الفهرس 
Mature B-Lymphoid NeoplasmsOnly 14 pages are availabe for public view
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Abstract
B-CLPDs are a heterogeneous group of clonal (neoplastic) diseases that share the single characteristic of arising as the result of a somatic mutation(s) in a B-lymphocyte progenitor. Any site of the lymphatic system can be the primary site of origin of the disorder, including LNs, gut-associated lymphatic tissue, skin, or spleen.
The diagnosis and classification of lymphopoietic malignancies are extremely complex and have undergone successive changes over the course of time. Recognition of the various disease entities is essential because of major implications for prognosis and patient management. The diagnosis of the B-CLPDs requires use of multiple technologies including cell morphology, immunological markers and, in some cases, histopathology of the affected tissues and molecular and cytogenetic investigations. However, it has become evident that the overlapping morphologic and immunophenotypic features of various B-CLPDs can make diagnosis difficult.
Flow cytometric IPT studies are indispensable for the diagnosis of mature B-cell lymphoid neoplasms. It is considered to be an integral part of the diagnostic process pointing to the correct classification of the individual diseases and to the choice of the most suitable type of therapy through the identification of phenotypically abnormal cells belonging to the B-cell lineage and recognition of phenotypes characteristic of separate disease entities.
The aim of this study was to perform a comprehensive flow cytometric immunophenotypic analysis of B-CLPDs with the use of an extended panel of MoAbs to provide data on the pattern of expression of various immunological markers in different B-CLPDs, trying to resolve the immunophenotypic overlap, hence, clarifying the role of immunophenotyping in the precise diagnosis of these disorders.
This study was conducted on eighty two newly diagnosed B-CLPDs patients. They were subjected to complete history taking, clinical examination and abdominal ultrasonography for organomegaly and lymphadenopathy. Diagnostic workup for B-CLPDs cases included: CBC, BM aspiration and trephine biopsy, flow cytometric IPT using an extended panel of MoAbs. In certain cases, additional diagnostic tools were performed to reach/confirm the diagnosis i.e. FISH for the t(11;14) and t(14;18) and Cy D1 immunohistochemical staining on BM trephine biopsy. LN biopsy reports were obtained from patients’ records. Accordingly, patients were classified into: forty CLL, seven aCLL, eight B-PLL, six MCL, ten HCL, four HCL-V, three SLVL, two FL and two LPL patients.
In this study, the age of the patients varied between different studied groups being highest in HCL-V and lowest in SLVL. The mean ages in HCL and SLVL groups were significantly lower than that in CLL, aCLL, B-PLL and HCL-V groups (p<0.05). It is of interest that out of the forty CLL cases one case was 27 years old. No clinical, morphologic or immunophenotypic differences were detected when compared to CLL in elderly.
Most of the studied patients had splenomegaly; though of varying degree. LN enlargement was observed in a variable percentage of patients ranging from 100% in aCLL and FL, to none in HCL-V. Hepatomegaly was a finding in most of the patients. The male to female ratio showed predominance for males except SLVL where females were predominant.
As regards the routine laboratory investigations, the mean TLC and absolute lymphocytic count were increased in all studied groups except HCL. The highest increase was detected in aCLL group. The mean TLC was significantly lower in HCL compared to that in CLL, aCLL and B-PLL groups, while in HCL-V group, only aCLL and B-PLL were statistically higher (p<0.05). Other groups showed statistically insignificant differences as regards mean TLC (p>0.05). The mean hemoglobin level and platelet count were decreased in all patient groups with the lowest levels being observed in HCL group.
In CLL group, CD5 was expressed in 38 out of 40 cases (95% expression) while CD43 was expressed in 38 out of 39 (97.4% expression). CD43 expression is useful in CLL diagnostic panel as it correctly classified two CD5 negative CLL cases.
Seven aCLL cases were included in this study. Four cases with atypical morphology demonstrated high CLL scores (4 and 5). No significant differences as regards clinical and immunophenotypic markers expression were found between aCLL and CLL groups except for moderate expression of CD5 and CD20 in aCLL compared to dim expression in CLL. As regards cases with typical CLL morphology and low CLL scores, two cases showed positive expression of FMC7, moderate intensity of CD79b and CD22, and the third showed moderate intensity of kappa light chain. CD43 was expressed in all aCLL cases. This adds to the value of CD43 in the diagnosis of CLL especially in cases with borderline scores (i.e. aCLL).
In MCL group, CD5 and CD43 were expressed in 100% and 83.3% respectively. CD43 had no role in differentiating MCL from CLL; however, it has a major role in differentiating CLL and MCL groups on one hand from other B-CLPDs on the other hand especially when positivity of CD5 was detected. CD5 was expressed in 4 out of 8 (50%) of B-PLL cases and 2 out of 3 (66.6%) of SLVL and 1 out of 2 (50%) of LPL cases while CD43 was negative in every case.
CD23 is a very useful marker to distinguish CLL from MCL and B-PLL since CD23 expression is characteristic of CLL and is generally absent in MCL and B-PLL. CD23 was expressed in 97.5% of CLL cases compared to 25% and 16.7% in B-PLL and MCL respectively. Thus, CLL can be reliably differentiated from MCL only if CD23 is negative. However, due to the heterogeneous nature of these diseases, MCL remains a disease in which, in some cases, the diagnosis must be further refined by Cy D1 by IHC and/or molecular analysis for t(11;14).
As regards CD22 and CD79b, we found that replacement of CD22 by CD79b significantly increased the potential of the CLL scoring system to discriminate between CLL and other B-CLPDs. Hence, CD22 antigen analysis in routine CLL IPT panel seems to be unnecessary.
The monoclonal antibody FMC7 recognizes an epitope of the multimeric CD20 complex. In our study analysis of FMC7 and CD20 expression in different studied groups showed that FMC7 was found to be of greater diagnostic value for distinguishing CLL from other B-CLPDs than CD20. On the other hand, in HCL CD20 is of great diagnostic value in differentiating HCL from other B-CLPDs in particularly lymphoid malignancies with villous lymphocytes (HCL-V and SLVL). CD20 showed bright expression (the most intense antigenic expression in HCL) compared to moderate expression in HCL-V and SLVL. Also, CD20 expression was brighter than CD22, the combination of CD20/CD11c may be even more characteristic than CD22/CD11c in the diagnosis of HCL.
CD11c is not specific for HCL, being positive in other B-CLPDs (100% in HCL-V and SLVL, 27.5% in CLL, 28.6% in aCLL and 33.5% in MCL). The low specificity of monoclonal antibodies CD11c in the diagnosis of HCL can be overcome by considering its MFI. CD11c is brightly expressed in HCL group compared to moderate expression in HCL-V and dim expression in SLVL and other B-CLPDs.
As regards CD25 and CD103 expression, they showed great differentiating power among HCL, HCL-V and SLVL. They were expressed in all cases of HCL. Absence of CD25 with positive expression of CD103, favors the diagnosis of HCL-V. On the other hand, absence of CD103 with positive CD25 expression favors the diagnosis of SLVL. Yet, there is still more to come in the diagnosis of these overlapping entities. The overlap between HCL-V and SLVL as regards clinical data, morphologic and immunophenotypic features is highlighted in the recently published WHO classification of B-cell neoplasms. A provisional group designated as “Splenic B-cell lymphoma/leukemia, unclassifiable” was proposed delineating their strong developmental and diagnostic overlap and that their precise diagnostic criteria are not fully established.
Two other important markers for differentiation of HCL on one hand from HCL-V and SLVL on the other hand are CD123 and CD27. In HCL group, CD123 was expressed in all HCL patients while it was negative in all other B-CLPDs. In contrast, CD27 was negative in all HCL patients compared to 100% positivity in all B-CLPDs studied. Their role in HCL diagnosis was magnified by their ability to confirm the diagnosis (together with bone marrow trephine biopsy histology) in one of our cases (Appendix-Table 14, case no. 4) which showed low moderate expression of CD11c and CD22 together with dim expression of CD25 and CD103. This is unusual of classic HCL where these markers are reported to be typically high moderate or bright. However, the diagnosis of classic HCL was based on BM trephine biopsy pathology together with the positivity of CD123 and the negativity of CD27. Thus positivity of CD123 with negativity of CD27 can safely be regarded as diagnostic of HCL.
As regards CD10, it showed 100% expression in FL with moderate intensity compared to 2.5% expression with dim intensity in CLL and absent expression in other B-CLPDs studied.
Moderate CD10 intensity in FL seems to be of value when differentiating FL from CD10+ CLL together with the absence of CD5, CD43 and CD23 observed in all FL cases. Yet in FL cases with non typical morphological features and/or dimly expressed CD10, molecular cytogenetic studies to evaluate specific translocation as t(14;18) should be considered.
As for SmIg light chain expression in different studied groups, κ restriction was more frequent than λ in all groups except for MCL where λ restriction was predominant. Lambda light chain restriction together with coexpression of CD5/CD43/CD19 with negative CD23, favor the diagnosis of MCL.
Accurate classification of chronic B-cell neoplasms is sometimes difficult. Some diseases as CLL and HCL have well characterized phenotypes while others as MCL do not. It is important to aware that, in those with characteristic phenotypes, there are still variations creating areas of overlap between these disorders. However, even when the phenotype is not conclusive, the comprehensive interpretation of the pattern of reactivity to an extended panel of monoclonal antibodies including CD43, CD123, CD27, can still provide clues that might help resolve the overlap or at least limit the differential diagnosis and probably suggest the appropriate investigation that may be required to reach a definite diagnosis. These investigations may be histopathology of bone marrow, LN, spleen and FISH analysis for specific cytogenetic abnormalities.
Despite the high diagnostic value of flow cytometric immunophenotyping in B-CLPDs demonstrated in this work, it is crucial to recognize the importance of approaching each case systematically taking into consideration all the available information. Data should always be interpreted in the context of one another as when it comes to B-CLPDs, no single investigation is always sufficient for accurate diagnosis.