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2. Tumours of Mature Peripheral B-cells

•  2.1 Follicular Lymphoma
•  2.2 Diffuse Large B-cell Lymphoma
•  2.3 Burkitt Lymphoma
•  2.4 B-cell Chronic Lymphocytic Leukaemia
•  2.5 Mantle Cell Lymphoma
•  2.6 CD5+/CD23- LPD NOS
•  2.7 Systemic Marginal Zone Lymphoma
•  2.8 Extranodal Marginal Zone Lymphoma
•  2.9 Hairy Cell Leukaemia
• 2.10 CD5 negative B-cell LPD NOS

2.1 Follicular Lymphoma

Follicular lymphoma is a tumour of the germinal centre cell that in lymph nodes shows a follicular growth pattern.

Diagnostic Criteria

Typical Cases

In lymph node biopsy specimens the following features must be present:

1. The tumour must contain a mixture of cells with the morphology of centrocytes and centroblasts
2. The tumour must have a germinal centre phenotype:
   • Immunocytochemistry: CD20⁺,CD79⁺,CD10⁺,bcl-6⁺,CD23^variable, IRF-4 (MUM-1)^variable
   • Flow Cytometry: clonal sIgM or sIgG, CD19⁺,CD10^+/-,CD38⁺,CD5^-, and bcl-6 and CD10 demonstrated by immunocytochemistry
3. bcl-2 expression by the neoplastic cells and/or t(14;18) by FISH or PCR
4. Partially or wholly follicular growth pattern

Variants

1. As above but bcl-2 and t(14;18) negative; clonality should be demonstrated by PCR, flow cytometry or unequivocal evidence of bone marrow infiltration should be present. 3q27 rearrangement will be assessed by interphase FISH in these cases.
2. Diffuse growth pattern: If typical phenotype and cytology use diffuse follicle centre lymphoma. Some of these cases have the phenotype CD10^-, IRF4⁺, BCL6⁺. The significance of this is currently unknown.
3. Aberrant phenotype. A proportion of cases have a follicular growth pattern, BCL2 expression and phenotype CD10^-, IRF-4⁺, BCL6^variable. These appear to lack the t(14;18). These cases are currently diagnosed as follicular lymphoma; common type with a comment about the aberrant phenotype, however little is currently known about these cases.
4. FL with marginal zone differentiation. Demonstration of a follicular component with above phenotype is essential for distinction from marginal zone lymphoma.
5. Presentation in skin and other extranodal sites. Follicular lymphoma is only diagnosed if all above criteria are met. Nature of 'primary cutaneous follicular lymphoma' remains undefined.

Prognostic Factors

1. Estimating the proportion of follicular and diffuse areas is of no clinical value and will not be used
2. Grading by WHO criteria is poorly reproducible and is of minimal significance except for the large cell variant. A comment should be made in the body of the report on the overall cellular composition. Cases in which the follicles consist of a monomorphic population of large lymphoid cells will be reported as follicular lymphoma, large cell type. In some cases it may be difficult to distinguish this from DLBCL with a nodular growth pattern. In these cases the distinction will be based on the presence of follicular dendritic cell networks
3. The significance of a range of cytogenetic and molecular prognostic factors is under evaluation
4. The prognostic significance of bcl-2 / t(14;18) negative cases is not yet known

Transformation

Transformation is defined by diffuse areas of tumour that lack a FDC network and consist mainly of large lymphoid cells with a high rate of cell proliferation (>30% Ki67) in patients with a history of follicular lymphoma. These cases should be diagnosed as DLBCL with underlying follicular lymphoma. This term should be used in all cases with a known previous follicular lymphoma regardless of FDCs and also in cases of DLBCL in a tissue biopsy and follicular lymphoma in the bone marrow. Make a comment if phenotype is different to underlying follicular lymphoma.

Bone Marrow Infiltration

As part of staging: Presence of paratrabecular/nodular/diffuse infiltrates with typical morphology confirmed by the demonstration of a clonal CD5^- B-cell population by flow cytometry. In some cases a diagnosis can be made without flow cytometry if the morphology is typical and a paratrabecular pattern of infiltration is evident. All other cases must be confirmed by immunocytochemistry as a B-cell infiltrate. If no evidence of an infiltration use term 'no evidence of lymphoma'.

If these features are present in a patient known to have DLBCL this is considered as indicative of a transformed follicular lymphoma. The presence of DLBCL in the lymph node should be commented on in the body of the report.

At presentation: In the absence of other tissue biopsies the diagnosis requires confirmation by flow cytometry and the presence of t(14;18) by FISH or PCR. If these criteria are not met the diagnosis should be considered to be provisional and a lymph node biopsy should be advised.

Follow up: Bone marrow is reported as positive if infiltration is found by morphology, flow cytometry or PCR, irrespective of the extent of disease. If no evidence of bone marrow involvement after treatment use 'remission marrow' if previously infiltrated. These terms apply to all staging/post-treatment marrows.

Differences from WHO classification

1. Grading and subdivision by follicular/diffuse component not used because of lack of reproducibility and clinical significance
2. More stringent phenotypic/molecular criteria

Terms to be used

2.2 Diffuse Large B-cell Lymphoma

This is a tumour that shows diffuse replacement of normal nodal architecture by a population of large B-lymphoid cells.

Diagnostic Criteria

Typical Cases

1. Diffuse infiltrate of large lymphoid cells
2. Proliferation fraction of at least 30%
3. Immunophenotype may be:

   a. Germinal centre type:

   CD10⁺, BCL6^variable, MUM-1^variable; OR CD10^-, BCL6⁺, MUM-1^-

   b. Post germinal centre / activated phenotype:

   CD10^-,BCL6^-, MUM-1^variable; OR CD10^-,BCL6⁺, MUM-1⁺

   c. CD5⁺, bcl-1 / t(11;14)^-ve (rare)

Variants

1. Anaplastic morphology should be regarded as DLBCL
2. T-cell rich variant. Greater than 50% T-cells and a B-cell population with morphology and phenotype as above. This should be commented on in the body of the report. No definitive evidence of prognostic significance.
3. Mediastinal large B-cell lymphoma. This is a distinct entity. Diagnosis requires mediastinal mass, clear cell morphology, pericellular fibrosis, CD10^-, MUM-1⁺, IgM and IgG negativity, CD21^-, CD30^{+ or -}. Cases not meeting these criteria are DLBCL
4. Extranodal. Cases should be classified as above unless clear evidence of underlying marginal zone lymphoma
5. Intravascular. Cellular features as above but tumour cells mainly within vessels adherent to the endothelium
6. Plasmablastic type. This diagnosis should only be made for soft tissue tumours with no evidence of myeloma. Diagnosis requires plasmablastic morphology, CD138⁺, cCD79^+/-, CD20^-, cIg^+/-, Pax5^-, MUM1⁺ and high proliferation. Alk positivity will be assessed, and noted in the final diagnosis. In cases of plasmablastic-type DLBCL an HIV test should be suggested.

Prognostic Factors

1. DLBCL with adverse cellular features. Any of the following:
   • bcl-2 expression and/or deregulated P53 (P53⁺P21^-) in non-GC phenotype
   • t(14;18)
   • 3q27 rearrangements

   Cases with t(8;14) and t(14;18) or complex cytogenetics have a very poor outcome. These cases will be diagnosed as DLBCL with adverse prognostic features, and if there is evidence of underlying follicular lymphoma, this should be commented on in the body of the report. If there is a previously diagnosed follicular lymphoma or follicular lymphoma in bone marrow change to DLBCL with underlying follicular lymphoma.

2. DLBCL with favourable cellular features
   • Germinal centre phenotype with none of the above

NB. These final diagnoses should only be made if all tests have been completed.

Differences from WHO classification

1. Subdivision by phenotype and prognostic features

Terms to be used

1. Used as primary diagnosis pending further tests (unless a Burkitt lymphoma phenotype is demonstrated), and as a final diagnosis if none of the other criteria are met.
2. To be used when a Burkitt phenotype is demonstrated and FISH is pending.
3. "The prognostic effect of the cellular features should be interpreted in the context of the IPI and other biological prognostic factors" will be appended onto the bottom of the report automatically by HILIS. These terms should only be used when results of FISH studies are complete

2.3 Burkitt Lymphoma

Burkitt lymphoma is defined as a germinal centre cell lymphoma with c-myc deregulation and absence of BCL2 deregulation.

Diagnostic Criteria

Typical Cases

1. Large B-cell lymphoma with round nuclei, central nucleoli and vacuolated cytoplasm
2. Germinal centre phenotype: CD10⁺,bcl-6⁺, (variable MUM-1) by immunocytochemistry with bcl-2^-
3. A hyperproliferative state demonstrated by Ki67 approaching 100%, p53⁺, p21^- and evidence of apoptosis
4. t(8;14) or variants demonstrated by FISH or cytogenetics must be present. If not demonstrated a diagnosis of DLBCL should be made.

Variants

1. Leukaemic type. Primary marrow and blood involvement with cellular features as above
2. HIV associated

Prognostic factors

1. Cases with t(8;14) and t(14;18) or complex cytogenetics have a very poor outcome regardless of underlying follicular lymphoma. These cases will be diagnosed as DLBCL with adverse prognostic features, unless there is previous clinical evidence of a follicular lymphoma or follicular lymphoma in the bone marrow - in this case DLBCL with underlying follicular lymphoma will be used.

Differences from WHO

1. Diagnosis based on phenotype/cytogenetics

Terms to be used

1. To be used when FISH is pending

2.4 B-cell Chronic Lymphocytic Leukaemia

This is a chronic leukaemia of CD5⁺ B-cells. The term includes cases presenting as lymphadenopathy, formerly known as small lymphocytic lymphoma.

Diagnostic Criteria

Typical Cases

1. Consists mainly of small lymphocytes with clumped heterochromatin, although considerable morphological variation can occur
2. Lymph node biopsies show a diffuse infiltrate usually with pseudofollicle formation
3. Immunophenotype: CD5⁺,CD19⁺,CD20^wk,CD79b^wk, FMC7^-,CD38^-,CD23⁺,weak sIg (sIgM⁺/sIgD⁺ or sIgM^-/sIgD⁺)
4. Cases with low level of peripheral blood involvement, i.e. Cells with a CLL-phenotype present below 5x10⁹/l with otherwise normal haematological parameters are reported as Monoclonal B-lymphocytosis (B-CLL phenotype).

Variants

1. Class switching with IgG, this should be commented on but the significance is not certain
2. CD23^-,bcl-1 / t(11;14) negative. In house data suggests this is an adverse prognostic group with outcome similar to mantle cell lymphoma
3. Solitary lymph node disease is very rare
4. Expression of kappa and lambda light chain (apparently biclonal) with otherwise typical phenotype. This should be reported but is of uncertain signficance

Prognostic Factors

1. B-CLL with favourable cellular features
   • Mono-alleleic deletion of 13q14 as sole abnormality with a typical phenotype
2. B-CLL with adverse cellular features
   • 11q (ATM) or 17p (P53) deletions by interphase FISH
   • Germline IgH genes

Transformation

This diagnosis can only be made on a tissue biopsy and requires cohesive sheets of large lymphoid cells sufficient for the diagnosis of DLBCL in a patient with a documented history of CLL. The term Richter's transformation will not be used.

Differences from WHO

1. Incorporation of prognostic data
2. No morphological subtyping

Assessment of involvement

These terms should appear in the body of the report. Comments 1 - 5 should be made only when aspirate and trephine specimens are available at the same time and processed by HMDS. The aspirate must be assessed as adequate by flow cytometry.

1. Extensive infiltration (>50% neoplastic cells on flow and/or trephine)
2. Moderate infiltration (10 - 50% neoplastic cells on flow and/or trephine)
3. Low level infiltration (<10% neoplastic cells on flow and/or trephine)
4. Minimal residual disease (Flow/PCR⁺, normal morphology)
5. No evidence of disease (Flow and/or PCR^-, normal morphology)
6. Extent of disease not assessable (Flow <10% with no trephine or referred aspirate slide)
7. Morphological remission, but the presence of residual disease cannot be excluded (to be used when there is no flow or the flow is deemed to be inadequate)

Assessment of response

Response to treatment is defined as a change of 2 or more categories as defined above. Reduced level of involvement should appear in the body of the report.

Terms to be used

To be used as primary diagnosis, before FISH results are available and if neither favourable or adverse cellular factors are demonstrated:

To be used only when FISH studies are complete:

1. Additional terms are available for assessment of involvement / treatment response - see section above. These terms should appear in the body of the report.
2. "The absolute CLL count is below the level required for a diagnosis of CLL by NCI criteria in the absence of other features" will be appended onto the bottom of the report automatically by HILIS
3. "The prognostic effect of the cellular features should be interpreted in the context of other biological and clinical prognostic factors" will be appended onto the bottom of the report automatically by HILIS

2.5 Mantle Cell Lymphoma

Diagnostic Criteria

Typical Cases

1. Monomorphic population of small to intermediate sized B-cells
2. Phenotype: sIg^++/+++ (IgM & IgD), CD5⁺, CD23^-, CD20⁺
3. bcl-1 expression / t(11;14)

Variants

1. Blastic or large cell variant associated with aggressive clinical course

Terms to be used

2.6 CD5+/CD23- LPD NOS

This term should be used in the absence of FISH / cytogenetics results, where the B-lymphoproliferative disorder has the phenotype CD5⁺, CD23^-. If a t(11;14) or BCL-1 staining by immunocytochemistry is demonstrated, the final diagnosis should be changed to Mantle cell lymphoma. If negative for the t(11;14) and /or BCL-1, and CD20 is weak, sIg (CD79b) is moderate/weak by flow cytometry and/or CLL-associated FISH abnormalities are demonstrated, a diagnosis of B-CLL can be made, with a comment about the lack of CD23 in the comments. If neither of these criteria are met, the diagnosis should be left as CD5⁺/CD23 ^- LPD NOS.

2.7 Systemic Marginal Zone Lymphoma

This includes WHO categories of lymphoplasmacytic lymphoma/Waldenstroms macroglobulinaemia, splenic marginal zone lymphoma, nodal marginal zone lymphoma. There is currently no objective basis for separation of these entities other than site of origin.

Diagnostic Features

Typical Cases

1. Cellular composition includes small lymphocytes, centrocyte-like cells, 'monocytoid' B-cells, plasmacytoid cells
2. Pattern of infiltration (a trephine biopsy or other tissue biopsy is always required for this diagnosis):
   • Bone Marrow: nodular, interstitial, diffuse
   • Spleen: marginal zone infiltration
   • Nodes: interfollicular expansion with replacement of germinal centres
3. Immunophenotype: CD5^-,CD10^-,CD19⁺,CD20⁺,CD23^-,sIgM ⁺,bcl-6^-,sIgD^{+ or -}. MUM-1 in plasma cells.
4. Absence of a t(14;18)

Transformation

The criteria as for follicular lymphoma. The significance of increased large lymphoid cells is uncertain in otherwise typical cases.

Differences from WHO

Terminology: see above.

Terms to be used

When tumour is in a peripheral node comment should be made on the possibility of occult extranodal disease

2.8 Extranodal Marginal Zone Lymphoma

Diagnostic Criteria

Typical Cases

1. Cellular composition includes small lymphocytes, centrocyte-like cells, 'monocytoid' B-cells, plasmacytoid cells
2. Invasion of epithelial structures and existing germinal centres
3. CD5^-, CD10^-,CD19⁺,CD20⁺,CD23^-,sIgM⁺,sIgD ^{+ or -}
4. Extranodal site

Prognostic Factors

Extranodal marginal zone lymphoma with t(11;18). In the stomach this may be resistant to helicobacter eradication.

Assessment of residual disease: The definitive diagnosis of residual disease requires the same criteria as at presentation. Solitary or multiple B-cell aggregates without germinal centres and cellular morphology suggestive of marginal zone lymphoma should be reported as suspicious and further follow up advised.

Transformation

The criteria as for follicular lymphoma. The significance of increased large lymphoid cells is uncertain in otherwise typical cases.

Terms to be used

FISH (for rearrangement of MALT) will be requested automatically by HILIS when this diagnosis is entered in a presentation sample.

To be used once FISH has been reported. If MALT-1 is not rearranged - leave as Extranodal marginal zone lymphoma

2.9 Hairy Cell Leukaemia

Diagnostic Criteria

Typical Cases

1. Typical morphology in blood or marrow
2. Interstitial infiltrate with vascular congestion and myeloid suppression in marrow
3. Flow Phenotype: CD19⁺,CD22⁺⁺⁺,CD25⁺,CD11c⁺,CD103⁺, CD5^-,CD23^-. Multiple Ig heavy chains may be found.
4. Immunocytochemistry: CD20⁺,DBA44⁺

If a trephine only is received, confirmation of diagnosis requires PB or bone marrow for flow cytometry.

Variant

1. Otherwise typical cases that lack CD25

Terms to be used

2.10 CD5 negative B-Cell LPD NOS

This diagnostic category should be used in PB samples with a CD5- lymphoproliferative disorder or in bone marrow, where a diagnosis of follicular lymphoma or marginal zone lymphoma cannot be made on the basis of the pattern of infiltration in a trephine sample, and if there is no tissue biopsy.

If FISH/PCR demonstrate a t(14;18), the final diagnosis can be changed to follicular lymphoma. If FISH for the t(14;18) is negative, the sample was regarded as adequate, and CD10 and CD23 are negative by flow, the final diagnosis can be changed to marginal zone lymphoma, where the presenting sample is PB or bone marrow with no evidence of nodal disease. If FISH failed or was regarded as inadequate, the diagnosis should be left as CD5^- B-cell LPD NOS.

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