B-cell Chronic Lymphocytic Leukemia / Small Lymphocytic Lymphoma

(B-cell CLL)

Epidemiology:

• Adults – rare before 40y
• Common
• Familial predisposition in 5-10% of patients with CLL
• 2-7x higher risk with 1^st degree relative
• Also increased risk of other lymphoid neoplasms
• As many as 30 genomic loci related to inherited susceptibility to CLL

Common sites:

• Peripheral blood
• bone marrow
• lymph nodes
• liver
• spleen
• occasionally:
• skin
• breast
• ocular adnexae

Gross features:

Histologic features:

• diffuse architecture
• almost entirely small lymphocytes
• look exactly like interfollicular lymphocytes
• very little cytoplasm
• round to oval nuclei
• little irregularity usually
• condensed chromatin
•  prolymphocytic areas (pseudofollicles or proliferation centres)
• continuum of small, medium, and large cells
• prolymphocytes:
• medium-size
• dispersed chromatin
• small central nucleolus
• paraimmunoblasts:
• medium to large size
• round to oval nuclei
• dispersed chromatin
• central eosinophilic nucleolus
• slightly basophilic cytoplasm
• cells with more cytoplasm
• prominent central nucleoli
• CLL with increased prolymphocytes (CLL/PL):
• 10% < prolymphocytes < 55%
• clinically aggressive
• Other findings associated with progression/transformation:
• Increase in size of proliferation centres (broader than a 20x field or becoming confluent)
• Increase in proliferation rate of proliferation centres
• ki-67 index > 40% (data limited)
• > 2.4 mitoses in proliferation centres (data limited)
• transformation to diffuse large B cell lymphoma (Richter syndrome) (3.5%):
• confluent sheets of large cells
• often centroblast or immunoblast-like
• may resemble paraimmunoblasts
• only 50% arise from the CLL clone – the remainder may be a second neoplasm
• may see:
• plasmacytoid differentiation
• scattered Hodgkin / Reed-Sternberg cells in a background of CLL (association with Hodgkin disease)
• cytology:
• small lymphocytes
• clumped chromatin
• nucleoli indistinct or absent
• scant cytoplasm
• clear to lightly basophilic
• regular outline
• smudge or basket cells typically (blood smear)
• prolymphocytes:
• typically <2% on blood film - if more correlates with more aggressive course, p53 abnormalities and trisomy 12
• spleen:
• white pulp involvement prominent
• red pulp also involved
• pseudofollicles less conspicuous than in lymph nodes
• bone marrow:
• nodular, interstitial, diffuse, or combo of patterns
• diffuse pattern correlates with advanced disease and bone marrow failure
• pseudofollicles less common
• NOT typically paratrabecular aggregates
• Mu heavy chain disease variant:
• Defective mu heavy chain lacking a variable region is produced
• Characteristic vacuolated plasma cells admixed with small round lymphocytes in the bone marrow
• Slowly progressive course
• Hepatosplenomegaly
• Absence of lymphadenopathy

Immunophenotype:

Marker:	Sensitivity:	Specificity:
CD5	>95%
CD23 (receptor for IgE)	>95%	Rare cases of mantle may partially express CD23
Surface IgM +/- IgD (dim)	Some my have strong sIg; exceptional cases may express IgG or IgA
cIg	-/+
CD19 (strong)
CD20 (weak)
CD22 (neg or weak)	+/-
CD79a
CD43
CD11c (weak)
FMC7 (neg or weak)	85%
CD79b (neg)	usually
p53	10%
CD38	Some (worse prognosis)
Cyclin D1 (neg)	May be seen in cells of proliferation centres
CD10 (neg)
ZAP70	Some (poor prognosis)

• CD38 correlates without Ig V_H mutations, trisomy 12, and atypical morphology (worse survival)
• Immunophenotype scoring system for the diagnosis of CLL (1 point each – most CLL cases score 4 to 5, and rare CLL cases score 3):
• CD5+
• CD23+
• FMC7-
• sIg weak (not moderate or strong)
• CD22/CD79b weak or negative
• CD5+ cases with a score < 3, with morphology of atypical CLL, should have FISH for trisomy 12 and t(11;14)

Molecular features:

• No specific genetic markers
• Similar to B-PLL cytogenetically
• rearrangement of Ig heavy and light chain genes
• Immunoglobulin VH genes (mutation status of IGHV gene):
• 30-50% - unmutated (>= 98% identity with the germline) (poor prognosis)
• no somatic mutations of variable region genes (like naïve B cells)
• BCR signaling more active
• 50-70% - mutated (< 98% identity with the germline)
• somatic mutations present (like post-germinal centre B cells)
• response resembling anergy
• Background (from WHO 2017 book):
• B lymphoblasts undergo IGH VDJ gene rearrangement and differentiate into pre-B cells (cytoplasmic mu heavy chains) and immature IgM+ B-cells into mature surface immunoglobulin (sIg)-positive (IgM+, IgD+) naïve B cells (often CD5+)
• Naïve B cells circulate in the peripheral blood and also occupy primary lymphoid follicles and follicle mantle zones (so-called recirculating B cells)
• Many cases of mantle cell lymphoma are thought to correspond to CD5+ naïve B cells
• Mostly somatically unmutated, but somatically mutated variants also exist
• Negative for: bcl-6
• Some CLL has DNA methylation epigenetic expression resembling naïve B cells (mainly unmutated IGHV)
• Upon encountering antigen that fits their sIg receptors, naïve B cells undergo transformation, proliferate, and ultimately mature into antibody-secreting plasma cells and memory B cells
• May mature directly into plasma cells that produce the early IgM antibody response to antigen
• Postulated normal cell counterpart of CLL is an antigen-experienced mature CD5+ B cell with mutated or unmutated IGHV genes
• Other antigen-exposed B cells migrate into the centre of a primary follicle, proliferate, and fill the follicular dendritic cell meshwork, forming a germinal centre
• Germinal centre centroblasts express low levels of sIg and switch off expression of BCL2; therefore, they and their projeny are susceptible to apoptosis.
• Centroblasts express CD10 as well as BCL6
• In the germinal centre, somatic hypermutation occurs in the IGV genes; these mutations can result in a non-functional gene or a gene that produces antibody with lower or higher affinity for antigen.
• Ongoing IGV gene mutation with intraclonal diversity is a hallmark of germinal centre cells
• Also in the germinal centre, some cells switch from IgM production to IgG or IgA production (higher affinity antibodies of the late primary or secondary immune response)
• BCL6 also undergoes somatic mutation in the germinal centre, but at a lower frequency than do the IG genes
• Both IGV and BCL6 mutations serve as markers of cells that have been through the germinal centre.
• Most DLBCLs have mutated IGV genes
• Burkitt lymphoma cells are BCL6+ and have mutated IGH genes, and are therefore also thought to correspond to a germinal centre blast cell
• MYC plays an important role in germinal centre formation, and is upregulated by BCL6
• Centroblasts mature into centrocytes
• Centrocytes are seen predominantly in the light zone of the germinal centre
• Express sIg that has an altered antibody-combining site compared with that of their progenitors, due to both somatic mutations and heavy-chain class switching.
• Centrocytes with mutations that result in increased affinity are rescued from apoptosis and re-express BCL2
• Follicular lymphomas are centrocytes and centrolbasts in which the germinal centre cells fail to undergo apoptosis, due to mechanisms that prevent the normal switching off of BCL2 expression
• Centrocytes switch off BCL6 expression and differentiate into either memory B cells or plasma cells
• IRF4 (MUM1) and BCL6 are reciprocally expressed, with IRF4 being positive in late centrocytes and plasma cells
• Post-germinal centre memory B cells circulate in the peripheral blood and account for at least some of the cells in the follicular marginal zones of lymph nodes, spleen, and mucoas-associated lymphoid tissue (MALT).
• Typically express pan-B-cell antigens and surface IgM (with only low levels of IgD), and lack both CD5 and CD10.
• Mutated IGV genes (but not continuing to undergo mutation)
• B cells that arise in MALT tend to return there, whereas B cells that arise in the lymph nodes home ot nodal sites and bone marrow
• probably through surface integrin expression
• marginal zone lymphomas of the MALT type, splenic type, and nodal type correspond to post-germinal centre memory B cells of marginal zone type that derive from and proliferate specifically in extranodal, splenic, and nodal tissues, respectively.
• Postulated normal cell counterpart of CLL is an antigen-experienced mature CD5+ B cell with mutated or unmutated IGHV genes
• Plasma cells produced in the germinal centre enter the peripheral blood and home to the bone marrow
• Predominantly IgG and IgA
• Lack sIg and CD20
• Express IRF4, CD79a, CD38, and CD138
• Mutated IGV genes (but not continuing to undergo mutation)
• Plasma cell myeloma corresponds to a bone marrow-homing plasma cell
• Gene mutations:
• TP53 (very poor prognosis)
• May be seen initially as a subclone, but these also show a poor survival (over time they become the predominant population)
• Associated with DLBCL transformation
• NOTCH1 (9q34.3) (poor prognosis)
• Associated with +12
• Associated with DLBCL transformation
• SF3B1 (2q33.1) (intermediate or poor prognosis / intermediate risk)
• BIRC3 (very poor prognosis)
• May be associated with chemorefractory CLL (fludarabine)
• Mutations and deletions rarely detected at diagnosis, but detected at 24% of fludarabine-refractory CLL patients
• Mutually exclusive to TP53 abnormalities
• MYD88 (no apparent prognostic effect)
• associated with 13q-
• ATM (poor prognosis)
• POT1
• Cytogenetics:
• 80% have abnormal karyotypes (after appropriate stimulation by CD40-ligand or CpG oligonucleotide and Interleukin-2) (40-50% by conventional methods):
• >80% have abnormal FISH result
• MLPA is also commonly used
• Normal karyotype and FISH (intermediate risk)
• 13q14 deletion (up to 50%) (good prognosis as a sole abnormality)
• predominantly mutated immunoglobulin variable region genes
• longer survival
• Type I (low risk but shorter overall survival than type I):
• Larger deletions often including RB1 gene
• Type II (very good prognosis as a sole abnormality):
• < 2 Mb in size encompassing the minimally deleted region (MDR) (DLEU7, miR-15a, miR-16-1)
• Note: percentage of cells with 13q deletion also affects prognosis (like 65-90%)
• Biallelic in 30% of those with 13q-
• Characteristically small and not involving RB1
• may be cryptic on karyotype
• trisomy 12 (15-20%) (intermediate to poor prognosis)
• prognosis affected by other factors (CD38, ZAP70, IGHV mutation status, NOTCH1 and other mutations)
• predominantly unmutated immunoglobulin variable region genes
• correlates with atypical morphology
• 10-20% have partial trisomy 12
• 12q13 is minimal common gained region
• Associated recurrent aberrations:
• t(14;19)(q32;q13) (IGH-BCL3)
• t(14;18)(q32;q21) (IGH-BCL2)
• del(14)(q24q32) involving IGH locus
• other recurrent CLL deletions (13q, 11q, 17p)
• +19
• +18
• Associated with mantle cell-like morphology maybe
• 11q22-23 deletion (5-20%) (poor prognosis)
• ATM deleted in all cases (11q22.3)
• BIRC3 deleted in 60% of cases
• somatic mutations in other allele in these cases
• increased copy number alterations (genomic instability)
• extensive lymphadenopathy
• poor survival
• may be cryptic on karyotype
• 17p13 deletion (p53 locus) (5-10%) (very poor prognosis)
• Patients with de novo 17p- have a longer median OS (4-5 years) than those who acquired 17p- during clonal evolution (1-1.5 years)
• Percentage of abnormal nuclei by FISH also stratifies the risk
• Frequently associated with TP53 mutation in the remaining allele
• Unmutated IGHV association
• Higher genomic complexity
• Atypical immunophenotype:
• CD20 higher intensity
• FMC7 higher intensity
• CD79b higher intensity
• Surface Ig higher intensity
• CD38 increased
• ZAP-70 increased
• Underexpression of TP53, CCND3, BCL2, SYK, ATM, TCL, PI3K, CCND1, and AID
• Overexpression of P2,MYC, and AICL
• Poor response to fludarabine-cyclophosphamide (FC) and fludarabine-cyclophosphamide-rituximab (FCR) regimens
• Poor response to alkylating agents and purine analogs
• may be cryptic on karyotype
• CDKN2A deletion
• Associated with DLBCL transformation
• Complex Karyotype (CK) / Genomic Complexity (3 or more > 5 Mb CNAs by microarray, 3 or more chromosomal abnormalities by karyotype) (~35%) (independent risk factor for disease progression)
• Often associated with other known high-risk features
• 11q- and 17p- in particular
• Chromothripsis (2%) (poor prognosis in one study)
• Alternating regions of gains, losses, and normal copy number status involving a chromosome
• at least 10 switches in copy number states in one chromosome
• Reflection of genomic instability
• Mainly unmutated IGHV
• Frequent association with TP53 mutation
• 6q21 deletion (5%)
• High heterogeneity in size and location, cannot define a minimally deleted region
• 2p gain (7%) (associated with high-risk genomic changes, likely poor prognosis, increased risk of transformation)
• MYCN, REL, BCL11A
• 8p loss (?relative to 8q)
• 8q gain (?MYC) (5%) (shorter PFS, likely poor prognosis)
• Association with 17p-
• 18 gain (1%)
• 19 gain (2%)
• 15q15 loss (MGA gene)
• 10q24 loss (NFKB2 gene)
• 4p deletion
• Association with 17p-
• 18p deletion
• Association with 17p-
• 20p deletion
• Association with 17p-
• 22q11 deletion (submicroscopic) (15%)
• 20q13.12 gain (submicroscopic) (19%)
• CNLOH
• May be associated with a homozygous mutation of TP53 for example
• Balanced translocations:
• IGH (4-9%)
• t(14;19) (BCL3)
• associated with trisomy 12 and complex cytogenetics, unmutated IGHV, atypical CLL morphology, poor prognosis
• t(14;18) (BCL2) (2%)
• as a secondary change
• usually witih mutated IGHV
• t(8;14) (MYC) (<1%) (poor prognosis; associated with transformation to DLBCL)
• Unbalanced translocations
• Quite heterogeneous
• Many result in microdeletions of TSGs (most common is TP53)