MicroRNA Signature Obtained From the Comparison of Aggressive With Indolent Non-

Hodgkin Lymphomas:

Potential Prognostic Value in Mantle-Cell Lymphoma

R. S. Goswami, S Kamel-Reid et al

JCO, 10 August, 2013 MOHSIN

MAQBOOL

27-11-2013

IntroductionNon-Hodgkin’s Lymphoma

Non-Hodgkin’s lymphomas (NHL) - heterogeneous group of malignant lymphomas

many different subtypes, every few years the classification is updated

morphology, immunophenotype, molecular, cytogenetics, and other techniques are used for diagnosis

Treatment generally depends on the aggressiveness of the disease (indolent, aggressive, or very aggressive)

Introduction

Mantle Cell Lymphoma (MCL)

Unique & aggressive lymphoma from "mantle zone" of the lymph node

6% MCL(NHL), ( 3 % India)

Age>50y ,M:F ratio = 3:1

poor long-term prognosis ,Typically advanced stage

involvement: BM, blood, liver, GI, CNS

morphology,

immunophenotype,

and cyclin D1 overexpression

incidence is increasing,

and survival varies

1 to 10 years

Armitage JO. Management of Mantle Cell Lymphoma. Oncology (Willston Park). 1998.Romaguera JE, et al. Cancer. 2003;97:586-591.

young patient (<65) elderly patient (>65) compromised patientFirst line treatment

conventionalimmuno-chemotherapy

(e.g. R-CHOP)

Rituximab maintenance ?radioimmunotherapy ?

watch & wait ?Rituximab

monotherapyChlorambucilBendamustin

1. relapse

high tumor load:immuno-chemotherapy

(e.g. R-FC)

allo-transplant ?radioimmunotherapy ?

Rituximab maintenance ?

immuno-chemotherapy(e.g. R-FC,

R-Bendamustin)

molecular approaches ? autologous PBSCT

radioimmunotherapy ? Rituximab maintenance ?

immuno-chemotherapy

(e.g. R-Bendamustin)

molecular approaches

higher relapse

molecular approaches: Bortezomib, CCI-779, Thalidomide/Lenalidomide, Flavopiridol (preferable in combination)

repeat previous therapy (long remissions) mTor inhibitor ( temsirolimus)

dose-intensifiedimmuno-chemotherapy

(either sequential:

e.g. R-CHOP =>PBSCTor R-Hyper-CVAD)

Dreyling ASCO 2006

Treatment options for MCL

Introduction (MCL)……

Adding intensive therapy and ASCT in first remission may improve survival- not curative

various clinical & pathologic parameters –used to prognosticate - t(11;14)(q13;q32)

- overexpression of cyclin D1

- CD5+, CD23 -, CD19/20/22+

(Myc, secondary genetic alterations,topoisomerase II, PRAD1/cyclin D1 , CCND1gene)

No approach has significant predictive potential

Introduction (MCL)……

The MCL-IPI (M-IPI) may identify patients with varying outcomes using available clinical variables

Informative biomarkers distinguishing patient populations with favorable versus unfavorable biology is needed

E Hoster Blood 2008

Micro RNAs (miRNA)

microRNA (miRNA/ miRs) short (19-25 (22) ribonucleic acid(RNA)  in eukaryotic cells

Initially discovered by Victor Ambros in 1993 in the C. elegans (worm)

mi RNA – control many genes, role in development, growth, & human cancer and other diseases (cardiac, Neuro etc)

C. elegans V Ambros

mi RNA (miRs)

miRNA Oncogenes or Tumor Suppressor Genes

Croce Nat Rev Genet. 2009 Oct;10(10):704-14

AIM

AIM

To evaluate and examine the miRNA signature in MCL

To study its prognostic impact and role in distinguishing the disease status

Materials and Methods

Total 346 fresh frozen paraffin embedded (FFPE) samples collected Univ health network, British Columbia Cancer agency , Kingston general Hospital & Cross Cancer Institute ( all samples obtained before treatment)

3-5 separate 10 µ m sections kept under Rnase-free conditions for RNA All tumor samples contained 80% tumor cells, confirmed by pathologists- immunophenotyping and FISH

Samples

Method…..

RNA from FFPE samples extracted using the kit [Recover All total Kit(Ambion)]

MCL samples stained with Ki-67 antibody , percentage positivity assessed by two pathologists

MCL cases were scored as low (10% cells positively stained), intermediate (10% to 30% cells positively stained), or high (30% cells positively stained) for Ki-67

RNA extraction

Immunohistochemistry

miR expression profiling

miRNA (miRs) expression performed by Taqman Human microRNA array

Bioinformatics tools were used for data analysis of mi RNA expression and Validation of miR signature

miR expression profiling

Statistical Analysis

Log rank test – for bivariate analysis

Kaplan-Meier – OS & miRNA expression

Multivariable Cox proportional hazards regression analysis – for miRNAs associated with OS

Results Set of miRs Can Distinguish Between Indolent and

Aggressive NHL

Taqman Arrays run for 365 miRNAs (miRs) on a group of 43 NHL ( 19 indolent, 24 aggressive) and 20 benign lymph nodes

314 (86%) of 365 miRs were detected and expressed

Unsupervised clustering of benign and malignant nodes and Venn diagram analysis of the differentially expressed miRs performed – miRs expression of benign nodes different than malignant

14 differentially expressed miRs (p<0.001) for validation in an independent set of NHL

Set of miRs Can Distinguish Between Indolent andAggressive NHL

44 separate validation set NHL FFPE (25 Indolent, 19 aggressive NHLs) and five benign lymph nodes used to validate the same set of miRs by miR array and Taqman probe RQ-PCR

Eleven of the 14 miRs were differentially expressed between aggressive and indolent NHL

11 validated miRs considered to be signature defining the NHL aggressiveness

miRs Derived From the NHL AggressivenessSignature Are Associated With MCL Prognosis and

Outperform Clinical Prognostic Indicators

MCL samples from the four participating – divided into two groups

- 119 cases (training group) & 114 cases ( validation group)

miRs in MCL

miRs in NHLs

Two of the validated miRs within NHL aggressiveness signature miR-127-3p (P=.0006), miR-615-3p(P=0.0001)- significantly associated with OS among the MCL patients

Two more miRs ( miR -26a & miR-198) in MCL with ( P< .2) were also taken for survival analysis

miRs Derived From the NHL AggressivenessSignature Are Associated With MCL Prognosis and

Outperform Clinical Prognostic Indicators

Multivariable analysis demonstrated that only miR-127-3p and miR-615-3p , significantly associated with OS

Further analysis (recursive partitioning analysis) of the two miRs (miR-127-3p and miR-615-3p) was done to associate role with survival

Comparison of patient outcome on expression levels of both miR-127-3p and miR-615-3p – demonstrated both miRs can distinguish groups with different survival outcome ( P= .0033) and outperformed Ki-67 cut-offs ( P= .0231)

Analysis of mantle-cell lymphoma (MCL) overall survival (OS) through the use of microRNAs (miRs) and Ki-67

A = KM curve : association of miRs-127-3p and -615-3p with OS in a training set of 119 patients with MCL

B= KM curve : demonstrating association of Ki-67 with OS in a training set

A= miR-127-3p < 0.9238

B = miR-127-3p ≥ 0.9238 and miR-615-3p < 3.488

A = iKi-67 10%

B Ki-67 10% to < 30%

C Ki-67 > = 30%

C = miR-127-3p ≥ 0.9238 and miR-615-3p ≥ 3.488

Training set of 119 patients with MCL. Training set of 119 patients with MCL.

Analysis of mantle-cell lymphoma (MCL) overall survival (OS) through the use of microRNAs (miRs)

and Ki-67

C = KM curve demonstrating association of miRs-127-3p and -615-3p with OS in a validation set of 114 patients with MCLD = KM curve demonstrating association of Ki-67 with OS in a

validation set of 114 patients with MCL

validation set of 114 patients with MCL. validation set of 114 patients with MCL.

A= miR-127-3p < 0.9238

B = miR-127-3p ≥ 0.9238 and miR-615-3p < 3.488

A = Ki-67 10%

B Ki-67 10% to < 30%

C Ki-67 ≥ 30% C = miR-127-3p ≥ 0.9238 and miR-615-3p ≥ 3.488This analysis demonstrates that the use of miRs-127-3p

and -615-3p is superior to that of Ki-67 using cut-offs reported in the literature and that the miRs demonstrate their utility in predicting OS in patients with MCL within the

training set as well as a separate, independent validation set

Model to Predict MCL Prognosis Can Be Created byCombining miR Expression and Clinical Indicators

Multivariable Cox modeling revealed that combining miR expression with Ki-67 – more informative (P=.001) than using the Ki-67(P= .497) or miR expression (P=.178) alone

- new Ki-67 cutoffs used as part of new model, <39.5% ; 39.5 to 91.5 % ; > 91.5%

Using further recursive partitioning analysis , Ki-67 and miR-127-3p & miR-615-3p expression were used to develop prognostic model for MCL patients

similar models was created using the miR expression and M-IPI and miR-615-3p here had better effect than mi-127-3p

KM curve demonstrating utility of model combining Ki-67 scores and miR-127-3p expression in combined cohort of patients with MCL

KM curve demonstrating utility of model combining MCL International Prognostic Index (M-IPI) scores and miR-615-3p expression expression in combined cohort of patients with MCL

Ki-67<39%, OS= 46.3Mo GOOD

Ki-67= 39% to 91.5%, OS= 18.8 Mo INTERMEDIATE

Ki-67> 91.5%, POOR

M-IPI< 6.65 , OS= 55.3 Mo GOOD

M-IPI= <6.65 to 7.45, miR-615 <1.14 OS= 36 Mo INTERMEDIATE

M-IPI > 7.45, miR-615 > 1.14 Os=13 Mo POOR

Discussion miR-127-3p & miR-615-3p expression can be combined with current

clinical indicators to yield prognostic information in MCL

Results demonstrate that current ki-67 and M-IPI cut-offs can be adjusted to yield more accurate prognostic information about the MCL patients

Earlier studies examining MCL prognosis and global miRNA expression have shown miR-29, with poor prognosis (n=30),

miR-20b underexpression associated with good prognosis; miR-129-3p, mi135a, miR222 , mi-424, mi-450 as good and poor prognostic factors with OS of 4 years and 2 years

Discussion…..

This study is novel – hypothesis driven, independent validated NHL groups and large sample size regarding the prognostic utility of miRs identified

Followup (median= 33.8Mo) [2.8 Yrs], limited in terms of miRs analysed, treatment heterogenous , and samples archived

Set of 11 miRs define aggressiveness in NHL, have role in pathogenesis , cell cycle, proliferation and apoptosis, but still other molecular pathways leading to neoplasia may be involved

Discussion….

This study has uncovered new miRs in the disease severity that deserve further study to know role into cellular pathways that control lymphomagenesis

Two miRs ---miR-127-3p & miR-615-3p – closely associated with MCL and can distinguish between different prognostic groups and real utility realized when combined with current clinical prognostic factors

Conclusion

miRs have role in Lymhomagenesis and will be useful adjuncts to current clinical prognostic indicators

Expression of miR-127-3p, miR-615-3p is superior to Ki-67 at stratifying prognostic outcome in patients with MCL

This clinical model can be adopted in prospective trials for new MCL treatment

Validation in Prospective trials can stratify patients to facilitate treatment decisions and more novel and effective treatment options can be used for different prognostic MCL groups

Thank you …!!

Thank you ..!

Background: Cancer Classification

No general approach has been made for identifying new cancer classes (class discovery) and assigning tumors to known classes(class prediction).

A generic approach to cancer classification based on gene expression monitoring by DNA microarrays is described and applied to acute human leukemia as a test case.

http://www.nejm.org/action/showMediaPlayer?doi=10.1056%2FNEJMoa1301689&aid=NEJMoa1301689_attach_1&area=

http://compbio.cs.brown.edu/aml_tcga/