Diagnosis of lymphoproliferative disorders

Question 1

Lymphoma

Answer 1

Neoplastic disease of lymphocytes

Burden of disease primarily in the lymph nodes - can infiltrate other organs

Categorised into large cell lymphomas - more aggressive clinical behaviour, or small cell lymphoma - more slowly progressive

Question 2

Leukaemia

Answer 2

Neoplastic proliferation of leukocytes/haematopoietic precursors

Burden of disease is predominantly within the bone marrow

May be of lymphoid or myeloid origin

Usually associted with increased numbers of circulating neoplastic cells, but may be normal

Stage V lymphoma can present similarly

Question 3

Diagnostic approach to lymphoma

Answer 3

History

Clinical exam

Clin path - haem and biochem

FNA of enlarged LNs - cytology

Adjunctive tests
- LN biopsy and histopath
- PCR for antigen receptor rearrangements (PARR)

Flow cytometry - to immunophenotype

Question 4

Usual lymphoma history

Answer 4

Vague
Lethargy
Anorexia
Weight loss

Can have
PUPD (hypercalcaemia)
V+D+
Dyspnoea
Pyrexia

Question 5

Clinical signs of lymphoma

Answer 5

Lymphadenopathy

Enlargements of other organs - liver, spleen, kidney

Question 6

Clin path abnormalities in lymphoma

Answer 6

Mild non-regenerative anaemia (anaemia of inflammatory/neoplastic disease)

If stage V lymphoma may also have other cytopaenias

Hypercalcaemia - due to PTHrP secretion by some neoplastic cells

Others depend on organs involved

Serum globulins may be high with B cell lymphoma

Question 7

Differentials for lymphadenopathy

Answer 7

Reactive lymphoid hyperplasia - inflammation in the area drained by the node

Lymphadenitis - infiltration of the node by inflammatory cells, reflects inflammation in the area drained by the node

Lymphoma - infiltration of the LN by neoplastic lymphocytes, efface the normal lymphoid architecture

Metastatic neoplasia - infiltration by neoplastic cells from a distant tumours, usually via lymphatic system

Benign lymphoid hyperplasia of young cats

Question 8

Normal LN cytology

Answer 8

Mostly (>90%) small lymphocytes with low numbers of intermediate and large sized lymphocytes. Occasional histiocytes (macrophages).

Compare sizes to neurtophils, ignore any with no cytoplasmic border

Question 9

Cytology of lymphoid hyperplasia

Answer 9

Clinically enlarged and palpable

Predominant population still small lymphocytes, but 10-50% intermediate and large cell lymphocytes - mixed/pleomorphic population of lymphocytes

Increased numbers of plasma cells, some which may have become Mott cells (plasma cells which contain large numbers of Russel bodies with immunoglobulins)

Can be mildly increased neutrophils, eosinophils, and macrophages

Indicates the presene of an immune stimulus or inflammation in the area drained by this lymph node

Question 10

Cytology of lymphadenitis

Answer 10

3 categories
- Neutrophilic: >5% neutrophils
- Eosinophilic: >3% eosinophils
- Histiocytic: increased macrophages (fungal or protozoal infections)

Question 11

Cytology of lymphoma

Answer 11

Lymphadenopathy

Predominance (>50%) of intermediate to large cell lymphocytes

Increased mitotic figures if high grade

Will be tangible body macrophages present

Further tests for confirmation - clonality testing such as PARR, biopsy, immunophenotyping

Small cell (indolent) more difficult to diagnose - suspect due to enlarged lymph node with no evidence of reactive hyperplasia, sometimes subtle morphological changes such as ‘hand mirror’ appearance

Question 12

Benign lmyphoid hyperplasia of young cats

Answer 12

This is a condition reported in young cats, in which there is marked peripheral lymphadenopathy.

Lymph nodes appear consistent with lymphoma, however this condition regresses spontaneously without treatment.

Therefore, care should be excised when diagnosing lymphoma in a young cat.

In the previous reports, the majority of cats were FeLV positive (do an FeLV test to check before diagnosing lymphoma).

Question 13

Cytology of metastatic disease within the lymph node

Answer 13

Indicated by the presence of cells that would not normally be expected e.g. epithelial cells or mesenchymal cells - WITH criteria of malignancy

Rule out accidental aspiration of other structures

if mast cells diagnosis can only be made if you see clusters of 3-5 mast cells and/or they show criteria of malignancy

Question 14

Lymph node biopsy

Answer 14

Ideally take the whole node rather than incisional biopsy

Question 15

PARR

Answer 15

PCR for antigen receptor rearrangements

Performed on cytology slides

Clonal population of lymphocytes is highly suggestive of lymphoproliferative neoplasia

Occasional false positives in dogs with Ehrlichia (uncommon)

Occasional false negatives if low cell number or mutations where the primers would anneal

Unrealiable for immunophenotyping due to cross linneage rearrangments

Question 16

Flow cytometry for lymphoma

Answer 16

Used after a diagnosis of lymphoma has been made on cytology, histo, or PARR - cannot be used to make a diagnosis

Performed on unstained slides to identify a panel of markers (needs to be fresh)
- serum from EDTA tube
- take LN aspirate into the serum
- also submit aspirate cytology smears

Identify then as T or B cell origin, and identify additional markers that can be helpful for sub-classifying

Question 17

Prognoses of various lymphomas

Answer 17

Large B cell lymphomas have a longer survival time than large T cell lymphomas

CD8+ T cell lymphomas have superior survival times compared to CD4+ T cell lymphomas

Lymphomas in which MHC class II expression is high will live longer than those with low MHC II expression (for both large B and large T cell lymphomas).

Question 18

Immunocytochemistry

Answer 18

Alternative way to immunophenotype lymphoma

Can be performed on cytology slides that have been pre-stained

Considerable subjectivity in interpretation and only a limited panel of markes

Question 19

Diagnostic protocol for lymphoma

Answer 19

Suspect lymphoma

FNA

-> Diagnosed on FNA

-> Flow cytometry to immunophenotype

OR

-> suspicion of lymphoma on FNA

-> PARR
OR
-> LN biopsy

-> Flow cytometry

Question 20

Diagnostic approach to leukaemia

Answer 20

History

Clinical exam

Diagnostic techniques
- Haematology
- Acute phase proteins
- Infectious (FeLV, FIV)

Flow cytometry
OR
PARR

Question 21

Classic history of animals with leukaemia

Answer 21

Vague clinical signs

Anorexia

Lethargy

Weight loss

May have none

Question 22

Signs of leukaemia on bloods

Answer 22

Marked lymphocytosis or high numbers of circulating atypical cells (could be seen in stage V lymphoma)

Mild to moderate lymphocytosis (<40x10^9 cells/L) may or may not have it, need further testing

Question 23

Differentials for mild lymphocytosis (up to 2x upper end)

Answer 23

Excitement

Normal in young animals

Long standing/chronic inflammation

Some inflammatory disorders e.g Ehrlichia, mycoplasma

Leukaemia

Question 24

Flow cytometry in leukaemia

Answer 24

Used to identify the immunophenotype

if there is a mixed immunophenotype of cells detected, then PARR can be used to confirm if the lymphocytes present are clonal - cannot be used to confirm presence of clonal population of myeloid cells

Question 25

Leukaemia subtypes

Answer 25

Acute -> lymphoblastic - T cell - B cell -> myeloid Chronic -> lymphocytic - T cell - B cell -> myeloid Distinction made by flow cytometry -PARR no reliable

Question 26

Acute leukaemia

Answer 26

Neoplastic proliferation of immature leukocytes - large in size Confirmed by finding CD34 positive leukocytes in circulation Tend to be younger animals Often see other cytopaenias

Question 27

Chronic lymphocytic leukaemia

Answer 27

neoplastic proliferation of mature lymphocytes - cells seen are often indistinguishable from normal lymphocytes (small to intermediate in size, i.e. smaller than neutrophils). The diagnosis of CLL is based on the finding of a lymphocytosis without lymphadenopathy (although T cell CLL is often associated with splenomegaly), and the lymphocytes do not express CD34 on flow cytometry. On bone marrow aspirate cytology, a significant infiltrate (>20-30%) of small lymphocytes is seen.

Question 28

Chronic myeloid leukaemia

Answer 28

Chronic myeloid leukaemias are very rare, but when present they are usually a neoplastic clonal proliferation of mature neutrophils, therefore these animals will have a marked neutrophilia. Diagnosis of chronic myeloid leukaemia is a diagnosis of exclusion of other causes of marked neutrophilia (leukaemoid response), primarily inflammatory or paraneoplastic disorders.

Question 29

DIagnostic protocol for leukaemia

Answer 29

Increased lymphocyte/atypical cell count (>10x10^9/L) Consider diagnosis of stage V lymphoma if lymphadenopathy found RUle out other causes of lymphocytosis Flow cytometry of blood <40x10^9 cells/L -> if mature (small) and/or mixed phenotype then consider PARR to confirm neoplasia -> if not then leukaemia and immunophenotype confirmed >40x10^9cells/L -> leukaemia and immunophenotype confirmed