The patient had a mild splenomegaly but no obvious lymph node enlargement. The consensus phenotype obtained from part one of the exercise was:
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1 Case History An 86 year old male was admitted to hospital with chest infection. Haematological examination subsequently revealed the following: Hb g/dl; WBC x 10^9/l; PLT- 99 x10^9/l; RBC x10^12/l; HCT ; MCV fl; RDW- 17.4%. The patient had a mild splenomegaly but no obvious lymph node enlargement. Immunophenotype The consensus phenotype obtained from part one of the exercise was: Negative Antigens (value less than 30% as defined by BCSH guidelines): CD2, surface CD3, CD7, CD10, CD11c, CD13, CD25, CD79b, CD103, FMC-7 Positive Antigens (value of 30% or greater as defined by BCSH guidelines): CD5, CD19, CD20, CD22, CD23, CD38, CD43, CD45, HLA-DR Molecular and The patient had a clonal IgH gene re-arrangement and deletion of 11q22-23 on chromosome analysis. Peripheral Blood Morphology Morphology comments from Dr Wendy Erber This blood film shows EDTA-related changes. There is a peripheral blood lymphocytosis of small size and some of the small-intermediate sized. The lymphocytes have mature coarsely clumped chromatin and basophilic cytoplasm around half to one-third of the nucleus. The larger cells have more irregular nuclear shape, open chromatin and some have a nucleolus. There are large numbers of smear cells. Neutrophils appear normal. The red cells are normochromic and normocytic and the platelet count appears normal. Comment: Blood film features of chronic lymphocytic leukaemia. The blood film shows EDTA related artefactual changes. Although prolymphocytes are present, there are insufficient to suggest disease transformation or progression.
2 Examples of Digital Images Used Figure 1: Peripheral Blood x50 magnification (Romanowsky) Figure 2: Peripheral Blood x100 magnification (Romanowsky)
3 Exercise Conclusion/Case Discussion Initial examination of the blood film and the immunophenotype namely the expression of CD5 and CD19 would immediately suggest chronic lymphocytic leukaemia or possibly Mantle Cell Lymphoma. This is reflected in the conclusions provided by 94.1% of participants with Mantle cell lymphoma suggested by 3.6%. B-cell prolymphocytic leukaemia and Diffuse large B-cell lymphoma (DLBCL), NOS were submitted by 1.8% and 0.5% of participants respectively. Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is the most common leukaemia in the western world. The incidence rate is 2-6 cases per persons per year. In biopsies, CLL/SLL accounts for between 6-7% of NHL cases 1 and has a male to female ratio of 1.5-2:1. Although the majority patients are asymptomatic, some patients present with symptoms related to splenomegaly, hepatomegaly, auto immune haemolytic anaemia (AIHA), lymphadenopathy or extra nodal infiltrates. Morphology of Chronic lymphocytic leukemia/small lymphocytic lymphoma In both the BM and PB the small lymphocytes are present which have clumped chromatin and scanty cytoplasm. The proportion of prolymphocytes in CLL/SLL is usually <10%. An increased proportion is consistent with a more aggressive disease with >55% prolymphocytes favouring a diagnosis of prolymphocytic leukaemia 2. The immunophenotype of CLL/SLL is the expression of CD20, CD22, CD5, CD19, CD79a, CD23, CD43 and CD11c (weak). CD10 is not expressed with FMC 7 and CD79b being weakly or not expressed. The Matutes scoring system for the diagnosis of CLL/SLL is based on the common marker profile of the strong expression of CD5 and CD23, negativity of FMC7, the weak to moderate expression of SmIg and the negative or weak expression of CD79b. A score of 1 is assigned if the antigen expression is typical for CLL/SLL whether it be positive or negative. A score of 4 or 5/5 is strongly supportive of a diagnosis of CLL. If the score falls below this the diagnosis is less certain 3,4. The Matutes score in this case was CD5 (97%) - +1 CD23 (74%) - +1 CD79b (7%) - +1 FMC 7 (0%) - +1
4 As can be seen this case has a score of 4 therefore suggesting CLL/SLL. Although SmIg was not reported, retrospective analysis showed clonality, therefore increasing the score to 5. FISH detects the presence of genetic abnormalities in 80% of cases. The deletion of 13q14.3 is seen in 50% of cases with 20% showing trisomy 12. Less common deletions are the deletion of 11q22-23,17p13 and 6q21. The patient in this case had a clonal IgH gene re-arrangement and deletion of 11q22-23 on chromosome analysis. Mantle Cell lymphoma (MCL) comprises 3-10% of NHL. It occurs in middle to old age and has a marked male predominance of approximately 2:1. Sites of involvement include lymph nodes, the spleen and bone marrow. There may or may not be peripheral blood involvement. Most patients present with stage 3 or 4 disease which include lymphadenopathy, hepatosplenomegally. At this stage there is bone marrow involvement and peripheral blood involvement is common. A marked lymphocytosis is seen in some patients mimicking prolymphocytic leukaemia. Morphology of Mantle Cell lymphoma (MCL) The lymphoma cells are small to medium in size. They are variable in shape and nuclear:cytoplasmic ratio. Some have a clefted or irregular nuclei. The chromatin condensation is less than in CLL and some cells appear blastic. The immunophenotype of MCL is positivity for CD5, FMC 7 and CD43 and negativity of CD10. CD23 is weakly or not expressed. The mutation which is considered to be the primary genetic event in MCL is the t(11;14)(q13;q32) between IGH and the cyclin D1 gene and is seen in the majority of cases. As mentioned above the patient had a clonal IgH gene re-arrangement and deletion of 11q22-23 on chromosome analysis. This mutation is one of the less common genetic alterations seen in CLL/SLL and although reported in MCL it is not considered to be a primary genetic event. Therefore, this has been clarified as a minor error because t(11;14) was not reported as the primary cytogenetic finding which is present in the majority of cases of MCL. B prolymphocytic leukaemia (B-PLL) is a B cell neoplasm which affects the spleen, bone marrow and peripheral blood and is a rare disease which accounts
5 for about 1% of lymphocytic leukaemias. The majority of patients are over 60 with a similar occurrence rate between men and women. Most patients present with splenomegally with little or no lymphadenopathy. A rapidly rising lymphocyte count with counts >100x10 9 /l is commonly seen. 65% of patients will present with anaemia with 35% presenting with thrombocytopenia 5. Morphology of B prolymphocytic leukaemia (B-PLL) By definition, the number of circulating prolymphocytes dictates classification of B-PLL. If there are less than 10% then the leukaemia is classified as CLL. If CLL is in transformation then between 11% and 55% of the circulating lymphoid cells are prolymphocytes. A level greater then 55% is classed as B-PLL. These cells are twice the size of a lymphocyte. They have a round nucleus, moderately condensed chromatin and a predominant central nucleolus. There is a small amount of cytoplasm which has a faintly basophilic appearance 2. The immunophenotype in B-PLL is the expression of CD19, CD20, CD22, CD79a and b and FMC 7. CD5 and CD23 are positive in approximately 25% and 15% of cases respectively. The t(11;14)(q13;q32) translocation which is seen in 20% of B-PLL cases is now considered to be a leukaemic variant of MCL 6,7. In this case, although prolymphocytes are present, they are not in sufficient number to suggest B-PLL. Diffuse large B-cell lymphoma (DLBCL), NOS constitutes 25-30% of adult NHLs in the west. The median age is 70 but can occur in children and young adults, although this is uncommon. The incidence is slightly higher in males than females. This lymphoma is usually a primary cancer but can also occur as a secondary of a less aggressive lymphoma, for example CLL/SLL or Follicular lymphoma. A significant risk factor for DLBCL, NOS can be the presence of an underlying immunodeficiency. The majority of patients with DLBCL, NOS are asymptomatic but those who do present with symptoms usually have stage I or II disease which is usually a rapidly increasing tumour mass at a single or multiple extranodal sites. There is nodal and extranodal involvement in this disease with the most common extranodal site being the gastro intestinal tract although it can be found in many other areas such as the liver, spleen, thyroid and kidney. There is bone marrow involvement in 11 27% of cases with malignant cells circulating in the PB in approximately 1/3 of these patients. The immunophenotype of DLBCL, NOS is the expression of CD19, CD20, CD22 and CD79a but one or more of these may be lacking. There may be demonstration of surface and/or cytoplasmic immunoglobulin in between 50 and
6 75% of cases. CD38 and CD138 are rarely expressed in CD20 positive cases, and CD5 and CD10 may be expressed in 10% and 30-60% of cases respectively. The most common abnormalities seen in DLBCL, NOS include t(14;18)(q32;q21), t(3;14)(q27;q32), t(1;3)(q34;q27), t(2;3)(q35;q27), t(3;12)(q27;q13), t(3;22)(q27;q11) and inv(3)(q13q27). The immunophenotype and cytogenetics in this case do not suggest DLBCL, NOS. EXERCISE CONSENSUS DIAGNOSIS (Classified as Correct) Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) Other Differential Diagnoses (Classified as Minor Errors): Mantle Cell lymphoma (MCL) Other Differential Diagnoses (Classified as Major Errors): B prolymphocytic leukaemia (B-PLL) Diffuse large B-cell lymphoma (DLBCL), NOS References 1. A clinical evaluation of the International Lymphoma Study Group classification of non-hodgkins lymphoma. The Non-Hodgkins Lymphoma Classification Project. Blood 89: MeloJV, Wardle J, ChetyM, et al. The relationnship between chronic lymphocytic leukaemia and prlymphocytic leukaemia III: evaluation of the cell size by morphology and volume measurements. Br J Haematol. 1986;64: Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A, Que TH, Catovsky D. The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia. Oct;8(10): Moreau EJ, Matutes E, A'Hern RP, Morilla AM, Morilla RM, Owusu- Ankomah KA, Seon BK, Catovsky D. Improvement of the chronic lymphocytic leukaemia scoring system with the monoclonal antibody SD8 (CD79b). Am J of Clin Path; 108:
7 5. MeloJV, Wardle J, ChetyM, et al. The relationnship between chronic lymphocytic leukaemia and prlymphocytic leukaemia III: evaluation of the cell size by morphology and volume measurements. Br J Haematol. 1986;64: Siebert R, Matthiesen P, Harder S, Zhang Y, Borowski A, Zühlke-Jenisch R, Plendl H, Metzke S, Joos S, Zucca E, Weber-Matthiesen K, Roggero E, Grote W, Schlegelberger B. Application of interphase cytogenetics for the detection of t(11;14)(q13;q32) in mantle cell lymphomas. Ann Oncol. 1998;9: Vaandrager JW, Schuuring E, Zwikstra E, de Boer CJ, Kleiverda KK, van Krieken JH, Kluin-Nelemans HC, van Ommen GJ, Raap AK, Kluin PM. Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multicolor DNA fiber fluorescence in situ hybridization. Blood :
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