidence intervals of 10-month
survival. Relative risks and confidence intervals of survival at 10 months after the
withdrawal of the two studies that caused the asymmetry, associated with the non-
detection/detection of the immunophenotypic markers in each study and its meta-
analytical measurements.
Figure 3. Forest Plot of relative risks and confidence intervals of 20-month
survival. Relative risks and confidence intervals of survival at 20 months associated
with the non-detection/detection of the immunophenotypic markers in each study and
their meta-analytical measurements.
51
Tables Table 1: Contingency table (2x2).
Outcome
Total
Alive Dead
Negative Marker a b a + b
Positive Maker b d c + d
a + c b + d a + b + c + d
Table 2: Main characteristics of the individual studies analyzed on the systematic review and meta-analysis.
MARKER ARTICLE JCR LOCATION DESIGN AIM OF THE STUDY STROBE
CD82 Nishioka et al. (Int J
Cancer 2013) 5.085 Japan
Cross-sectional
observational
Analyze the protein expression profile of CD34+/CD38- AML cells and compare it with the expression profile of their CD34+/CD38+ counterparts using isobaric tags for relative and absolute quantitation (iTRAQ) and explored the
function of CD82 in CD34+/CD3- AML cells in vitro as well as in vivo.
17 (77.8%)
CD82 Nishioka et al. (Int J
Cancer 2014) 5.085 Japan
Cross-sectional
observational Explore the regulation of STAT5/IL-10 by CD82 and its impact on the survival of CD34+/CD38- AML cells.
17 (77.8%)
CD87 Atfy et al. (Med Oncol
2012) 2.634 Germany Cohort
Assess the prognostic significance of pretreatment detection of CD87 and the prevalence of its expression and value as a predictor for survival.
20 (91%)
CD93 Iwasaki et al (Cell Stem
Cell 2015) 22.268 USA
Cross-sectional
observational
Report that the cell surface lectin CD93 is a functional marker of LSCs in a specific genetic subtype of AML with rearrangements of the MLL gene.
20 (91%)
CD135 Sharawat et al
(Cytometry B 2013) 2.398 India Cohort Evaluate clinical significance of FLT3 (CD135) and c-KIT (CD117) coexpression on myeloblasts in AML.
21 (95.4%)
CXCR4 Mannelli et al (Cytometry
B 2014) 2.398 Italy Cohort Investigate the expression of connexins in primary human AML cells derived from unselected patients
21 (95.4%)
CD133 Tolba et al (Med Oncol
2013) 2.634 USA Cohort
Assess CD133 expression in patients with acute myeloid or lymphoblastic leukemia and to evaluate its correlation with the different clinical and laboratory data as well as its relation to disease outcome.
20 (91%)
TRAILR2 (CD262) Schmohl et al
(Anticancer research 2015)
1.826 Germany Cohort Evaluate the association of co-expression of TRAILR1-3, TNFR1 and FAS on AML blasts at first diagnosis with different
AML subtypes and risk groups and to combine with clinical data in order to evaluate their prognostic and clinical significance.
21 (95.4%)
TRAILR3 (CD263)
TNFR1
ILT3 Dobrowolska et Al
(Cytometry B 2013) 2.398 USA
Cross-sectional
observational
Investigated ILT3 expression by normal and leukemic myeloid precursors. We report that ILT3 expression identifies normal hematopoietic precursors committed to the monocytic lineage and that ILT3 is a reliable marker that
distinguishes AML with monocytic differentiation from other types of AML
19 (86,4%)
hMICL Larsen et Al (Cytometry
B 2012) 2.398
United Kingdom
Case control Based on data from stem cell research, they hypothesized that the human inhibitory C-type lectin like receptor (hMICL)
might represent a novel diagnostic and prognostic vehicle in a standard flow cytometry (FCM) setting. 20 (91%)
CD90 Chávez-gonzález et al.
(Arc Med Res 2014) 2.645 Mexico Case control
Analyze the expression of CD90, CD96, CD117, and CD123 on CD34+ CD38- cells, CD34+ CD38+ cells and CD34- CD38+ cells.
21 (95,4%)
CD96
AML: Acute myeloid leukemia; JCR: journal citation reports; iTRAQ: isobaric tags for relative and absolute quantification; STAT5: signal transducer and activator of transcription 5; IL-5: interleukin 5; LSCs: leukemic stem cells; MLL gene: mixed lineage leukemia gene; FLT3: fms related tyrosine kinase 3; c-KIT: receptor tyrosine kinase protein; TRAILR1-3: Tumor necrosis factor-related apoptosis-inducing ligand 1-3; TNFR1: Tumor necrosis factor receptor 1; FAS: cell surface death receptor; ILT3: immunoglobulin-like transcript 3; HSC: hematopoietic stem cell.
52
Table 3: Main disease and treatment features of the individual studies included on the systematic review and meta-analysis.
AML: Acute myeloid leukemia; TRAILR2: Tumor necrosis factor-related apoptosis-inducing ligand 2; TRAILR3: Tumor necrosis factor-related apoptosis-inducing ligand 3; TNFR1: Tumor necrosis factor receptor 1; hMICL: human myeloid inhibitory C-type lectin-like receptor; MDS: myelodysplastic syndrome; NR: not related; ATEDox: cytarabine, 6-thioguanine, etoposide, doxorubicin; EFS: Event-Free Survival; OS: Overall survival;
MARKER PATIENTS
(N) CLASSIFICATION TREATMENT PROGNOSIS
FOLLOW-UP
SURVIVAL GENE MUTATION
CD82 12 AML with
myelodysplasia changes: 4
NR POOR. NR NR NR
CD82 14 M4: 4
MDS transformed to AML: 4
NR POOR NR NR NR
CD87 110 M4: 36
Double-induction therapy with thioguanine, cytosine arabinoside (AraC), and daunorubicin (TAD) followed by high-dose Ara-C and mitoxantrone (HAM). M3 cases received therapy protocols that contained all-trans-
retinoic-acid (ATRA).
POOR 17 months High expression of CD87
predict shorter overall survival.
NR
CD93 36 Normal: 11 NR POOR NR NR NR
CD135 115 M2: 66 “3+7 “(Daunorubicin and cytosine arabinoside) with Daunorubicin at 60
mg/m2 for 3 days. POOR
15.5 months
High expression of CD135 predicted poor EFS and OS
FLT3 ITD – 17%
CXCR4 142 M2: 38 M4: 38
“3 + 7” (Cytarabine 100 mg sqm21 over 3-h intravenous infusion bid on days 1–7; Idarubicin 12 mg sqm21 30 min intravenous infusion on days
1–3). POOR 20 months
High expression of CXCR4 predict shorter overall
survival.
FLT3 ITD – 23,9% NPM1 MUTATED – 39,4% CEBPA MUTATED – 11,3%
CD133 30 NR ‘‘3 + 7’’ induction chemotherapy protocol: doxorubicin (30 mg/m2/ day)
for 3 days and cytarabine (100 mg/m2/day as a continuous 24-h intravenous infusion) for 7 days.
POOR 12
months.
Increased CD133 leads to decrease the survival by the
time. NR
TRAILR2
(CD262)
46 M2: 17
POOR
55 - 120 months
Cut-off analyses for TRAILR2 showed
significantly shorter overall survival
NR
TRAILR3
(CD263)
Twenty-six patients received an anthracycline-based induction therapy (idarubicin or daunarubicin), the remaining patients received other
approved therapies or supportive therapy GOOD
Cut-off analyses for TRAILR3 showed a increase in survival.
NR
TNFR1 POOR Cut-off analyses for TNFR1 showed significantly shorter
overall survival NR
ILT3 37 M4/M5: 18 NR POOR NR NR NR
hMICL 55 M4: 7 M2: 7
ND: 29 NR POOR NR NR NR
CD90 12
M2: 4
Following the first course of induction (ATEDox 5 cytarabine, mercaptopurine, doxorubicin), children without evidence of residual
disease were allowed to recover before subjected to a second identical induction course
UNDETERMINED
4-45moths
NR NR
CD96 POOR NR NR
53
54
Table 4: Basic features of each antigen analyzed on this systematic review and meta-analysis
Antigen Molecular Group Function Frequency
in AML
Association with specific disease
features
Prognostic Impact
CD82 A member of
tetraspanin superfamily Cell adhesion NR NR Poor
CD87 Urokinase plasminogen
activator receptor Conversion of
plasminogen to plasmin 72.2% FAB M4 and M5 Poor
CD93 C-type lectin
transmembrane receptor
Phagocytosis, inflammation, and cell
adhesion NR
Leukemia Stem Cells in AML with rearrangements
of the MLL gene Poor
CD135 Tyrosine kinase
receptor
Promote the growth and differentiation of primitive
hematopoietic cells 82% NR Poor
CXCR4 Receptor for stromal-
derived factor 1 (SDF1)
Cell adhesion and hematopoietic
stem cell niche regulation 50%
Hepato-splenomegaly and
extra-hematological disease
Poor
CD133 A novel five
transmembrane molecule
Regeneration, proliferation and differentiation of
Steam cells 56% FAB M4 and M5 Poor
CD262 Death receptor Apoptosis 21,7% Monocytic subtipes, AML
FAB M5 and M6 Poor
CD263 Death receptor Inhibition of cell death through competitive
binding activity NR AML FAB M0 Good
CD120a Death receptor
Mediation of cytotoxicity; signaling of fibroblast growth, endothelial activation/adhesion
21,7% AML FAB M2 and M5 Poor
ILT3 Immunoglobulin-like
transcript (ILT) 3
Inhibitory receptor: down-regulation of immune
responses. 83%
AML with monocytic differentiation and
microgranular acute promyelocitic leukemia
Poor
hMICL A glycosylated
transmembranal C-type lectin
Control of myeloid cell activation during
inflammation 89%
Poorly characterized CD34 negative patient
group Poor
CD90 Cell-surface glycoprotein
Proliferation and expansion processes
NR NR Undetermined
CD96 Member of the
immunoglobulin superfamily
Adhesive interactions of activated T and NK cells during the late phase of the immune response
NR NR Poor
FAB: French-American-British Classification; AML: Acute myeloid leukemia; TRAILR2: Tumor necrosis factor-related apoptosis-inducing ligand 2; TRAILR3: Tumor necrosis factor-related apoptosis-inducing ligand 3; TNFR1: Tumor necrosis factor receptor 1; hMICL: human myeloid inhibitory C-type lectin-like receptor; NK: Natural Killer; NR: not related.
55
ANEXO A – SUPPLEMENTARY INFORMATION
Supplementary figure S 1: Forest plot with relative risks and confidence intervals of survival in 10 month.
Relative risks and confidence intervals of survival at 10 months associated to the non-detection/ detection of the immunophenotypic markers in each study and its meta-analytical measurements before the withdraw of the two studies found to be responsible for the asymmetry.
56
Supplementary figure S 2: Funnel plot of studies on survival in 10 months.
Publication bias potential of 10-month survival associated with the non-detection / detection of the immunophenotypic markers in each study and its meta-analytical measurements before the withdraw of the two studies found to be responsible for the asymmetry.
57
Publication bias potential of 10-month survival associated with the non-detection / detection of the immunophenotypic markers in each study and its meta-analytical measurements after the withdraw of the two studies found to be responsible for the asymmetry.
Supplementary figure S 3: Funnel plot of studies on survival in 10 months.
58
Supplementary figure S 4: Funnel plot of studies on survival in 10 months.
Publication bias potential of 20-month survival associated with the non-detection / detection of
the immunophenotypic markers in each study and its meta-analytical measurements.
59
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it may be appropriate to combine sections. Figure legends are limited to 350 words.
As a guideline references should be limited to 60 (this is not strictly enforced).
Footnotes should not be used.
We suggest that Articles contain no more than 8 display items (figures and/or tables).
In addition, a limited number of uncaptioned molecular structure graphics and
numbered mathematical equations may be included if necessary. To enable typesetting
of papers, the number of display items should be commensurate with the word length
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as figures.
Authors must provide a competing financial interests statement within the manuscript
file.
Submissions should include a cover letter, a manuscript text file, individual figure files
and optional supplementary information files. For first submissions (i.e. not revised
manuscripts), authors may incorporate the manuscript text and figures into a single file
up to 3 MB in size; the figures may be inserted in the text at the appropriate positions,
or grouped at the end. Supplementary information should be combined and supplied
as a single separate file, preferably in PDF format.
ONLY the following file types can be uploaded for Article text:
txt, doc, docx, tex
A submission template is available in the Overleaf template gallery to help you prepare
a LaTeX manuscript within the Scientific Reports formatting criteria.
64
Scientific Reports is read by scientists from diverse backgrounds. In addition, many
are not native English speakers. Authors should, therefore, give careful thought to how
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should be avoided and clearly explained where its use is unavoidable.
Abbreviations, particularly those that are not standard, should also be kept to a
minimum. Where unavoidable, abbreviations should be defined in the text or legends
at their first occurrence, and abbreviations should be used thereafter. The background,
rationale and main conclusions of the study should be clearly explained. Titles and
abstracts in particular should be written in language that will be readily intelligible to
any scientist. We strongly recommend that authors ask a colleague with different
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The format requirements of Scientific Reports are described below.
Scientific Reports uses UK English spelling.
Cover Letter
Authors should provide a cover letter that includes the affiliation and contact
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Format of manuscripts
In most cases we do not impose strict limits on word counts and page numbers, but
we encourage authors to write concisely and suggest authors adhere to the guidelines
below. For a definitive list of which limits are mandatory please visit the submission
65
checklist page.
Articles should be no more than 11 typeset pages in length. As a guide, the main text
(not including Abstract, Methods, References and figure legends) should be no more
than 4,500 words. The maximum title length is 20 words. The Abstract (without
heading) - which must be no more than 200 words long and contain no references -
should serve both as a general introduction to the topic and as a brief, nontechnical
summary of the main results and their implications.
The manuscript text file should include the following parts, in order: a title page with
author affiliations and contact information (the corresponding author should be
identified with an asterisk). The main text of an Article can be organised in different
ways and according to the authors' preferences, it may be appropriate to combine
sections.
As a guideline, we recommend that sections include an Introduction of referenced text
that expands on the background of the work. Some overlap with the Abstract is
acceptable. This may then be followed by sections headed Results (with subheadings),
Discussion (without subheadings) and Methods.
The main body of text must be followed by References, Acknowledgements (optional),
Author Contributions (names must be given as initials), Additional Information
(including a Competing Financial Interests Statement), Figure Legends (these are
limited to 350 words per figure) and Tables (maximum size of one page). Footnotes
are not used.
For first submissions (i.e. not revised manuscripts), authors may choose to incorporate
the manuscript text and figures into a single file up to 3 MB in size - the figures may be
inserted within the text at the appropriate positions, or grouped at the end.
Supplementary Information should be combined and supplied as a separate file,
preferably in PDF format. The first page of the Supplementary Information file should
include the title of the manuscript and the author list.
Authors who do not incorporate the manuscript text and figures into a single file should
66
adhere to the following: all textual content should be provided in a single file, prepared
using either Microsoft Word or LaTeX; figures should be provided as individual files.
The manuscript file should be formatted as double-spaced, single-column text without
justification. Pages should be numbered using an Arabic numeral in the footer of each
page. Standard fonts are recommended and the 'symbols' font should be used for
representing Greek characters.
TeX/LaTeX - Authors submitting LaTeX files may use the standard ‘article’ document
class (or similar) or may use the wlscirep.cls file and template provided by Overleaf.
Non-standard fonts should be avoided; please use the default Computer Modern fonts.
For the inclusion of graphics, we recommend graphicx.sty. Please use numerical
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Please see this help article on Overleaf for more details. Alternatively ensure that the
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Manuscripts published in Scientific Reports are not subject to in-depth copy editing as
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Methods
Where appropriate, we recommend that authors limit their Methods section to 1,500
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67
their work. Descriptions of standard protocols and experimental procedures should be
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when the source is critical to the outcome of the experiments. Sources for kits should
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should be included. The systematic name of the compound and its bold Arabic numeral
are used as the heading for the experimental protocol. Thereafter, the compound is
represented by its assigned bold numeral. Authors should describe the experimental
protocol in detail, referring to amounts of reagents in parentheses, when possible (eg
1.03 g, 0.100 mmol). Standard abbreviations for reagents and solvents are
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References
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References should be numbered sequentially, first throughout the text, then in tables,
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that have been published or accepted by a named publication or recognized preprint
server should be in the numbered list; preprints of accepted papers in the reference
list should be submitted with the manuscript. Published conference abstracts and
numbered patents may be included in the reference list. Grant details and
acknowledgments are not permitted as numbered references. Footnotes are not used.
BibTeX (.bib) bibliography files cannot be accepted. LaTeX submission must either
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Scientific Reports uses standard Nature referencing style. All authors should be
included in reference lists unless there are six or more, in which case only the first
68
author should be given, followed by 'et al.'. Authors should be listed last name first,
followed by a comma and initials (followed by full stops) of given names. Article titles
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capitals. Journal names are italicized and abbreviated (with full stops) according to
common usage. Volume numbers and the subsequent comma appear in bold. The full
page range should be given (or article number), where appropriate.
Published papers:
Printed journals
Schott, D. H., Collins, R. N. & Bretscher, A. Secretory vesicle transport velocity in living
cells depends on the myosin V lever arm length. J. Cell Biol. 156, 35-39 (2002).
Online only
Bellin, D. L. et al. Electrochemical camera chip for simultaneous imaging of multiple
metabolites in biofilms. Nat. Commun. 7, 10535; 10.1038/ncomms10535 (2016).
For papers with more than five authors include only the first author’s name followed by
‘et al.’.
Books:
Smith, J. Syntax of referencing in How to reference books (ed. Smith, S.) 180-181
(Macmillan, 2013).
Online material:
Manaster, J. Sloth squeak. Scientific American Blog Network
http://blogs.scientificamerican.com/psi-vid/2014/04/09/sloth-squeak (2014).
69
Acknowledgements
Acknowledgements should be brief, and should not include thanks to anonymous
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be acknowledged here.
Author contributions
Scientific Reports requires an Author Contribution Statement as described in the
Author responsibilities section of our Editorial and Publishing Policies.
Competing financial interests
A competing financial interests statement is required for all accepted papers published
in Scientific Reports. If there is no conflict of interest, a statement declaring this will still
be included in the paper.
Supplementary Information
Any Supplementary Information should be submitted with the manuscript and will be
sent to referees during peer review. It is published with the online version of accepted
manuscripts. We request that authors avoid "data not shown" statements and instead
include data necessary to evaluate the claims of the paper as Supplementary
Information. Supplementary Information is not edited, typeset or proofed, so authors
should ensure that it is clearly and succinctly presented at initial submission, and that
the style and terminology conform to the rest of the paper. Authors should include the
title of the manuscript and full author list on the first page.
The guidelines below detail the creation, citation and submission of Supplementary
Information - publication may be delayed if these are not followed correctly. Please
note that modification of Supplementary Information after the paper is published
requires a formal correction, so authors are encouraged to check their Supplementary
Information carefully before submitting the final version.
1. Where possible, Supplementary Information (text, tables and images) should be
combined and supplied as a single file, preferably in PDF format. If necessary,
we can also accept supplementary videos, spreadsheets or data files as
70
separate files.
2. Designate each item as Supplementary Table, Figure, Video, Audio, Note,
Data, Discussion, Equations or Methods, as appropriate. Number
Supplementary Tables and Figures as, for example, "Supplementary Table S1".
This numbering should be separate from that used in tables and figures
appearing in the main article. Supplementary Note or Methods should not be
numbered; titles for these are optional.
3. Refer to each piece of supplementary material at the appropriate point(s) in the
main article. Be sure to include the word "Supplementary" each time one is
mentioned. Please do not refer to individual panels of supplementary figures.
4. Use the following examples as a guide (note: abbreviate "Figure" as "Fig." when
in the middle of a sentence): "Table 1 provides a selected subset of the most
active compounds. The entire list of 96 compounds can be found as
Supplementary Table S1 online." "The biosynthetic pathway of L-ascorbic acid
in animals involves intermediates of the D-glucuronic acid pathway (see
Supplementary Fig. S2 online). Figure 2 shows..."
5. Remember to include a brief title and legend (incorporated into the file to appear
near the image) as part of every figure submitted, and a title as part of every
table.
6. File sizes should be as small as possible, with a maximum size of 50 MB, so
that they can be downloaded quickly.
Further queries about submission and preparation of Supplementary Information
should be directed to email: [email protected].
Figure Legends
Figure legends begin with a brief title sentence for the whole figure and continue with
a short description of what is shown in each panel in sequence and the symbols used;
methodological details should be minimised as much as possible. Each legend must
71
total no more than 350 words. Text for figure legends should be provided in numerical
order after the references.
Tables
Please submit tables in your main article document in an editable format (Word or
TeX/LaTeX, as appropriate), and not as images. Tables that include statistical analysis
of data should describe their standards of error analysis and ranges in a table legend.
Equations
Equations and mathematical expressions should be provided in the main text of the
paper. Equations that are referred to in the text are identified by parenthetical numbers,
such as (1), and are referred to in the manuscript as "equation (1)".
If your manuscript is or will be in .docx format and contains equations, you must follow
the instructions below to make sure that your equations are editable when the file
enters production.
If you have not yet composed your article, you can ensure that the equations in your
.docx file remain editable in .doc by enabling "Compatibility Mode" before you begin.
To do this, open a new document and save as Word 97-2003 (*.doc). Several features
of Word 2007/10 will now be inactive, including the built-in equation editing tool. You
can insert equations in one of the two ways listed below.
If you have already composed your article as .docx and used its built-in equation editing
tool, your equations will become images when the file is saved down to .doc. To resolve
this problem, re-key your equations in one of the two following ways.
1. Use MathType to create the equation. MathType is the recommended method
for creating equations.
2. Go to Insert > Object > Microsoft Equation 3.0 and create the equation.
If, when saving your final document, you see a message saying "Equations will be
converted to images", your equations are no longer editable and we will not be able to
72
accept your file.
General figure guidelines
Authors are responsible for obtaining permission to publish any figures or illustrations
that are protected by copyright, including figures published elsewhere and pictures
taken by professional photographers. The journal cannot publish images downloaded
from the internet without appropriate permission.
Figures should be numbered separately with Arabic numerals in the order of
occurrence in the text of the manuscript. When appropriate, figures should include
error bars. A description of the statistical treatment of error analysis should be included
in the figure legend. Please note that schemes are not used; sequences of chemical
reactions or experimental procedures should be submitted as figures, with appropriate
captions. A limited number of uncaptioned graphics depicting chemical structures -
each labelled with their name, by a defined abbreviation, or by the bold Arabic numeral
- may be included in a manuscript.
Figure lettering should be in a clear, sans-serif typeface (for example, Helvetica); the
same typeface in the same font size should be used for all figures in a paper. Use
'symbols' font for Greek letters. All display items should be on a white background, and
should avoid excessive boxing, unnecessary colour, spurious decorative effects (such
as three-dimensional 'skyscraper' histograms) and highly pixelated computer
drawings. The vertical axis of histograms should not be truncated to exaggerate small
differences. Labelling must be of sufficient size and contrast to be readable, even after
appropriate reduction. The thinnest lines in the final figure should be no smaller than
one point wide. Authors will see a proof that will include figures.
Figures divided into parts should be labelled with a lower-case bold a, b, and so on, in
the same type size as used elsewhere in the figure. Lettering in figures should be in
lower-case type, with only the first letter of each label capitalized. Units should have a
single space between the number and the unit, and follow SI nomenclature (for
example, ms rather than msec) or the nomenclature common to a particular field.
Thousands should be separated by commas (1,000). Unusual units or abbreviations
should be spelled out in full or defined in the legend. Scale bars should be used rather
73
than magnification factors, with the length of the bar defined on the bar itself rather
than in the legend. In legends, please use visual cues rather than verbal explanations
such as "open red triangles".
Unnecessary figures should be avoided: data presented in small tables or histograms,
for instance, can generally be stated briefly in the text instead. Figures should not
contain more than one panel unless the parts are logically connected; each panel of a
multipart figure should be sized so that the whole figure can be reduced by the same
amount and reproduced at the smallest size at which essential details are visible.
Figures for peer review
At the initial submission stage authors may choose to upload separate figure files or to
incorporate figures into the main article file, ensuring that any inserted figures are of
sufficient quality to be clearly legible.
When submitting a revised manuscript all figures must be uploaded as separate figure
files ensuring that the image quality and formatting conforms to the specifications
below.
Figures for publication
Each complete figure must be supplied as a separate file upload. Multi-part/panel
figures must be prepared and arranged as a single image file (including all subparts;
a, b, c, etc.). Please do not upload each panel individually.
Please read the digital images integrity and standards section of our Editorial and
Publishing Policies. When possible, we prefer to use original digital figures to ensure
the highest-quality reproduction in the journal. For optimal results, prepare figures to
fit A4 page-width. When creating and submitting digital files, please follow the
guidelines below. Failure to do so, or to adhere to the following guidelines, can
significantly delay publication of your work.
Authors are responsible for obtaining permission to publish any figures or illustrations
that are protected by copyright, including figures published elsewhere and pictures
taken by professional photographers. The journal cannot publish images downloaded
74
from the internet without appropriate permission.
Springer Nature remains neutral with regard to jurisdictional claims in published maps
and institutional affiliations.
1. Line art, graphs, charts and schematics
For optimal results, all line art, graphs, charts and schematics should be supplied in
vector format, such as EPS or AI, and should be saved or exported as such directly
from the application in which they were made. Please ensure that data points and axis
labels are clearly legible.
2. Photographic and bitmap images
All photographic and bitmap images should be supplied in a bitmap image format such
as tiff, jpg, or psd. If saving tiff files, please ensure that the compression option is
selected to avoid very large file sizes.
Please do not supply Word or Powerpoint files with placed images. Images can be
supplied as RGB or CMYK (note: we will not convert image colour modes).
Figures that do not meet these standards will not reproduce well and may delay
publication until we receive high-resolution images.
3. Chemical structures
Chemical structures should be produced using ChemDraw or a similar program. All
chemical compounds must be assigned a bold, Arabic numeral in the order in which
the compounds are presented in the manuscript text. Structures should then be
exported into a 300 dpi RGB tiff file before being submitted.
4. Stereo images
Stereo diagrams should be presented for divergent 'wall-eyed' viewing, with the two
panels separated by 5.5 cm. In the final accepted version of the manuscript, the stereo
images should be submitted at their final page size.
Statistical guidelines
75
Every article that contains statistical testing should state the name of the statistical test,
the n value for each statistical analysis, the comparisons of interest, a justification for
the use of that test (including, for example, a discussion of the normality of the data
when the test is appropriate only for normal data), the alpha level for all tests, whether
the tests were one-tailed or two-tailed, and the actual P value for each test (not merely
"significant" or "P < 0.05"). It should be clear what statistical test was used to generate
every P value. Use of the word "significant" should always be accompanied by a P
value; otherwise, use "substantial," "considerable," etc.
Data sets should be summarized with descriptive statistics, which should include the n
value for each data set, a clearly labelled measure of centre (such as the mean or the
median), and a clearly labelled measure of variability (such as standard deviation or
range). Ranges are more appropriate than standard deviations or standard errors for
small data sets. Graphs should include clearly labelled error bars. Authors must state
whether a number that follows the ± sign is a standard error (s.e.m.) or a standard
deviation (s.d.).
Authors must justify the use of a particular test and explain whether their data conform
to the assumptions of the tests. Three errors are particularly common:
Multiple comparisons: When making multiple statistical comparisons on a single
data set, authors should explain how they adjusted the alpha level to avoid an
inflated Type I error rate, or they should select statistical tests appropriate for
multiple groups (such as ANOVA rather than a series of t-tests).
Normal distribution: Many statistical tests require that the data be approximately
normally distributed; when using these tests, authors should explain how they
tested their data for normality. If the data do not meet the assumptions of the
test, then a non-parametric alternative should be used instead.
Small sample size: When the sample size is small (less than about 10), authors
should use tests appropriate to small samples or justify their use of large-sample
tests.
76
There is a checklist available to help authors minimize the chance of statistical errors.
Chemical and biological nomenclature and abbreviations
Molecular structures are identified by bold, Arabic numerals assigned in order of
presentation in the text. Once identified in the main text or a figure, compounds may
be referred to by their name, by a defined abbreviation, or by the bold Arabic numeral
(as long as the compound is referred to consistently as one of these three).
When possible, authors should refer to chemical compounds and biomolecules using
systematic nomenclature, preferably using IUPAC. Standard chemical and biological
abbreviations should be used. Unconventional or specialist abbreviations should be
defined at their first occurrence in the text.
Gene nomenclature
Authors should use approved nomenclature for gene symbols, and use symbols rather
than italicized full names (for example Ttn, not titin). Please consult the appropriate
nomenclature databases for correct gene names and symbols. A useful resource is
LocusLink.
Approved human gene symbols are provided by HUGO Gene Nomenclature
Committee (HGNC), e-mail: [email protected]; see also www.genenames.org.
Approved mouse symbols are provided by The Jackson Laboratory, email:
[email protected]; see also www.informatics.jax.org/mgihome/nomen.
For proposed gene names that are not already approved, please submit the gene
symbols to the appropriate nomenclature committees as soon as possible, as these
must be deposited and approved before publication of an article.
Avoid listing multiple names of genes (or proteins) separated by a slash, as in
'Oct4/Pou5f1', as this is ambiguous (it could mean a ratio, a complex, alternative
names or different subunits). Use one name throughout and include the other at first
mention: 'Oct4 (also known as Pou5f1)'.
77
Characterization of chemical and biomolecular materials
Scientific Reports is committed to publishing technically sound research. Manuscripts
submitted to the journal will be held to rigorous standards with respect to experimental
methods and characterization of new compounds. Authors must provide adequate data
to support their assignment of identity and purity for each new compound described in
the manuscript. Authors should provide a statement confirming the source, identity and
purity of known compounds that are central to the scientific study, even if they are
purchased or resynthesized using published methods.
1. Chemical identity
Chemical identity for organic and organometallic compounds should be established
through spectroscopic analysis. Standard peak listings (see formatting guidelines
below) for 1H NMR and proton-decoupled 13C NMR should be provided for all new
compounds. Other NMR data should be reported (31P NMR, 19F NMR, etc.) when
appropriate. For new materials, authors should also provide mass spectral data to
support molecular weight identity. High-resolution mass spectral (HRMS) data are
preferred. UV or IR spectral data may be reported for the identification of characteristic
functional groups, when appropriate. Melting-point ranges should be provided for
crystalline materials. Specific rotations may be reported for chiral compounds. Authors
should provide references, rather than detailed procedures, for known compounds,
unless their protocols represent a departure from or improvement on published
methods.
2. Combinational compound libraries
Authors describing the preparation of combinatorial libraries should include standard
characterization data for a diverse panel of library components.
3. Biomolecular identity
For new biopolymeric materials (oligosaccharides, peptides, nucleic acids, etc.), direct
structural analysis by NMR spectroscopic methods may not be possible. In these
cases, authors must provide evidence of identity based on sequence (when
appropriate) and mass spectral characterization.
4. Biological constructs
78
Authors should provide sequencing or functional data that validates the identity of their
biological constructs (plasmids, fusion proteins, site-directed mutants, etc.) either in
the manuscript text or the Methods section, as appropriate.
5. Sample purity
Evidence of sample purity is requested for each new compound. Methods for purity
analysis depend on the compound class. For most organic and organometallic
compounds, purity may be demonstrated by high-field 1H NMR or 13C NMR data,
although elemental analysis (±0.4%) is encouraged for small molecules. Quantitative
analytical methods including chromatographic (GC, HPLC, etc.) or electrophoretic
analyses may be used to demonstrate purity for small molecules and polymeric
materials.
6. Spectral data
Detailed spectral data for new compounds should be provided in list form (see below)
in the Methods section. Figures containing spectra generally will not be published as a
manuscript figure unless the data are directly relevant to the central conclusions of the
paper. Authors are encouraged to include high-quality images of spectral data for key
compounds in the Supplementary Information. Specific NMR assignments should be
listed after integration values only if they were unambiguously determined by
multidimensional NMR or decoupling experiments.
Authors should provide information about how assignments were made in a general
Methods section.
Example format for compound characterization data. mp: 100-102 °C (lit. 99-101 °C);
TLC (CHCl :MeOH, 98:2 v/v): R = 0.23; [α] = -21.5 (0.1 M in n-hexane); H NMR (400
MHz, CDCl ): δ 9.30 (s, 1H), 7.55-7.41 (m, 6H), 5.61 (d, J = 5.5 Hz, 1H), 5.40 (d, J =5.5
Hz, 1H), 4.93 (m, 1H), 4.20 (q, J = 8.5 Hz, 2H), 2.11 (s, 3H), 1.25 (t, J = 8.5 Hz, 3H); C
NMR (125 MHz, CDCl ): δ 165.4, 165.0, 140.5, 138.7, 131.5, 129.2, 118.6, 84.2, 75.8,
66.7, 37.9, 20.1; IR (Nujol): 1765 cm- ; UV/Vis: λ 267 nm; HRMS (m/z): [M] calcd. For
C H C NO , 420.0406; found, 420.0412; analysis (calcd., found for C H C NO ): C
(57.16, 57.22), H (3.60, 3.61), Cl (16.87, 16.88), N (3.33, 3.33), O (19.04, 19.09).
79
7. Crystallographic data for small molecules
Manuscripts reporting new three-dimensional structures of small molecules from
crystallographic analysis should include a .cif file and a structural figure with probability
ellipsoids for publication as Supplementary Information. These must have been
checked using the IUCR's CheckCIF routine, and a PDF copy of the output must be
included with the submission, together with a justification for any alerts reported.
Crystallographic data for small molecules should be submitted to the Cambridge
Structural Database and the deposition number referenced appropriately in the
manuscript. Full access must be provided on publication.
8. Macromolecular structural data
Manuscripts reporting new structures should contain a table summarizing structural
and refinement statistics. Templates are available for such tables describing NMR and
X-ray crystallography data. To facilitate assessment of the quality of the structural data,
a stereo image of a portion of the electron density map (for crystallography papers) or
of the superimposed lowest energy structures (≳10; for NMR papers) should be
provided with the submitted manuscript. If the reported structure represents a novel
overall fold, a stereo image of the entire structure (as a backbone trace) should also
be provided.