MicroRNA Signature With Indolent Non-Hodgkin Lymphomas

1. 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

2. Introduction
Non-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)

3. 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.

4. young patient (<65) elderly patient (>65) compromised patient
First line treatment
conventional
immuno-chemotherapy
(e.g. R-CHOP)
Rituximab maintenance ?
radioimmunotherapy ?
watch & wait ?
Rituximab
monotherapy
Chlorambucil
Bendamustin
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-intensified
immuno-chemotherapy
(either sequential:
e.g. R-CHOP =>PBSCT
or R-Hyper-CVAD)
Dreyling ASCO 2006
Treatment options for MCL

5. 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

6. 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

7. 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)

8. miRNA Oncogenes or Tumor Suppressor Genes
Croce Nat Rev Genet. 2009 Oct;10(10):704-14

9. AIM
AIM
 To evaluate and examine the miRNA signature in MCL
 To study its prognostic impact and role in distinguishing the
disease status

10. 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

11. 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

12. 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

13. Statistical Analysis
 Log rank test – for bivariate analysis
 Kaplan-Meier – OS & miRNA expression
 Multivariable Cox proportional hazards regression analysis – for
miRNAs associated with OS

14. 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


15. Set of miRs Can Distinguish Between Indolent and
Aggressive 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

16. miRs Derived From the NHL Aggressiveness
Signature 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
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

17. miRs Derived From the NHL Aggressiveness
Signature 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)

18. 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.

19. 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 MCL
D = 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 ≥ 3.488
This 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

20. Model to Predict MCL Prognosis Can Be Created by
Combining 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

21. 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

22. 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

23. 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

24. 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

25.  Thank you …!!

26. 
Thank you ..!

27. 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/