OVEREXPRESSION OF MYC AND BCL2 IN DIFFUSE LARGE B CELL LYMPHOMA THROUGH IMMUNOHISTOCHEMICAL
TECHNIQUE TO PROVIDE PROGNOSTIC INFORMATION Dissertation submitted in
partial fulfilment of the requirements for the degree of
M.D. PATHOLOGY BRANCH – III
INSTITUTE OF PATHOLOGY MADRAS MEDICAL COLLEGE
CHENNAI – 600003
THE TAMIL NADU DR.M.G.R. MEDICAL UNIVERSITY CHENNAI
MAY 2020
CERTIFICATE
This is to certify that this Dissertation entitled “OVEREXPRESSION OF MYC AND BCL2 IN DIFFUSE LARGE B CELL LYMPHOMA THROUGH IMMUNOHISTOCHEMICAL TECHNIQUE TO PROVIDE PROGNOSTIC INFORMATION” is the bonafide original work of DR.T.JEMIMA EVANGELYN, in partial fulfilment of the requirement for M.D., (Branch III) in Pathology examination of the Tamilnadu Dr.M.G.R Medical University to be held in May 2020.
Prof.Dr.Rajavelu Indira, M.D., Professor of Pathology,
Kasthuribai Gandhi Hospital for Women and Children, Madras Medical College,
Chennai-3.
Prof.Dr.Bharathi Vidhya Jayanthi M.D., Prof.Dr.Jayanthi M.D.,FRCP(Glasg), Director & Professor of Pathology, Dean,
Institute of Pathology, Madras Medical College, Madras Medical College, Rajiv Gandhi Government General Chennai – 600003. Hospital, Chennai – 600003.
DECLARATION
I, DR.T. JEMIMA EVANGELYN, solemnly declare that the dissertation entitled “OVEREXPRESSION OF MYC AND BCL2 IN DIFFUSE LARGE B CELL LYMPHOMA THROUGH IMMUNOHISTOCHEMICAL TECHNIQUE TO PROVIDE PROGNOSTIC INFORMATION” is the bonafide work done by me in the Institute of Pathology, Madras Medical College under the expert guidance and supervision of PROF. DR. RAJAVELU INDIRA ,M.D., Professor of Pathology, Kasthuribai Gandhi Hospital for Women and Children and DR.VIJAYABHASKAR, M.D., Assistant professor of Pathology, Madras Medical College.The dissertation is submitted to the Tamilnadu Dr.M.G.R Medical University towards partial fulfilment of requirement for the award of M.D., Degree (Branch III) in Pathology.
Place: Chennai.
Date: DR.T.JEMIMA EVANGELYN
ACKNOWLEDGEMENT
I thank the Lord God for his grace and love. I express my sincere thanks to Prof. Dr. Jayanthi.R, MD FRCP (Glasg), Dean, Madras Medical College and Rajiv Gandhi Government General Hospital, for permitting me to utilize the facilities of the Institution.
I take the opportunity to express my gratitude to Prof. Dr. Bharathi Vidhya Jayanthi, M.D., Director and Professor, Institute of Pathology, Madras Medical College, Chennai for her keen interest, constant encouragement and valuable suggestions throughout the study.
I am extremely thankful to Prof.Dr.Rajavelu Indira,M.D.
Professor of Pathology, Kasthuribai Gandhi Hospital for Women and Children and Dr.Vijayabhaskar M.D.,Assistant professor of Pathology, Institute Of Pathology, Madras Medical College, for their valuable suggestions, constant support, advice and encouragements throughout the study.
I am thankful to Prof.Dr.Rama M.D., Prof.Dr.Padmavathi M.D., Prof.Dr. Geetha Devadas M.D DCP., Prof.Dr.Sudha Venkatesh M.D., Prof.Dr.M.P.Kanchana M.D., Prof. Dr.S.Pappathi M.D., Prof.
Dr.Rajavelu Indira M.D., Prof.Dr.Selvambigai M.D., and Prof.Dr.Sheeba M.D.,for their valuable suggestions and encouragement throughout the study.
I express my heartfelt sincere thanks to all my Assistant Professors for their help and suggestions during the study.
I am thankful to my colleagues, friends, technicians and staff of the Institute of Pathology, Madras Medical College, Chennai for all their help and support they extended for the successful completion of this dissertation.
My sincere thanks to all the patients and their families who were co-operative during the course of this study.
Finally ,I thank my family for being a pillar of support in my endeavours.
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ABBREVIATION
DLBCL- Diffuse Large B-cell Lymphoma
DLBCL, NOS- Diffuse Large B-cell Lymphoma, Not otherwise specified
DEL- Double expressor lymphoma
DHL- Double hit lymphoma
BCL2- B-cell lymphoma 2
MYC- Myelomatosis
BCL6- B cell lymphoma 6
WHO- World Health Organisation
R-CHOP- Rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone
R-EPOCH - Rituximab, etoposide, prednisolone, vincristine, cyclophosphamide, doxorubicin
HGBL- High grade B-cell lymphoma
GCB- Germinal centre B-cell origin
ABC- Activated B-cell origin
CD- Cluster of differentiation
CONTENTS
S .NO. TITLE PAGE NUMBER
1 INTRODUCTION 1
2 AIMS AND OBJECTIVES 3
3 REVIEW OF LITERATURE 4
4 MATERIALS AND METHODS 48
5 OBSERVATION AND RESULTS 53
6 DISCUSSION 66
7 SUMMARY 76
8 CONCLUSION 82
9 BIBLIOGRAPHY
10 ANNEXURE
11 MASTER CHART
1
INTRODUCTION
Having entered the biological era of genetics, the field of oncopathology is seeking to evolve into personalized diagnostics in providing better treatment and predicting prognosis of patients with aggressive lymphomas. The concurrent deregulation of MYC and B-cell lymphoma 2(BCL2) whether at genomic or protein level, has important implications in the treatment of diffuse large B cell lymphomas(DLBCL). Overexpression of MYC and BCL2 proteins , unrelated to underlying chromosomal rearrangements is found to have poor outcome in patients with DLBCL. DLBCLs those overexpresss these proteins are called Double expressor lymphomas(DELs).(1)
The current WHO uses 40% as cutoff for myc protein and 50% as cutoff for BCL2 protein to define overexpression.
Double expressor lymphomas have got poor survival rate with R- CHOP chemotherapy.(2) MYC protein expression is detected in 30% to 50% of DLBCL with concomitant expression of BCL2expression in 20% to 35% of cases. (3) That is, double expressors constitute about 20% to 35% of total DLBCL cases.
Thus it has been suggested in the WHO that double expression of both these proteins without molecular aberrations should be
2
considered a prognostic indicator in DLBCL,NOS. It is not a separate category. The WHO 2016 identifies the respective gene rearrangements in DLBCL. These are named Double hit lymphomas(DHL) .High grade B-cell lymphoma (HGBL) with rearrangements of MYC and BCL2 and/or BCL6 include double hit lymphomas also.(4) MYC rearrangement constitutes about 5% of DLBCL , NOS . It is frequently associated with BCL2 rearrangement.(3) the protein over expression is not always co- existent with gene rearrangements. The latter are more aggressive than the former type of lymphomas . Double hit lymphomas are classified as distinct entities .(5)
3
AIMS AND OBJECTIVES
• To identify the double expressor lymphomas using immunohistochemistry technique
• To find out the proportion of DLBCL,NOS that express MYC and BCL2
• To analyse the correlation of the current study with other similar studies conducted in various institutions
4
REVIEW OF LITERATURE
• Diffuse large B cell lymphoma
Definition Epidemiology Clinical features Microscopy Variants
Immunohistochemistry
• MYC oncogene
• MYC alterations in DLBCL
• BCL2 family of proteins
• BCL2 anti- apoptic protein
• Overexpression of BCL2 in DLBCL
• Cell of origin classification of DLBCL
• Double expressor lymphomas
• Double hit in DLBCL
• Prognostic significance of double expressor lymphomas
• Role of immunohistochemistry in assessing the prognosis of DLBCL
5
DIFFUSE LARGE B CELL LYMPHOMA
Diffuse large B cell lymphoma is an aggressive , rapidly growing neoplasm composed of intermediate to large lymphoid cells. They have nucleus comparable to or larger than that of a reactive histiocyte or more than twice the size of a normal lymphocyte.
EPIDEMIOLOGY:
Diffuse large B cell lymphoma occurs over a wide age range, with slight male preponderance.
CLINICAL FEATURES:
Diffuse large B cell lymphomas arise de novo . it can also transform from a low grade lymphoma such as follicular lymphoma, CLL/SLL, extranodal marginal zone lymphoma or lymphoplasmacytic lymphoma. Some cases arise in a setting of autoimmune disease or immunodeficiency.some extranodal cases can arise in a setting of local chronic inflammation or irritation such as lymphedema, metallic implants and long standing pyothorax.
Diffuse large B cell usually presents as lymphadenopathy . Extranodal presentationis also common. The tumors are rapidly growing. Constitutional symptoms are usually present in patients with high stage disease. Nearly half of the patients present with stageI or II disease.On staging , marrow examination may reveal
6
lymphomatous involvement in 16% cases. It is an aggressive neoplasm if untreated.
MICROSCOPIC FEATURES:
Diffuse large B cell lymphoma is characterized by diffuse destructive infiltration of the involved site. Perinodal tissue and blood vessel infiltration is more common. Some cases show sclerosis.
Necrosis is common. Some cases show a starry sky pattern due to reactive histiocytes. Epithelioid histiocytes, plasma cells and eosinophils can also be seen in the background.
Lymphoma cell can be centrobastic, immunoblastic or plasmacytoid. The nuclei can be round , indented, or irregularly folded. The chromatin patterns vary from vesicular to coarse granular. Small or large eosinophilic or basophilic nucleoi may be present. It may be central or membrane bound. Cytoplasm may beeosinophilic, basophilic or amphophilic. Multinucleated cells resembling Reed-Sternberg cells can be seen. Mitotic rate is high with presence of atypical mitosis and karyorrhexis.
7
FIGURE 1 DIFFUSE LARGE B-CELL LYMPHOMA
MORPHOLOGICAL VARIANTS:
1. MYXOID VARIANT:
These lymphomas have abundant myxoid matrix with round, spindle or stellate tumor cells embedded in it.
8
2. VARIANT WITH FIBRILLARY MATRIX AND ROSETTES:
These have abundant fibrillary matrix with or without rosettes.
3. SPINDLE CELL VARIANT:
Lymphoma cells grow in spindle cell fascicles.
4. SIGNET RING CELL VARIANT:
There is eccentric displacement of the nuclei by a clear vacuole or eosinophilic globule.
5. CLEAR CELL VARIANT:
Lymphoma cells have clear cytoplasm.
6. ANEMONE CELL/ FILIFORM/ MICROVILLOUS VARIANT:
Lymphoma cells have long bushy cell processes .
7. SINUSOIDAL VARIANT:
Some lymphomas infiltrate the lymph node in a pure sinusoidal pattern.
9
8. INTERFOLLICULAR VARIANT:
These grow predominantly in the interfollicular region of the lymphoid tissue.
9. MULTILOBATED VARIANT:
These are characterized by folding in nuclear membrane.
10.ANAPLASTIC VARIANT:
These lymphomas are composed of large cells with voluminous cytoplasm and large horsrshoe shaped or bizarre nuclei.
10
FIGURE 2 DLBCL – ANAPLASTIC VARIANT
11.VARIANT WITH PLASMA CELL DIFFERENTIATION:
Large neoplastic cells are admixed with plasma cells that are shown to exhibit the same immunoglobin light chain. The plasma cells may or may not exhibit atypia.
11
IMMUNOHISTOCHEMISTRY:
B – lineage markers like CD19, CD20, CD22, CD79a and PAX5 are positive in these lymphomas. Aberrant loss of one or more markers normally expressed by B cells can occur. The lymphoma cells often express cytoplasmic and/or surface immunoglobins. They can be negative also. CD10 is positive in 40% of cases. BCL6 is positive in 60%
of cases. BCL2 is positive in 50% of cases. Activation markers like CD25 and CD30 can be positive in some patient . Aberrant expression of CD43 is seen in 20% of cases. CD5 is positive in 10% of cases. The mean Ki67 index is 55% and it can reach upto 100%. Exceptionally , they are cyclinD1 positive , but such cases are CD5 and SOX11 negative and do not show CCND1 gene rearrangements.
12
MYC ONCOGENE:
MYC is a proto-oncogene that plays a vital role in oncogenesis through diverse mechanisms. The proto-oncogene lies at the crossroads of many signaling pathways and lies as downstream gene for many ligand-receptor membrane complexes. Its expression is tightly controlled by transcriptional regulatory motifs in the proximal promoter region.(6) MYC was also identified to be one of the genes including Sox2, Oct4 and KLF4 . These reprogram fibroblasts into a stem cell.(7)
FIGURE 3
13
Molecular functions of MYC:
The MYC mRNA generates MYC polypeptides that initiates at a CUG upstream of the canonical AUG start codon and another that starts at an internal AUG . The MYC protein translated from the canonical AUG contains an N-terminal transcriptional regulatory domain followed by nuclear localizationsignal and a C-terminal region with a basic DNA binding domain tethered to a helix- loop- helix- leucine zipper dimerization motif. MYC dimerizes with MAX to bind DNA and mediate many of the functions of DNA. (8) It is not known about the function of myc gene upstream CUG. That initiated from an internal AUG appears to play a role in stress response and perhaps serves as a dominant negative MYC protein. (9)
A cytoplasmic protein cleavage product of MYC known as MYC-nick can promote alpha-tubulin acetylation and cell differentiation in a non-transcriptional manner. (10) It lacks nuclear localization signal and DNA binding domain . Myc also appears to recruit DNA replication licensing factors to catalyze DNA replication. It also plays an important non-transcriptional role that stimulates cap-dependent translation .(11) Myc appears to function even in the absence of functional Max protein. It is not known whether Myc could homo-oligomerize or hetero-oligomerize with other helix-loop-helix proteins to regulate transcription in the absence of Max in cells.(12) It activates transcription
14
by mechanisms such as the recruitment of histone acetylase. A vast numbers of targets are repressed by Myc. A fraction of it is linked to Miz-1.(13)
Transforming growth factor – beta signaling shows a role for Myc-Miz-1 interaction. In the absence of this factor, the oncogene represses CDKN2B (p15INK4b) by binding Miz-1 and displacing Miz-1 cofactors so as to silence CDKN2B. With this groeth factor, the oncogene expression is suppressed, and the Smad transcription factor translocates and cooperates with Miz-1 to recruit NPM1 as a Miz-1 cofactor to stimulate CDKN2B transcription . as a result, cell cycle arrest is induced.(14)
Myc activates many ribosomal protein genes including Rpl23, that binds to and retains NPM1 in the nucleolus, thereby inhibiting the activity of Miz-1. NPM1 itself acts as a positive oncogene coactivator.
15
The oncogene mediates gene repression through its ability to activate microRNAs. The activation of the miR-17-92 cluster of microRNAs mediates a number of biological activities of Myc, including the attenuation of E2F1 activity. TGFβ signaling pathway is targeted by miR-17-92. (15) It also represses many more microRNAs resulting in increased gene expression at the protein level.
FIGURE 4
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FIGURE 5
17
Transcription : Upstream and downstream of MYC:
The MYC proto-oncogene is under tight transcriptional control as are the mRNA and Myc protein. MYC is not only regulated by a whole host of transcription factors, such as CNBP, FBP, and TCF that is downstream of the Wnt pathway, but it is also regulated by non-B DNA structures .(16) The FUSE (Far UpStream Element), melts when bound by FBP (FUSE binding protein), which relieves torsional stresses on DNA from ongoing transcriptional activity. (17)
TCF ,a transcription factor is found to play a role in deregulated MYC expression downstream of the WNT pathway. An example is with the loss of the tumor suppressor APC . Loss of which results in constitutive nuclear localization of the TCF co-factor β-catenin.
Genome wide association studies identified polymorphisms linked to multiple cancers near the oncogene. Such SNPs ( single nucleotide polymorphisms) lie in enhancers that involve TCF binding and DNA looping, which connects the enhancer to the MYC proximal promoter.(18) BET domain containing transcriptional regulator, BRD4.
Binding of the regulator to promoter region of myc plays a critical role in MYC expression in human cancer cells such that a drug-like BET domain chemical inhibitor could inhibit in vivo tumorigenesis.(19)
18
The MYC mRNA, which is short-lived, is affected by microRNAs. The Myc protein is post-translationally modified, ubiquitinated and degraded. It has a half life of 15–20 minutes. Mutations of residues that result in its proteasomal degradation in normal cells are related to tumorigenesis.(20)
The canonical Myc E-box 5′-CACGTG-3′ is one of the most frequently occurring DNA binding motifs in the human genome . It is bound by different transcription factors such as HIF-1and TFE3.
In non-proliferating cells, non-Myc E-box transcription factors regulate basal metabolism for cellular structural and functional integrity. When cells are stimulated to proliferate, Myc levels rise and occupy E-box driven genes normally bound by other transcription factors and activate a program of biomass accumulation . This increases energy production(21)
Myc binding alone is insufficient to induce changes in mRNA levels of target genes as supported by genome wide mapping of Myc binding sites and associated gene expression profiling . Binding by multiple transcriptional factors is necessary to activate a target gene.(22)
Metabolic and MYC oncogene addiction:
E-box genes are expressed by resting cells for homeostatic purposes. Lipogenesis in resting liver or fat cells is mediated by SREBP
19
gene. Mitochondrial function is stimulated in normal cells by NRF1.
Some genes are regulated in a circadian rhythm by Clock/Bmal.
Glycolytic and angiogenic genes can be activated by HIF-1 in response to hypoxia. When Myc is elevated, it binds to the same targets of other transcription factors. Thus Myc can co-opt the functions of other E-box transcription factors .this enables this oncogene to coordinate a growth program along with angiogenesis.(23) Deregulated oncogene stimulates many of the E-box genes to enable the cell to grow and then divide. Myc also drives angiogenesis.(24)
FIGURE 6
20
The loss of Myc was associated with profoundly diminished expression of genes involved in metabolism and the inability for normal primary T cells to grow and proliferate. Given that Myc drives biomass accumulation, the sensitivity of Myc-overexpressing cells to nutrient deprivation could reflect their deregulated growth which renders them dependent on and addicted to continual bioenergetic sources.
Myc-induced cancers are also addicted to it , such that conditional silencing of ectopic Myc expression causes tumor regression in experimental studies. The disturbanceto the tumor microenvironment and blood vessel formation proves MYC oncogene addiction.(25)
Inactivation of Myc can also restore the normal transforming groeth factor – beta regulatory activityy. The immune cells are critical components of the tumor microenvironment. They influence the oncogene’s ability to regulate angiogenesis and senescence. (26)
Suppression of MYC can lead to senescence. There is also imbalance between apoptotic and anti-apoptotic gene expression allowing cell death with its withdrawal.(27)
21
Metastasis and genomic instability induced by MYC:
Cancer cells can proliferate with disregard to extracellular cues . There are many other properties implicated to them. They are increased genomic instability, changes in morphology and function such as epithelial-mesenchymal transition (EMT) and an increased ability to metastasize.
Myc can repress genes involved in cell-cell and cell-matrix contact. The contact is diminished as normal cells detach from neighboring cells to undergo mitosis. Overexpressed Myc would elicit these phenotypes.(28) Myc has been linked to EMT and metastasis via its regulation of a microRNA miR-9 , and its ability to transactivate Bmi-1.
The micro RNA targets E-cadherin. Bmi-1 is linked to EMT .(29)(30)
Myc induces reactive oxygen species.This occurs through its induction of mitochondrial biogenesis and increased metabolism.It results in genomic instability . The normal cells with normal regulated Myc would have the appropriate compensatory mechanisms to detoxify free oxygen radicals. This does not occur in myc
activation .ROS insult on the genome causes instability. (31) Myc also interferes with telomere function and increase genome
instability.(32)It regulates a number of components of the mitotic
22
checkpoint. Its contribution to chromosomal instability is not fully known.(33)
Therapeutic opportunities:
Strategies have emerged in cancer treatment. It involves inhibition of its expression, Myc-Max dimerization, Myc-Max DNA binding, and to interfere with key Myc target genes.(34)(35)
MYC ALTERATIONS IN DLBCL:
MYC is expressed in mature B cells initiating germinal center formation and in a small subpopulation of B cells of the light zone of the germinal center. It is absent in the highly proliferative cells of the germinal center dark zone. Its expression in germinal center cells is repressed when BCL6 is upregulated. A subset of activated cells of the light zone have up-regulated NF-κB and express IRF4. BCL6 is down- regulated. The light zone positive cells are a subpopulation of B cells with high-affinity BCR. They reenter the dark zone for a subsequent round of proliferation if needed.
MYC-negative cells in the light zone will probably be the subset primed to exit the GC. The plasma cell differentiation program is
23
induced by BLIMP-1. It represses MYC expression by binding to its promoter.
TCF3 (E2A) is a potent transcription factor which is highly expressed in the dark zone. It induces its own negative inhibitor, ID3, a target of MYC. ID3 expression suppresses TCF3 program allowing the cell to move from the dark to the light zone. (36)
The oncogenic events leading to the up-regulation of MYC seem to overcome the inhibitory effect of physiological repressors such as BCL6 in germinal centre cells or BLIMP1 in terminally differentiated B cells. These aggressive lymphomas appear to have acquired additional oncogenic alterations that seem to cooperate with MYC dysregulation by counteracting especially its proapoptotic function.
24
TABLE 1
Approximately 5% to 14% of DLBCL cases have been reported to carry MYC translocations. DLBCL that show myc rearrangement may arise de novo. It may be due to transformation from low grade lymphomas. Complicon is amplification of translocated allele.
It is observed in some DLBCL cases.
GCB-DLBCL ABC-DLBCL
Normal cell counterpart
Light zone GC cell Light zone GC cell
MYC inhibitory physiological
mechanisms
BCL6 BCL6
MYC oncogenic activation
MYC translocation BCR/MYD88 activation
MYC cooperating mechanism
BCL2 translocation BCL2 overexpression (18q amplification)
25
MYC protein expression is seen in the majority of DLBCL cases .(37) Low copy number gains of MYC are more common in DLBCL.T there is high copy number of micro RNA expression(38) DLBCL with myc rearrangement shows high expression of micro RNAs. Myc protein expression is seen only in some cases with rearrangements. Mechanisms other than gene rearrangements are responsible for elevated protein expression in a majority of DLBCL cases.
MYC overexpression only when it is regulated along with Bcl2 is associated with shorter survival.(39) Immunohistochemical expression scores of these proteins are able to identify patients with poor prognosis even within IPI subgroups. (40)
FIGURE 7 C-MYC ABNORMALITIES IN IN DLBCL
26
BCL2 FAMILY OF PROTEINS:
Death of cells in many instances apoptosis. It is a mitochondrial pathway in which proteins of the BCL-2 family maintains the integrity of the outer membranes of mitochondria. The interactions among these proteins results in apoptosis.
Pro-apoptotic BCL-2 effector proteins, BAX and BAK are responsible for mitochondrial outer membrane permeabilization(MOMP) . When MOMP occurs, proteins present in the mitochondrial intermembrane space enter the cytosol .this is followed by activation of caspases, cysteine proteases that orchestrate the dismantling of the cell.
Death can never occur by a loss of mitochondrial function (mitochondrial catastrophe) if caspases are inhibited. (42) Some cells can resuscitate if glycolysis is maintained.(43) A small number of mitochondria persist to repopulate the cell. Defects in the mitochondrial pathway downstream of MOMP is seen in tumor cells.(44)
Anti-apoptotic BCL-2 proteins, such as BCL-2, BCL-W, BCL-xL, A1/Bfl1 and MCL-1 inhibit the permeabilization function of BAX and BAK.. Myc synergizes with the anti-apoptotic BCL-2 proteins to transform primary B lymphocytes in vivo by using this interaction.
27
(45) This synergy between Myc and the anti-apoptotic BCL-2 proteins is seen in other cells also . BH3 only proteins regulate the anti-apoptotic BCL-2 proteins and the pro-apoptotic effectors .the efficacy of the proteins differ. Some of these activate the pro-apoptotic proteins. (46)
MCL-1 is the most labile anti-apoptotic BCL-2 proteins. It has ashort half-life . the short half life is due to ubiquitin-dependent and - independent proteasomal degradation .(47) Concerning the cancer therapeutics, BH3-mimetics target only BCL-2 and BCL-xL. The resistance to these drugs is mainly due to MCL-1.(48) The inactivation of MCL-1 is due to binding of BH3 domain .(49) Anti-apoptotic effects are seen with increased expression of conventional MCL-1 (MCL-1L) . Apoptosis is seen with spliced variants, like MCL-1S and MCL1ES.(50)
28
FIGURE 8 : UBIQUITIN DEPENDENT AND INDEPENDENT PROTEASOMAL DEGRADATION OF MCL-1 PROTEIN
Cancer cells show upregulation of Bcl 2. This helps to overcome tumor suppression by pro-apoptotic proteins. Many malignant cells are “primed for death” . Therapeutic impairment of anti-apoptotic proteins can cause death of tumor cells. (51)
29
BCL2 ANTI- APOPTIC PROTEIN:
Human B-cell follicular lymphoma initially showed excess transcription of this gene.(52) it prevents mitochondrial apoptosis by binding both Bax and Bak . It occurs at the mitochondrial outer membrane and by inhibition of Bax/Bak oligomerization. It interferes with pore formation.(53) Bcl-2 can antagonize BH3-only activators Bim and Bid and sensitizers including Puma, Bad and Bik. It localizes to the membranes of multiple organelles in nonapoptotic cells. It includes mitochondrial inner and outer membranes, the ER membrane and the nuclear outer membrane.(54)
The protein mediates the rate of oxidative phosphorylation by interaction with mitochondrial ETC complex IV.(55) Overexpression of Bcl-2 increases oxide within mitochondria. Oxide increases the rate of electron flow through the respiratory chain. Bcl-2-overexpressing tumor cells shows increased activity of mitochondrial respiration rate and complex IV activity.(56)
The Bcl 2 protein physically interacts with Va subunit of complex IV. The extent of localization of this subunit to the mitochondrial inner membrane is in creased in Bcl-2 overexpressing
30
cancer cell lines. Thus Bcl-2 controls mitochondrial bioenergetic function. This happens by influencing the activity of the rate-limiting ETC enzyme, complex IV.
Gene silencing of Bcl-2 expression diminished oxide generation . it is due to reduced inner membrane localization of both Va and Vb complex IV subunits. The expression levels of either subunits is not affected by Bcl 2. This indicates that the effects of Bcl-2 on respiration rate and reactive oxygen species production are due to the role of Bcl-2 in complex IV assembly.(57) Bcl-2 regulates the rates of oxidative phosphorylation and reactive oxygen species production. It also plays role in the promotion of survival and oncogenic signaling in cancer cells.(58)
FIGURE 9 BCL2 TRANSLOCATION DETECTED IN FISH
31
OVEREXPRESSION OF BCL2 IN DLBCL:
Expression of Bcl2 in immunohistochemistry is a strong prognostic factor . It is independent of the IPI and MYC protein/rearrangement status in DLBCL treated with R-CHOP. (59) The ratio of BCL2-positive cases is range from 24% to 80% in studies using immunohistochemistry (IHC). The mutations of Bcl2 are mostly seen in in GCB DLBCL. It is associated with Bcl2 translocation in many cases.(60) Its overexpression is associated with drug resistance.(61)
FIGURE 10 BCL2 OVEREXPRESSION
32
CELL OF ORIGIN CLASSIFICATION OF DLBCL:
DLBCL has become curable in about two-thirds of patients, with the introduction of immunochemotherapy. The current researches in DLBCL have focused on determining which patients have poor prognosis to give them novel targeted-treatment strategies. Gene expression profiling (GEP) of DLBCL identified a subset of diffuse large B-cell lymphoma cases . They expressed genes associated with the germinal center B-cell reaction.Other group expressed genes involved in lymphocyte activation in the post-germinal center state. The germinal center group expresses genes including those encoding for CD10 and BCL6. The latter group, notably expresses IRF4 and BCL2. The former group was called , the germinal center B-cell like lymphomas (GCB) and the latter group was called the activated B-cell like (ABC) lymphomas.
(62)
Later group 3 or unclassifiable has been identified by gene expression profiling . It does not express genes characteristic of either ABC- or GCB-type cells.(63) The immunohistochemical (IHC) patterns are then developed to approximate gene expression profiling. The ABC and GCB subtypes provided a basis for some of the observed heterogeneity of DLBCL. On a molecular level, both these overexpress
33
the antiapoptotic protein BCL2. However nuclear factor kappa B (NF-κB) activation is associated with ABC-type DLBCL . It is due to the mutations in the B-cell receptor (BCR) signaling pathway.
Clinically, the ABC subtype is associated with inferior survival. (64) Owing to the significant molecular and clinical differences between ABC- and GCB-DLBCL subtypes, COO identification provides opportunities to select appropriate therapy. Several targeted therapies are under investigation, such as proteasome inhibitors that reduce NF-κB signaling and BCR pathway inhibitors for ABC-DLBCL.(65) Both subtypes may also be associated with additional molecular subsets which impact their prognosis. Double-expressor lymphoma (DEL) occurs primarily in the activated B- cell subtype. It is associated with high MYC and BCL 2 expression. Alternatively, double-hit lymphoma with concurrent translocations of MYC and BCL 2 is primarily found in the germinal center B- cell subtype. Despite the potential for MYC and BCL 2 expression to be considered as a prognostic biomarker, COO remains independently associated with DLBCL outcomes with prognostic significance. In 2016, the WHO classification of lymphoid neoplasms was revised to include a requirement to determine cell of origin at diagnosis for patients with DLBCL.(1)
34
METHODS OF DETERMINATION OF CELL OF ORIGIN:
GEP originally used Lymphochip competitive array and Affymetrix oligonucleotide microarray methods. But those methods wre time – consuming, expensive and needed fresh frozen biopsy tissue. Also concurrent morphological analysis could not be done. Now, immunochemistry staining and novel GEP methods using formalin- fixed, paraffin – embedded tisssues , help overcome the above difficulties.
IHC allows direct visualization of tumor , minimizing the possibility for misclassification due to the presence of nontumor tissue.
Further, protein levels are expected to correlate better with outcome than messenger ribonucleic acid expression.(66) However, IHC does not identify the unclassifiable subtype that can be determined using molecular techniques. IHC classifies cell of origin into GCB and non- GCB. Non-GCB includes ABC and unclassifiable subtypes. The possibility of inter- and intra- observer variation is also an issue in IHC classification.(67)
35
One of the earliest attempts to classify DLBCL by IHC using markers of B-cell differentiation, including CD10 and BCL6 for GCB expression patterns and IRF4/MUM1 and CD138 expression patterns, but did not predict clinical outcome.(68) The Hans algorithm has became the most commonly used method of IHC-based COO prediction thereafter.
The Hans method relies on expression of CD10, BCL6, and IRF4/MUM1 to distinguish GCB from non-GCB subtypes and has a 71%
and 88% overall agreement with GEP classification for the GCB and non- GCB subtypes, respectively. The chances that the algorithm correctly identifies an ABC subtype patient from a given sample are proportionally lower, thus potentially limiting the ability of IHC methods to retain the prognostic significance of GEP.(69) Additional proposed methods included the Muris algorithm, identification of LMO2, the Choi algorithm and the Tally algorithm. Later Lymph2Cx method was developed as an economical, robust and molecularly validated method to determine COO using GEP on FFPE tissue. It has low error rate compared to IHC methods.
Because immunohistochemical methods are widely available, in places where gene expression technologies are not available, then
36
immunohistochemical methods are considered as an alternative and the algorithm used has to be specified.(1)
DOUBLE EXPRESSOR LYMPHOMAS:
There is marked downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes in double expressor lymphomas. (70) MYC/BCL2 coexpression is a better predictor of prognosis in patients with DLBCL treated with R-CHOP than cell of origin.Furthermore, both the GCB and ABC subtypes of DLBCL as defined by GEP are heterogeneous and do not correlate well with IHC and contain biological subgroups that have different prognoses and may require different therapeutic approaches.
HANS ALGORITHM FIGURE 11
37
Hence dividing patients into subgroups that are biologically homologous and prognostically meaningful, and that are more predictive than the overall categories of GCB and ABC is needed, thereby facilitating therapeutic decisions. Despite their clinical aggressiveness, almost all cases of MYC/BCL2 double-hit lymphoma are of the GCB subtype, a generally favorable prognostic group, illustrating an important discordance between clinical behavior and cell-of-origin (COO) subtypes.
The concept of MYC/BCL2 double-hit lymphoma by assessing for MYC and BCL2 protein expression by IHC, the logic being that protein expression, regardless of mechanisms, may have prognostic significance. In 2 studies, Green et al and Johnson et al showed that DLBCL patients with MYC/BCL2 coexpression, with or without MYC or BCL2 gene rearrangements, have a poorer prognosis .(71) (72)
The availability of anti-MYC antibodies have made it suitable for IHC staining in paraffin-embedded tissues. The results show that MYC/BCL2 coexpression is associated with a poor prognosis . double expression is common in ABC subtype. MYC/BCL2 coexpression in DLBCL is also a strong predictor of poor prognosis in the 2 COO subtypes. Patients with DLBCL with MYC/BCL2 coexpression have
38
many clinicopathological features associated with adverse prognosis. The parameters include older age, advanced stage of disease, multiple extranodal sites of involvement, high IPI score, high proliferation index, and poor treatment response. Their prognosis is better than that of routine diffuse large B cell lymphomas , but worse than that of double hit lymphomas.
39
FIGURE 12 – DOUBLE EXPRESSION ( C-MYC >70% AND BCL2
>90%)
HAEMATOXYLIN
& EOSIN (40x)
40
DOUBLE HIT LYMPHOMAS:
Double hit lymphomas are a separate category of B-cell non- Hodgkin lymphomas. These include high grade lymphomas with myc and bcl2 rearrangements. They can be accompanied by bcl6 rearrangements and are called “triple hit lymphomas”. Morphologically it can be B – lymphoblastic lymphomas, burkitt lymphoma, diffuse large B cell lymphomas and lymphomas that are intermediate between Burkitt lymphoma and diffuse large B cell lymphoma.(1) Approximately 5% of DLBCL are double hit lymphomas , that is, they have translocations in both MYC and BCL2 genes. Prognosis is generally poor in double hit lymphomas.R-EPOCH appears to be an effective therapy for many patients. Patients with double-hit lymphomas have a poor prognosis when treated with standard chemoimmunotherapy. (5) They also have increased risk of central nervous system involvement .
There is no strategy yet developed as when to look for double hit status. Institutions where FISH is readily available and doing FISH does not pose additional cost of testing, FISH is done routinely on all aggressive lymphomas at diagnosis.(73) Studies have been published using immunohistochemistry to restrict FISH testing to selected cases.
This includes cell of origin determination using immunohistochemical
41
methods, high proliferation rate determined using Ki67 index and expression of c-myc protein expression. But double hit status could not be inferred by any of the immunohistochemical methods or patient related characteristics.(74)
R-CHOP is not sufficient in treating double hit lymphomas determined either by immunohistochemistry or FISH. High dose R- CHOP was suggested as an alternative, but most of the double hit occur in older patients, it is difficult for them to tolerate. Studies are going on
FIGURE 13
42
in evaluating dose adjusted R-EPOCH in treating these lymphomas. (75) Some retrospective studies has shown it to be effective . (76) (77) In relapsed and refractory double expressor or double hit diffuse large B cell lymphomas , autologous and allogeneic haematopoietic stem cell transplant have been tried as an alternative to immunochemotherapy . Allogeneic stem cell transplant has come up with better survival than autologous stem cell transplant.(78)
FIGURE 14 C-MYC AND BCL2 DOUBLE HIT
43
PROGNOSTIC SIGNIFICANCE OF DOUBLE EXPRESSOR LYMPHOMAS:
Double expressor lymphomas are identified by expression of c-myc in more than 40 percentage of cells and expression of bcl 2 in more than 50 percentage of cells as determined by immunohistochemistry. Median age, stage and presence of extra-nodal disease of DEL and non-DEL groups vary in different studies. In a study by Aggarwal et al, there was no significant difference in these parameters. In astudy by Johnson et al, the mean age of majority of the double expressor lymphomas was higher.
The International Prognostic Index score was high in DEL group when compared to non-DEL group. The mean LDH level was also high in DEL group compared to the non-DEL group. The relapse rate was high in DEL group following the conventional R-CHOP chemoimmunotherapy. DEL patients survived better with R-CHOP than with R-EPOCH. (79) Cell of origin classification has been studied as a prognostic marker in DLBCL.
It is not much significant because the activated B-cell subtype which is associated with poor prognosis and the germinal center B-cell subtype was found in double expressor lymphomas.(72)
44
STUDY TYPE OF DLBCL PROGRESSION FREE SURVIVAL(PFS)
OVERALL SURVIVAL
(OS)
JOHNSON ET AL(5 YEARS) (72)
Normal 66% 70%
Double expressor 44% 39%
GREEN ET AL (3 YEARS) (40)
Normal 82% 91%
Double expressor 52% 54%
TEOH ET AL (5 YEARS) (81)
Normal - 69%
Double expressor - 44%
45
ROLE OF IMMUNOHISTOCHEMISTRY IN ASSESSING THE PROGNOSIS OF DLBCL:
Immunohistochemistry in diffuse large B-cell lymphoma is gaining importance because of the tailored therapy given to patients .(67) Initially immunohistochemical stains were used for the diagnosis of lymphomas. CD20,CD79a and Ki 67 together with morphology helps in the diagnosis of diffuse large B cell lymphoma in majority of the cases.
However, different patterns exist.
Cell of origin determination using gene expression profiling has got better reproducibility. Since gene expression profiling is not widely available , cell of origin determination using immunohistochemical methods have come into practice.
The germinal center B-cell like subtype has got better prognosis than the non- germinal center B-cell like subtype .
Hans algorithm is the most commonly employed method. It uses CD10, BCL6 and MUM1.
46
Muris algorithm uses BCL2 instead of BCL6 .
Choi algorithm uses GCET1, MUM1, CD10, BCL6, and FOXP1.
Tally algorithm uses CD10, MUM1, GCET1, FOXP1 and LMO2.
FIGURE 15
A – Hans algorithm B –Muris algorithm C-Choi algorithm D-Tally algorithm
Additionally C-MYC and BCL2 are used to determine double expression which has got inferior prognosis than usual DLBCL. Double expressors have been discussed previously.
47
To remember is a small proportion of patients who do not express c- myc and bcl2 carry tanslocations involving c-myc and bcl 2 as areported by Johnson et al and Green et al.(80)
As of now immunohistochemistry does not completely replace molecular or cytogenetic studies in determining prognosis in DLBCL.
48
MATERIALS AND METHODS
This is a prospective and retrospective study conducted in the Department of Pathology , Madras Medical College between July 2017 and June 2019. The ethical approval was granted by the Institutional Ethics Committee of Madras Medical College. .Out of the total 136 non- Hodgkin lymphoma cases reported in 2 years, 118 cases were B-cell lymphomas and 18 were T-cell lymphomas. 34 DLBCL cases were reported under B-cell category. Both nodal and extra-nodal cases of DLBCL were taken for study excluding blocks with inadequate material.
The immunohistochemical examination was done in the chosen blocks.
CHART 1
118 18
NON-HODGKIN LYMPHOMAS
B-CELL NHL T-CELL NHL
49
DATA COLLECTION :
Case details such as age, gender, site, histopathological and immunohistochemical diagnosis were obtained from pathology registers.
Hematoxylin and eosin sections from the paraffin embedded tissue blocks were reviewed. Out of 34 cases of DLBCL cases, 4 blocks had very tiny material and were excluded, taking 30 cases for the study of immunohistochemical expression of c-myc and bcl2.
PROCEDURE OF IMMUNOHISTOCHEMISTRY:
Table 2
ANTIGEN VENDOR SPECIES (CLONE)
PATTERN OF STAINING
POSITIVE CONTROL
C-MYC Path insitu Rabbit monoclonal
Nuclear Colon
BCL 2 Path insitu Rabbit monoclonal
Cytoplasmic Reactive node
50
IMMUNOHISTOCHEMISTRY STEPS:
1. 4 Micron thick sections were cut from formalin fixed paraffin embedded tissue blocks and transferred to positively charged glass slides.
The glass slides were incubated at 37 degree celcius overnight 2.Deparaffinisation :
The slides were immersed in xylene for 15 minutes x 2 changes and immersed alcohol for 5 mins x 2 changes .after which the slides were kept in distilled water for 5 minutes
3.Retrival of antigen:
This was done using pressure cooker with the slides immersed in Tris Buffer which is preheated for 10 minutes at 160degree celcius.after preheated the slides were placed inside the tris buffer under pressure for 15 minutes at 130 degree celcius and then the temperature is reduced by maintaining 60 degree fOR 5 minutes and then gradually cooled to room temperature. The cool slides were then washed with distilled water for 5 mins x 2 changes . 4.Application of peroxide:
After distilled water wash the slides were washed with wash buffer prepared for 5 mins.hydrogen peroxide was added to the sections and washed twice after 10 minutes.
51
5.Application of primary antibody :
After placing the slides in wash buffer for 5 minutes ,the primary antibody ( c-myc and bcl2 each added to one slide) was added and incubated at room
temperature for 40 minutes. After 40 minutes the slides were washed in wash buffer for 5 mins x2 changes.
6.Application of secondary antibody :
Then HRP conjugated polymer was added and washed in wash buffer ( 2 changes each for 2 minutes) after 12minutes.
7.Application of chromogen:
The sections were covered with Diamino benzidine (DAB) chromogen .(DAB was prepared by diluting 1 drop of DAB chromogen in 1 ml of DAB buffer).
The slides were washed in distilled water after 5 minutes.
8.Counter staining with hematoxylin and washed in running tap water for 15 minutes ,the slides were dried and mounted .
52
STATISTICAL ANALYSIS
The collected data were analysed with IBM Statistical Package for the Social Science (SPSS) statistics software 23.0 Version.To describe about the data descriptive statistics frequency analysis, percentage analysis were used for categorical variables and the mean & S.D were used for continuous variables. To find the significance in categorical data Chi-Square test was used similarly if the expected cell frequency is less than 5 in 2×2 tables then the Fisher's Exact was used.
In all the above statistical tools the probability value less than 0.05 is considered as significant level.
53
OBSERVATION AND RESULTS
The current study on double expression of c-myc and bcl2 has yielded the following results. The observations are categorized into distribution according to age, gender, site, positivity of c-myc, positivity of bcl 2 , cell of origin and double expression. Furthermore, comparison is made between age and expression of c-myc, bcl 2 and double expression . Comparison of gender with the above parameters is also done.
DISTRIBUTION OF DLBCL ACCORDING TO AGE:
CHART 2
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
31-40 41-50 51-60 61-70 71-80 16.7
23.3
40.0
16.7
3.3
Percentage
Age
Age(years)
54
The median age of DLBCL in the current study is 53 years. The age range is 35 to 79 years. Maximum number of cases are reported in the 51 to 60 years of age . The percentage of cases reported in that age
2
5
4
2
1 3
2
8
3
0 0
1 2 3 4 5 6 7 8 9
31-40 41-50 51-60 61-70 71-80
Percentage
Age
Age(years) with Gender
Female Male
Age (years) Frequency Percent
31 – 40 5 16.7
41 – 50 7 23.3
51 – 60 12 40.0
61 – 70 5 16.7
71 - 80 1 3.3
Total 30 100
TABLE 3
CHART 3
55
group comes to 40%. The incidence is less after 70 years of age and there are no cases reported under 30 years .
DISTRIBUTION OF DLBCL ACCORDING TO GENDER:
CHART 4
TABLE 4
Gender Frequency Percent
Female 14 46.7
Male 16 53.3
Total 30 100
The current study shows reported cases of DLBCL to be slightly higher in males . percentage of DLBCL in males is 53.3%.
47%
53%
Gender
Female Male
56
DISTRIBUTION OF DLBCL ACCORDING TO SITE:
CHART 5
TABLE 5
Site Frequency Percent
Extranodal 11 36.7
Nodal 19 63.3
Total 30 100
The maximum number of cases reported are nodal in origin with the percentage of 63.3. The extranodal sites include gastrointestinal sites like stomach and ileum, salivary gland, testis, femur, back, skin and tonsil. Out of the 11 extranodal cases, 9 are in male and two in female with skin and femur being the sites reported in female patients.
37%
63%
Site
Extranodal Nodal
57
DISTRIBUTION OF DLBCL ACCORDING TO CELL OF ORIGIN:
CHART 6
TABLE 6
Cell of origin Frequency Percent Non-germinal center B-
cell like
6 24
Germinal center B-cell like
19 76
Total 25 100
Cell of origin was determined using Hans algorithm as germinal center B-cell like and non-germinal center B-cell like subtypes. Of the 30
24%
76%
Cell of Origin
Non-germinalcenter B-cell like Germinal centre B-Cell like
58
cases, cell of origin was analysed only in 25 cases. Out of the 25cases, 19 were germinal center B-cell like which comes to a percentage of 76%. Of the GCB subtype, 8 cases are extranodal.
DISTRIBUTION OF C-MYC POSITIVITY IN DLBCL:
TABLE 7
C-MYC FREQUENCY PERCENT
POSITIVE 3 10
NEGATIVE 27 90
TOTAL 30 100
Out of the 30 cases analysed for c-myc immunohistochemically, 3 showed positive expression of c-myc (more than 40%). Of the three, cell
POSITIVE NEGATIVE TOTAL
FREQUENCY PERCENT
CHART 7
59
of origin was known only in one case and it was non-germinal center B- cell like subtype.
COMPARISON OF AGE WITH C-MYC EXPRESSION:
COMPARISON OF GENDER WITH C-MYC EXPRESSION: s
0%
20%
40%
60%
80%
100%
N P
Percentage
c-MYC(%)
Age with c-MYC
31-40 41-50 51-60 61-70 71-80
0%
20%
40%
60%
80%
100%
N P
44.4%
66.7%
55.6%
33.3%
Percentage
c-MYC(%)
Gender with c-MYC
Female Male
CHART 8 P value
calculated using Fisher Exact method
p-value – 0.166 statistically not significant
CHART 9 P value
calculated using Fisher Exact method
p-value – 0.464 statistically not significant
60
DISTRIBUTION OF BCL2 EXPRESSION IN DLBCL:
CHART 10
TABLE 8
BCL2 FREQUENCY PERCENT
POSITIVE 14 46.6
NEGATIVE 16 53.4
TOTAL 30 100
Bcl 2 positivity, that is percentage of expression more than 50%, is observed in 14% of the cases, which comprises 46.6% of the 14 cases, cell of origin is determined in 10 cases with six cases coming under GCB subtype and four cases coming under ABC subtype.
POSITIVE NEGATIVE TOTAL
61
COMPARISON OF AGE WITH BCL 2 EXPRESSION:
COMPARISON OF GENDER WITH BCL 2 EXPRESSION:
0%
20%
40%
60%
80%
100%
N P
Percentage
BCL2(%)
Age with BCL2
31-40 41-50 51-60 61-70 71-80
50.0% 42.9%
50.0% 57.1%
0%
20%
40%
60%
80%
100%
N P
Percentage
BCL(%)
Gender with BCL2
Female Male
CHART 12 P value
calculated using Chi- square test p-value – 0.696 statistically not significant
CHART 11 P value
calculated using Fisher Exact method
p-value – 0.342 statistically not significant
62
DISTRIBUTION OF DOUBLE EXPRESSION IN DLBCL:
TABLE 9
DOUBLE EXPRESSION
FREQUENCY PERCENT
POSITIVE 3 10.0
NEGATIVE 27 90.0
TOTAL 30 100
Double expression is seen in 3 out of 30 cases which comprises 10%. Of the three cases, cell of origin is determined for one case and it is of non-germinal center like B-cell subtype. Two cases are extranodal . Two cases are of female gender. C-myc expression is 40% in two cases
90%
10%
Double Expression
Negative Positive
CHART 13
63
and 45% in one case. Bcl 2 expression is 90% in two cases and 60% in one case.
COMPARISON OF AGE WITH DOUBLE EXPRESSION:
COMPARISON OF GENDER WITH DOUBLE EXPRESSION:
0%
20%
40%
60%
80%
100%
N P
Percentage
Double expression
Age with Double Expression
31-40 41-50 51-60 61-70 71-80
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
N P
Percentage
Double expression
Gender with Double expression
Female Male
CHART 15 P value
calculated using Fisher Exact method
p-value – 0.586 statistically not significant
CHART 14 P value
calculated using Fisher Exact method
p-value – 0.166 statistically not significant
64
COMPARISON OF C-MYC WITH CELL OF ORIGIN:
COMPARISON OF BCL 2 WITH CELL OF ORIGIN:
0 5 10 15 20 25
POSITIVE NEGATIVE
ABC GCB
0 2 4 6 8 10 12 14 16 18 20
POSITIVE NEGATIVE
ABC GCB
CHART 16
P value calculated using Fisher Exact method
p-value – 0.24 statistically not significant
CHART 17
P value calculated using Fisher Exact method
p-value – 0.18 statistically not significant
65
COMPARISON OF DOUBLE EXPRESSION WITH CELL OF ORIGIN:
0 5 10 15 20 25
POSITIVE NEGATIVE
ABC GCB
CHART 18 P value
calculated using Fisher Exact method
p-value – 0.24 statistically not significant
66
DISCUSSION
Double expression in diffuse large B cell lymphoma is associated with inferior prognosis than non-double expressing lymphomas. The inferior prognosis of this category makes it essential to opt for an alternative therapy to the standard R-CHOP chemoimmunotherapy. Among the 30 cases selected for the study , the median age at diagnosis is 53 years; the range being 31 to 80 years. The age group pattern is different from the selected cohort in the study by Johnson et al and Green et al.
TABLE 10:
STUDY MEDIAN AGE AGE RANGE
Johnson et al (n=307) 63 17 to 92
Green et al (n=193) 64 16 to 91
Current study (n=30) 53 35 to 79
The median age of the current study is 10 years less than that in the study by Johnson et al and 11 years less than that in Green et al’s.
Owing to the large difference in sample size,the age range is also less than that in both these studies. The majority of the cases in the study are male but the difference is narrow.
