1. Molecular Biology of
Lymphoma
Dr Ramesh Purohit
Acharya Tulsi Regional Cancer Treatment &
Research Centre, Bikaner

2. Lymphoma classification
(based on 2001 WHO)
• B-cell neoplasms
– Precursor B-cell neoplasms
– Mature B-cell neoplasms
– B-cell proliferations of uncertain malignant potential
• T-cell & NK-cell neoplasms
– Precursor T-cell neoplasms
– Mature T-cell and NK-cell neoplasms
– T-cell proliferation of uncertain malignant potential
• Hodgkin lymphoma
– Classical Hodgkin lymphomas
– Nodular lymphocyte predominant Hodgkin lymphoma

3. Thirty diseases, one name:
Try to imagine a single type of cell giving rise to nearly thirty different
types of cancer - all with one name. Lymphoma - simply in the nodes, a
mass in your brain, a disease of your stomach, or lesions all over your
skin. It's not simply a matter of location. The behavior changes with the
type and so does the treatment and the outcome.
The microscope isn't enough:
Even a couple of decades back, what the pathologist saw under the
microscope with simple stains was all that we had to identify the type of
lymphoma. And there were only a few types of lymphoma that could be
distinguished. However, it often turned out that the behavior of the
same type of tumor was different in different individuals. Clearly, we
were missing something.
The clue is in the molecules: MOLECULAR BIOLOGY (including
Immunophenotyping and Genetic studies)

4. Use of Immunophenotyping and Genetic Studies in
the Diagnosis of Lymphoid Neoplasms
 The lymphoid neoplasms each have a characteristic morphology,
which is sometimes sufficient to permit diagnosis and classification if
well-prepared adequately sized sections are available.
 However, there are many pitfalls in the histologic diagnosis of
malignant lymphoma, immunophenotyping and genetic studies are
extremely useful for resolving differential diagnostic problems.
 Immunophenotyping and genetic studies are also developing key
roles in patient management beyond diagnosis, including
• identification of prognostic molecules,
• detection of minimal residual disease, and
• assessment of appropriate molecules for targeted therapy.

5. Need for Molecular Diagnosis
 Rule out other disorders associated with lymphocytosis
 If lymphoproliferative disorder remains a significant possibility after
clinical evaluation, cell surface phenotyping of lymphocytes should be
performed.
 Usually performed on peripheral blood using flow cytometry.
 Technique provides percentage of lymphocytes positive for a
particular antigen and density of antigens.
 Normal peripheral blood lymphocytes consist of approximately 10%
B-cells, 80% T-cells and 10% NK-cells

6. Flow Cytometry
to see markers on the surface of
the cells.
This is a test that uses
fluorescent antibodies to tag
molecules on the surface of cells.
The flow cytometer has a teeny
tube that allows the cells to flow
one at a time past a laser beam.
In addition to telling what kinds of
markers a cell has, you can also
sort cells by size and complexity.

7. CD Markers
The “CD” stands for “cluster designation / cluster of differentiation”
It’s just a way of referring to the different molecules on the surface of cells
so that instead of having all kinds of different names for these molecules,
there is just one name (a number, actually) for each molecule.
It’s used for lots of different purposes,
one of the most common (in hospital practice, anyway) being to find out
what markers are on the surface of cells.
e.g. In a g leukemia case, the cells expressed CD 13 and CD 33, you’d
know the cells were myeloid, and that it was most likely an acute myeloid
leukemia.
sometimes it’s the absence of a marker that helps you with the diagnosis.
e.g. if you have a lymphoid neoplasm in which the cells are small and
mature looking, and by flow those cells are CD5 positive but CD23
negative, you’d be able to rule out chronic lymphocytic leukemia and lean
towards a diagnosis of mantle cell lymphoma.
Flow cytometry is super helpful with making a specific diagnosis.

8. CD Markers
 Most commonly used markers (CD = cluster designation)
 B-cell – CD10, CD19, CD20, CD22, CD23, CD24, CD79b,
CD103, Pax-5, kappa, lambda, CD200, cytoplasmic kappa,
cytoplasmic lambda
 T-cell – CD1, CD2, CD3, CD4, CD5, CD7, CD8, TCR α-β, TCR
γ-δ, cytoplasmic CD3
 Myeloid/monocyte – CD11b, CD13, CD14, CD14, CD15, CD33,
CD64, CD117, myeloperoxidase
 Miscellaneous – CD11c, CD16, CD25, CD30, CD34, CD38,
CD41, CD42b, CD45, CD56, CD57, CD61, HLA-DR, glycophorin,
TdT, bcl-2

9. CD Markers
CD1a, CD207: Langerhan cell histiocytosis cells
CD2, CD3, CD4, CD5, CD7, CD8: T cells
CD10: Early pre-B cells (immature B cells)
CD11c, CD25, CD103, CD123: Hairy cell leukemia cells
CD13, CD33, CD117: Myeloid cells
CD14, CD64: Monocytic cells (positive in AML-M4 and AML-M5)
CD15 :Reed-Sternberg cells, neutrophils
CD16, CD56: Natural killer cells
CD19, CD20, CD21, CD22 : B cells
CD23 and CD5 : Chronic lymphocytic leukemia/small lymphocytic
lymphoma
CD23 negative and CD5 positive: Mantle cell lymphoma cells

10. CD Markers
CD30 and CD15: Reed-Sternberg cells
CD30 positive and CD15 negative: Anaplastic large cell lymphoma
CD31: Endothelial cells (positive in angiosarcoma)
CD33: Myeloid cells and precursors
CD34: Stem cells (also positive in angiosarcoma)
CD41, CD61: Megakaryocytes and platelets (positive in AML-M7)
CD45 : All leukocytes (except Reed-Sternberg cells!)
CD45 RO: Memory T cells
CD45 RA: Naive T cells
CD68: Histiocytes (positive in malignant fibrous histiocytosis)
CD99: Ewings sarcoma cells
CD117: Gastrointestinal stromal tumor (GIST) cells, mast cells
(positive in mastocytosis), myeloid cells

11. Stages of Maturation/Differentiation
Lineages
Lymphoid Myeloid
• cells are defined by lineage and stage of maturation/differentiation
• regulated by signaling pathways and transcription factors
•cell “identity” may be determined using morphology, immunophenotyping and
molecular/genetic studies

12. ALL CLL
AML
CML
Lymphomas
Lymphomas

13. Lymphocyte Differentiation

14. B-Lineage Lymphopoiesis
Morphology / Immunophenotyping / Molecular Studies
Markers are helpful in determining:
1. Lineage (ex. CD19)
2. Maturation (ex. TdT, CD34, CD10)
3. Both (ex. sIg)
Status of immunglobulin genes (i.e., germline, rearranged,
somatic mutations) has implications for both
lineage and maturation.

15. B-cell lymphoproliferative disorders
Probable if immunoglobulin light chain restriction is demonstrated by surface typing of
kappa or lambda
B-cell CLL or mantle cell lymphomas (MCL) are suspected if CD5 is positive and
CD10 is negative
Circulating MCL can be mistaken morphologically for B-cell CLL or B-cell
prolymphocytic leukemia (B-PLL)
MCL considered in the following
CD20, CD19 – strong intensity
Surface immunoglobulin – strongly expressed
CD23 – absent
Diagnosis
Molecular and FISH testing
Requires t(11;14) translocation demonstration
CLL is more likely when
CD20 – weak intensity
Surface immunoglobulins – weakly expressed
CD23 – present
CD200 – present

16. B-cell lymphoproliferative disorders
Circulating germinal center cell-derived lymphoma is probable if CD10 is positive
and CD5 is negative
Germinal center lymphomas – follicular, Burkitt lymphoma, diffuse large B-cell
lymphoma (DLBCL)
Diagnosis
Some cases can be confirmed by demonstration of t(14;18) breakpoint by
PCR or FISH testing
PCR detects approximately 80% of t(14;18) translocations found in follicular
lymphoma
FISH is more sensitive for this translocation in fixed tissue
FISH can also detect an MYC or BCL6 rearrangement for BL or DLBCL
Marginal zone lymphoma should be considered if both CD5 and CD10 are negative
Hairy cell leukemia (HCL) has a characteristic phenotype that is CD5-, CD10-,
CD11c+, CD22+, CD25+, and CD103+
CD103 antigen (also known as B-ly7) is present in virtually all cases
CD11c and CD25 are less specific but present in almost all cases of hairy cell
leukemia
HCL variant can be considered in otherwise typical cases of HCL when CD25-

17. T-cell lymphoproliferative disorders
Most show abnormalities of pan T-cell antigens CD2, 3, 5, or CD7
T-cell disorders
Proliferating lymphocytes are usually positive for CD3
Most common form is large granular lymphocytosis
Usually show rearrangement of TCR locus
Clonality assessed by flow cytometry, PCR or next generation
sequencing (NGS)
Large granular lymphocytosis is suspected if percentage of CD16+,
CD56+, or CD57+ T cells is >50% or if absolute count of these cells
>2,000/μL
Angioimmunoblastic lymphoma has characteristic CD10+ and CD4+, and
CD52-, CD56-, and CD16-
Anaplastic large cell lymphoma – CD30+ and ALK(+)
Some pan T-cell antigens are frequently deleted
Sézary syndrome should be considered if CD4+, CD7-, and CD26-

18. Hodgkin’s
Lymphoma

19. Immunophenotyping in Hodgkin’s
Lymphoma
NLPHL is immunophenotypically distinct from other types of HL.
The lymphocytic and histiocytic (L&H) cells usually express
• LCA (CD45),
• immunoglobulin J chain,
• B-cell antigens (CD19, CD20, CD22, CD79A, and BCL-6),
• and epithelial membrane antigen (EMA) and are
 negative for CD15 and CD30 (Fig. 7-1 C and D ).
These results suggest that the L&H cells are B cells that arise from the
germinal center.
The L&H cells are negative for T-cell antigens but are often surrounded
by a rosette of small, reactive T cells that may be positive for pan–T-cell
antigens and CD57.
Epstein-Barr virus (EBV) is almost always absent in the L&H cells of
NLPHL

20. Immunophenotypic Findings in
Classical Hodgkin's Lymphoma
 positive for CD15 and CD30 and
 negative for LCA (CD45) and EMA .
 B-cell antigens—such as CD20, CD79A, PAX-5/BSAP, and
MUM1/IRF4—are expressed in a subset of cases.
 CD20 expression is often weak.
 T-cell antigens are usually not expressed by the neoplastic cells.
 BCL-2 is positive in up to half the cases and has been correlated
with poorer prognosis.
 EBV is common in the Reed-Sternberg and Hodgkin cells of
classic HL

21. Hodgkin's Lymphoma and Cell Lineage
 both NLPHL and classical types of HL, the neoplastic cells arise from B-cell
precursors .
 neoplastic cells of HL carry monoclonal immunoglobulin (Ig) gene
rearrangements.
 In NLPHL, the Ig gene rearrangements are usually functional, and Ig mRNA
transcripts can be identified in most L&H cells. The Ig gene variable regions
also carry somatic mutations. As the process of somatic mutation is restricted
to the germinal center of secondary lymphoid follicles, the presence of somatic
mutations suggests that NLPHL arises from germinal center B cells.
 In classical HL, over 95% of cases carry monoclonal Ig gene
rearrangements, with somatic mutations in the variable regions suggesting
germinal center B-cell origin. However, unlike the case in NLPHL, there are
defects in Ig transcription, and thus Ig mRNA transcripts are often absent. In
25% of cases, the mutations are extensive or involve stop codons, so-called
"crippling mutations.”

22. NHL

23. Follicular Lymphoma
 FL is a neoplasm of mature B-cell lineage
 Most grade 1 and 2 tumors express immunoglobulin, but a subset of FLs,
mostly grade 3, may be immunoglobulin-negative.
 All FLs express pan–B-cell markers, and typically express immunoglobulin
and B-cell antigens at high density ("bright" immunofluorescence by flow
cytometry).
 These neoplasms also express the germinal center-associated markers
CD10 and BCL-6 and are negative for T-cell antigens.
 BCL-2 is expressed in 80 to 90% of FLs and is most often negative in
grade 3 neoplasms.
 As BCL-2 is negative in reactive germinal centers, this marker is helpful in
differential diagnosis

24. Follicular Lymphoma
 The cytogenetic hallmark of FL is the t(14;18)(q32;q21), which is
identified in 80 to 90% of neoplasms.
 However, a small subset of FLs lack the t(14;18) including
• grade 3B nodal FL
• FLs arising in extranodal sites, such as skin,
• and FLs occurring in children.
 Other cytogenetic abnormalities have been reported in FL. Of these,
trisomy 7 and 18, abnormalities of 3q27-28 and 6q23-26, and 17p
deletions are most frequent.
 Abnormalities of 3q27-28 involve the bcl-6 gene and most often occur
in the form of translocations .

25. Diffuse large B-cell lymphoma
 DLBCLs are of mature B-cell lineage. Approximately two-thirds of cases
express monotypic immunoglobulin (Ig);
 approximately one-third of DLBCLs are Ig-negative.
 These tumors express pan-B-cell antigens, 60 to 70%
express BCL-2, and a subset is positive for CD10 and BCL-6.
 Most DLBCLs have a high proliferation rate.
 Diffuse large B-cell lymphomas are heterogeneous at the molecular
level.
 A subset of cases carries the t(14;18) involving the bcl-2 gene,

26. Diffuse large B-cell lymphoma
 Another subset of DLBCLs has translocations or other
abnormalities involving the bcl-6 gene at chromosome 3q27.
 The bcl-6 gene is rearranged in approximately 20 to 40% of
DLBCLs, more often in tumors arising in extranodal sites
 Gene-expression profiling studies performed in recent years have
suggested that DLBCLs can be divided into three groups:
o germinal center cell type,
o activated B-cell type, and a third,
o noncharacteristic group.
 Patients with the germinal center type of DLBCL have a better
prognosis independent of the IPI

27. Mantle cell lymphoma
 Immunophenotypic studies have shown that MCLs express monotypic Ig light chain
(more often Ig λ), IgM, IgD, pan-B-cell antigens, BCL-2, alkaline phosphatase, and CD5
(23).
 Unlike CLL/SLL, MCL is often positive for CD79B and FMC-7 and typically is negative
for CD10, CD23, and BCL-6. However, approximately 10% of MCLs can be CD23-
positive.
 The t(11;14)(q13;q32) is present in virtually all cases of MCL (100). In this
translocation the ccnd-1 gene (also known as PRAD1 and bcl-1) on 11q13 is juxtaposed
with the Ig heavy chain gene on 14q32, resulting in overexpression of cyclin D1. Cyclin
D1 facilitates cell cycle transition from G1 to S phase (101).
 Although the t(11;14) is central to the pathogenesis of MCL, the t(11;14) is not
sufficient to cause lymphomagenesis. Other molecular abnormalities are also required
like mutations in the atm, p16, and p53 genes.

28. Burkitt lymphoma
Burkitt's lymphomas of endemic, sporadic, and AIDS-associated
types are of mature B-cell lineage
They express Ig, pan-B-cell antigens, CD10, and BCL-6.
Burkitt's lymphomas have a very high proliferation rate, >99%,
using an antibody specific for Ki-67.
These tumors are negative for IgD, CD21, CD23, lymphocyte
homing receptors, and T-cell antigens. They are usually negative
for BCL-2.

29. Burkitt lymphoma
 C-myc translocations are characteristic of Burkitt's lymphoma.
 Approximately 80% of cases carry the t(8;14)(q24;q32),
 the remaining cases having one of two variant translocations,
t(2;8)(p11;q24) or
t(8;22)(q24;q11).
 Common to each of these translocations is involvement of
chromosome region 8q24, the site of the c-myc gene, which is
deregulated.
 Via these translocations, c-myc is juxtaposed with the Ig heavy
chain on the derivative chromosome 14, or with the Ig κ and
Ig λ genes on the derivative chromosome 8.

30. stem
cell
lymphoid
precursor
progenitor-B
pre-B
immature
B-cell
mature
naive
B-cell
germinal
center
B-cell
memory
B-cell
MZL
CLL
plasma cell
DLBCL,
FL, BL, HL
LBL, ALL
CLL
MCL
MM

31.  Genetic alterations
 Infection
 Antigen stimulation
 Immunosuppression

32.  NHL: A heterogeneous group of diseases
deriving from
 Mature B cells 85% and
 T cells 15%.
 Among B-NHL, most histologic subtypes arise
from germinal center (GC) or post-GC B cells,

33.  In contrast with neoplasms of precursor
lymphoid cells, chromosomal translocations
associated with mature B and T-cell
malignancies do not generally lead to coding
fusions between two genes.
 They juxtapose the proto-oncogene to
heterologous regulatory sequences derived
from the partner chromosome.

34. REG REG
CODING REG CODING REG
CODING CODING
Proto-oncogene
Proto-oncogene
TRANSLOCATION TRANSLOCATION
CODING ING
REG = regulatory sequence.
TRANSCRIPTIONAL
DEREGULATION
FUSION
PROTEIN
REG
REG
COD

35.  Two exceptions to the deregulation
model of NHL translocations:
 t(2;5) of T-cell anaplastic large cell lymphoma
and
 t(11;18) of MALT lymphoma,
 These cause gene fusions coding for chimeric
proteins.

37. Oncogene Protein Translocation Disease
bcl-1 Cyclin D1 t(11;14) MCL
bcl-2 BCL2
(antiapoptosis) t(14;18) FL
myc Transcription factor t(8;14) Burkitt’s NHL
bcl-6 Zinc-finger
transcription factor t(3;14) DLBCL
.

38. Molecular Testing in Lymphoma
1. Establishing a diagnosis of lymphoma
•What is the significance of clonality?
2. Classification of lymphoma
3. Discovery and future developments
•Refining prognostic and diagnostic categories
•Developing new therapeutic regimens

39. In the presence of antigen T- and B-lymphocytes
B
T
combine to produce:
B
B B
B
Plasma cells/specific antibody
An expanded
clone of
memory B-cells

40. A reactive lymphocyte proliferation is polyclonal;
Each expanded clone has different gene re-arrangement

41. A neoplastic lymphocyte proliferation is clonal
•Same gene rearrangement
•Same chromosomal abnormality

42. Polymerase Chain Reaction for IGH chain gene
(and TCR gene) re-arrangement can be used to
determine pattern of clonality within a lymphoid
infiltrate
•Implication:
clonality = malignancy
primers
Products:
Same size in monoclonal population
Different sizes in polyclonal population

43. Limitations and Pitfalls of Molecular Clonality Studies
1. Limited sensitivity
2. Clonality does not equate with malignancy
3. Ig & TCR re-arrangements are not markers of lineage
4. Pseudoclonality
5. Oligoclonality
6. False positive results
7. False negative results

44.  Disruption of TS loci in NHL:
leads to biallelic inactivation, through deletion
of one allele and mutation the other.
 The TS genes in NHL: p53, p16, and ATM.

45. IGH gene rearrangement
DEATH
No encounter with antigen
Naïve B-cell
Encounter with appropriate antigen
SURVIVAL
CD34+
Progenitor B cell
Pre-B cell
L gene rearrangement
Immature B cell:
IgM+/IgD-IGK+/-
Mature B cell:
IgM+/IgD+
Immunoglobulin gene rearrangements

46. ALL MCL, CLL Burkitts, FL, DLBCL WM MM
Stem cell Pre-B Early B Mature B Activated B Plasmacytoid B
Plasma
Germinal center
Type of B cell lymphoma is a function of:
1) Where the cell was in development/maturation when it went “bad”
2) What molecular derangement occurred

48. B-cell Lymphoma
Type CD5 CD10 CD19 CD20 CD22 CD23 CD43 CD79a sIg cIg
Follicular 1 3 4 4 4 2 1 4 4 0
Nodal marginal
1 1 4 4 4 1 2 4 M4, D1 2
zone
MALT 1 1 4 4 4 1 2 4 M4 2
Splenic Marginal
zone 1 1 4 4 4 1 0 4 M4 2
CLL/SLL 4 0 4 4 4 4 4 4 D3 2
Lymphoplasmacytic
Waldenstroms 1 1 4 4 4 0 3 4 M4, D2 4
Mantle Cell 4 1 4 4 4 1 4 4 M&D 4 0
Precursor B-cell
(lymphoblastic) 4 3 4 4 4 0 0 4 0 0
Diffuse large B-cell 2 2 4 4 4 0 1 4
Mediastinal large
cell 2
Burkitt's 1 4
Footnote: 0 = negative, 1 = <10% positive, 2 10-50% positivity, 3 = 50-90% positivity and 4
= >90% positive

49. T-cell CD markers
Type CD3 CD5 CD7 CD4 CD8 CD30 NK16/56
T-prolymphocytic leukaemia + - + +(-) -(+) - -
T-large granular lymphoproliferative + - + - + - +(-)
Mycosis Fungoides + + + - -(+) -(+) -
Cutaneous ALCL + +(-) +(-) +(-) - ++ -(+)/-(+)
Primary systemic ALCL +(-) +(-) +(-) -(+) -(+) ++ -
Peripheral T-cell lymphoma,
unspecified +(-) +(-) -(+) +(-) -(+) -(+) -(+)/-(+)
Subcutaneous panniculitis-like T-cell
+ + + -(+) +(-) -(+) -/-(+)
Hepatosplenic T-cell lymphoma + - + - - - +/+(-)
Angioimmunoblastic T-cell
lymphoma + + - +(-) -(+) - -
Extranodal NK/Tcell lymphoma S -, C + - -(+) -(+) - - -/+
Enteropathy-associated T-cell
lymphoma + + + -(+) +(-) +(-) -
Adult T-cell leukaemia/lymphoma + + - +(-) -(+) +(-) -
Footnote: + = >90% positive: +(-) = >50% positive; -(+) = <50% positive; - = <10% positive.
ALCL-Anaplastic large cell lymphoma; C=Cytoplasmic; S-Surface.

50. Question: Identify the disease by this IHC report