Noraidah Masir

BCL2 protein expression in follicular lymphomas with t(14;18) chromosomal translocations

The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein overexpression in most follicular lymphomas. However the expression of BCL2 is not always homogeneous and may demonstrate a variable degree of heterogeneity. This study analysed BCL2 protein expression pattern in 33 cases of t(14;18)‐positive follicular lymphomas using antibodies against two different epitopes (i.e. the widely used antibody BCL2/124 and an alternative antibody E17). 16/33 (49%) cases demonstrated strong BCL2 expression. In 10/33 (30%) cases, BCL2 expression was heterogeneous and in some of these, its loss appeared to be correlated with cell proliferation, as indicated by Ki67 expression. Double immunofluorescence labelling confirmed an inverse BCL2/Ki67 relationship, where in 24/28 (86%) cases cellular expression of BCL2 and Ki67 was mutually exclusive. In addition, seven BCL2 ‘pseudo‐negative’ cases were identified in which immunostaining was negative with antibody BCL2/124, but positive with antibody E17. Genomic DNA sequencing of these ‘pseudo‐negative’ cases demonstrated eleven mutations in four cases and nine of these were missense mutations. It can be concluded that in follicular lymphomas, despite carrying the t(14;18) translocations, BCL2 protein expression may be heterogeneous and loss of BCL2 could be related to cell proliferation. Secondly, mutations in translocated BCL2 genes appear to be common and may cause BCL2 pseudo‐negative immunostaining.

Pseudonegative BCL2 protein expression in a t(14;18) translocation positive lymphoma cell line: a need for an alternative BCL2 antibody

Aim: The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein expression in most follicular lymphomas. However, a small number of cases lack BCL2 expression despite carrying the t(14;18)(q32;q21) translocation. This study aims to explore the mechanism accounting for the lack of BCL2 protein expression when the t(14;18) translocation is present. Methods: BCL2 expression in the t(14;18) positive cell lines FL18, Karpas‐422, SU‐DHL‐4 and SU‐DHL‐6, was analysed by Western blotting and by immunohistochemistry using two different antibodies. FISH analysis was performed to confirm the cytogenetic changes in the cell lines and real time quantitative PCR was used to evaluate the BCL2 mRNA level. Sequence analysis of translocated BCL2 was performed on FL18, Karpas‐422, SU‐DHL‐4 and SU‐DHL‐6 cell lines. Results: In FL18, Karpas‐422, and SU‐DHL‐4, the BCL2 mRNA level correlated with the BCL2 protein expression. In contrast, BCL2 protein was not detected in SU‐DHL‐6 line using standard anti‐BCL2 antibody (BCL2/124), despite the presence of the t(14;18) translocation and high level of mRNA. cDNA sequencing of translocated BCL2 showed three mutations in the SU‐DHL‐6 cell line, one of which resulted in an amino acid substitution (I48F) in the region recognised by the standard BCL2 antibody, whereas the other two were silent mutations at aa71 and aa72. Interestingly, when BCL2 expression was tested with an alternative antibody, E17, the protein was detected in SU‐DHL‐6, suggesting that the ‘negativity’ of SU‐DHL‐6 line for BCL2 using the standard antibody is spurious. Amino acid changes were found in Karpas‐422 (G47D, P59L) and SU‐DHL‐4 (P59T, S117R) but these did not affect BCL2 detection. Conclusions: This study suggests that some somatic mutations of the translocated BCL2 gene may prevent epitope recognition by BCL2 antibodies, and hence cause false negative expression using the standard antibody. It is recommended that in practice all BCL2 negative cases should routinely be stained with an alternative antibody to prevent false negativity.

Another look at follicular lymphoma: Immunophenotypic and molecular analyses identify distinct follicular lymphoma subgroups

The aim of this study was to analyse the immunophenotypic and molecular features of a large series of follicular lymphomas, focusing in particular on atypical cases that fail to express CD10 and/or bcl‐2. Such cases present diagnostic pitfalls, especially with regard to the differential diagnosis from follicular hyperplasia and marginal zone B‐cell lymphoma. Therefore, we also included an immunohistochemical evaluation of stathmin, which is strongly expressed by germinal centre B cells, as a putative new marker for follicular lymphomas, particularly those with an atypical phenotype.

RCL2, a potential formalin substitute for tissue fixation in routine pathological specimens

Masir N, Ghoddoosi M, Mansor S, Abdul‐Rahman F, Florence C S, Mohamed‐Ismail N A, Tamby M‐R & Md‐Latar N H 
(2012) Histopathology 60, 804–815

Variation in BCL2 protein expression in follicular lymphomas without t(14;18) chromosomal translocations

Aim: The hallmark of follicular lymphoma is the t(14;18)(q32;q21) chromosomal translocations that lead to deregulation of BCL2 expression in tumour cells. However, not all cases of follicular lymphoma express BCL2, nor is the t(14;18) translocation always present. Follicular lymphomas lacking the BCL2 rearrangement are less well studied with regards to their immunohistochemical and molecular features. This study aims to investigate the BCL2 protein expression pattern in t(14;18) negative follicular lymphomas. Methods: BCL2 protein expression pattern was analysed in 26 cases of t(14;18) negative follicular lymphoma [determined by fluorescence in situ hybridisation (FISH)], using antibodies against two-different epitopes, i.e., the widely-used antibody BCL2/124 and an alternative antibody E17. Results: Two of the t(14;18) negative cases showed evidence of BCL2 amplification and trisomy 18. A total of 13 cases (50%) lacked BCL2 expression. In 10 cases (38%) the expression was heterogeneous and in only three cases (12%) the BCL2 expression was strongly positive. These cases could thus be subdivided into three subgroups: Group I, normal BCL2 genes (i.e., no evidence of translocation or amplification), and BCL2 protein negative; Group II, normal BCL2 genes but BCL2 protein positive; and Group III, presence of other genetic alterations, i.e., BCL2 amplification and trisomy 18, and BCL2 protein positive. Conclusions: This study suggests that it may be possible on the basis of staining to predict that the t(14;18) translocation is absent if a case is either negative for BCL2 protein with different antibodies or has heterogeneous BCL2 expression, possibly acquired through a physiological process of differentiation.

The expression of Bcl‐2 by proliferating cells varies in different categories of B‐cell lymphoma

Masir N, Jones M, Lee A M, Goff L K, Clear A J, Lister A, Marafioti T & Mason D Y
(2010) Histopathology56, 617–626

Expression of DDR1 and DVL1 in Invasive Ductal and Lobular Breast Carcinoma does not Correlate with Histological Type, Grade and Hormone Receptor Status

BACKGROUND Invasive ductal (IDC) and lobular (ILC) carcinomas are the common histological types of breast carcinoma which are difficult to distinguish when poorly differentiated. Discoidin domain receptor (DDR1) and Drosophila dishevelled protein (DVL1) were recently suggested to differentiate IDC from ILC. OBJECTIVES To assess the expression of DDR1 and DVL1 and their association with histological type, grading and hormonal status of IDC and ILC. MATERIALS AND METHODS This cross sectional study was conducted on IDC and ILC breast tumours. Tumours were immunohistochemically stained for (DDR1) and (DVL1) as well as estrogen receptor (ER), progesterone receptor (PR) and C-erbB2 receptor. Demographic data including age and ethnicity were obtained from patient records. RESULTS A total of 51 cases (30 IDCs and 21 ILCs) were assessed. DDR1 and DVL1 expression was not significantly associated with histological type (p=0.57 and p=0.66 respectively). There was no association between DDR1 and DVL1 expression and tumour grade (p=0.32 and p=1.00 respectively), ER (p=0.62 and 0.50 respectively), PR (p=0.38 and p=0.63 respectively) and C-erbB2 expression (p=0.19 and p=0.33 respectively) in IDC. There was no association between DDR1 and DVL1 expression and tumour grade (p=0.52 and p=0.33 respectively), ER (p=0.06 and p=0.76 respectively), PR (p=0.61 and p=0.43 respectively) and C-erbB2 expression (p=0.58 and p=0.76 respectively) in ILC. CONCLUSIONS This study revealed that DDR1 and DVL1 are present in both IDC and ILC regardless of the tumour differentiation. More studies are needed to assess the potential of these two proteins in distinguishing IDC from ILC in breast tumours.

Systematic Epstein-Barr virus-positive T-cell lymphoproliferative disease presenting as a persistent fever and cough: a case report

IntroductionSystemic Epstein-Barr virus-positive T-cell lymphoproliferative childhood disease is an extremely rare disorder and classically arises following primary acute or chronic active Epstein-Barr virus infection. It is characterized by clonal proliferation of Epstein-Barr virus-infected T-cells with an activated cytotoxic phenotype. This disease has a rapid clinical course and is more frequent in Asia and South America, with relatively few cases being reported in Western countries. The clinical and pathological features of the disease overlap with other conditions including infectious mononucleosis, chronic active Epstein-Barr virus infection, hemophagocytic lymphohistiocytosis and natural killer cell malignancies. We describe the rare case of systemic Epstein-Barr virus-positive T-cell lymphoproliferative childhood disease in a 16-year-old Malay boy.Case presentationHe presented with a six-month history of fever and cough, with pulmonary and mediastinal lymphadenopathy and severe pancytopenia. Medium- to large-sized, CD8+ and Epstein-Barr virus-encoded RNA-positive atypical lymphoid cells were present in the bone marrow aspirate. He subsequently developed fatal virus-associated hemophagocytic syndrome and died due to sepsis and multiorgan failure.ConclusionsAlthough systemic Epstein-Barr virus-positive T-cell lymphoproliferative childhood disease is a disorder which is rarely encountered in clinical practice, our case report underlines the importance of a comprehensive diagnostic approach in the management of this disease. A high level of awareness of the disease throughout the diagnosis process for young patients who present with systemic illness and hemophagocytic syndrome may be of great help for the clinical diagnosis of this disease.

An interesting case of systemic lupus erythematosus presenting with hypercalcemia: A diagnostic dilemma

Background Hypercalcemia is common in primary hyperparathyroidism malignancies and even in tuberculosis. Interestingly, systemic lupus erythematosus (SLE) rarely presents with hypercalcemia. Case Report: We describe an interesting case of SLE in a patient who was otherwise thought to have either tuberculosis or a malignancy. The patient initially presented with feeling unwell, with generalized lymphadenopathy, bilateral pleural effusion, and bilateral corneal calcium deposits secondary to severe hypercalcemia. The diagnosis of SLE was made based on positivity of antinuclear antibodies (ANA) and anti-dsDNA, the presence of serositis, lymphadenopathy, autoimmune hemolytic anemia, and constitutional symptoms. She was treated with steroids, with tremendous improvement in her general well-being, resolution of lymphadenopathy and pleural effusion, and normalization of her hemoglobin and serum calcium. The atypical presentation of SLE with hypercalcemia with pleural effusion is discussed. Conclusions: SLE should be one of the differential diagnoses in patients presenting with severe hypercalcemia.

Role of co-expression of estrogen receptor beta and Ki67 in prostate adenocarcinoma

Purpose To evaluate the expression of estrogen receptor (ER)-beta and Ki67 in prostate cancer and study their relationship. Materials and Methods We analyzed 101 cases of prostate adenocarcinoma diagnosed from January 2011 to June 2015 in 100 patients. Immunohistochemical staining of ER-beta and Ki67 was analyzed according to Gleason score categorized into prognostic groups of 1 to 5. Double-immunofluorescent staining of ER-beta and Ki67 was performed in a total of 20 cases to study the co-expression and the relationship between these markers within the same tumor. Results A total of 53 of 101 cases (52.5%) were positive for ER-beta expression. There was a positive correlation whereby a high percentage of ER-beta expression was seen in the higher prognostic groups (groups 4 and 5; p=0.007). High Ki67 expression was observed in the higher prognostic group, whereas low Ki67 or negative expression was found in the lower prognostic group (p<0.001). The majority of cases evaluated with double-immunofluorescent staining (14/20) showed co-expression of ER-beta and Ki67 at the individual cell level. Conclusions ER-beta and Ki67 are independent tumor markers in high prognostic groups. Hence, co-expression of ER-beta and Ki67 indicates a more aggressive tumor with a poorer prognosis.