Oksana Moshynska

Clonal B cell populations in a minority of patients with Hashimoto's thyroiditis

Background: Hashimoto’s thyroiditis (HT) is a risk factor for thyroid lymphoma, and clonal B cell populations in HT support this link. The literature on B cell clonality in HT is controversial. Aims: To identify clonal B cell populations in HT and to assess their usefulness in differentiating HT from mucosa associated lymphoid tissue (MALT) lymphoma and predicting future development of lymphoma. Methods: DNA from formalin fixed, paraffin wax embedded blocks of thyroid specimens from 10 patients with HT and two thyroid MALT lymphomas was analysed for B cell clonality by seminested polymerase chain reaction (PCR) using FRIII/LJH and FRIII/VLJH primers to amplify the IgH gene VDJ region. In one case, PCR products were sequenced. Immunohistochemistry was performed by labelled streptavidin–biotin technique using antibodies to: CD45, CD45RO, CD3, CD20, and cytokeratin. Results: The histopathological and clinical findings were characteristic of HT. Clonal bands were seen in three and a polyclonal smear pattern was seen in seven cases. The clonal bands in HT were associated with a background smear, and could not be reproduced from other blocks from the same case or from deeper sections of the same block. The clonal bands in thyroid lymphomas were not associated with a background smear and were reproducible. None of the patients with clonal B cells has developed malignant lymphoma during a follow up of 10–13 years. Conclusions: B cell clonal bands in HT have different features from those in lymphoma (non-pure and non-reproducible) and do not predict future development of lymphoma.

Clonal relationship between Hashimoto thyroiditis and thyroid lymphoma

Background: Although Hashimoto thyroiditis (HT) is a predisposing factor for B-lineage thyroid lymphoma, clonal B-cell populations in HT are rare. Aim: To investigate whether there is a clonal relationship between HT and primary thyroid lymphoma. Methods: Clonalilty and sequence similarity was determined by PCR followed by sequencing and comparing immunoglobulin heavy chain (IgH) gene rearrangement sequences to germline sequences and to each other. Results: 12/20 patients with primary thyroid lymphoma had a previous history and histological diagnosis of HT. Clonal IgH bands associated with a polyclonal background were present in four of these 12 cases of HT; of these four, three had reproducible clonal IgH bands from the subsequently developed lymphoma. The range of similarity (homology) of multiple clonal bands in HT with the germline IgH varied from 90% to 96.3%. Multiple clonal bands in HT had sequence similarity (homology) of 62–100% with the clonal band in the lymphoma from the same patient. At least one clonal band in HT had more than 96% similarity (homology) with the clonal band of lymphoma in all three cases. Conclusion: Sequence similarity between the clonal bands in HT and subsequently developed thyroid lymphoma is supportive of the argument that primary thyroid lymphoma may evolve from HT.

Distinct B-cell clonal bands in Helicobacter pylori gastritis with lymphoid hyperplasia

Helicobacter pylori (Hp)‐associated gastritis is a risk factor for gastric mucosa‐associated lymphoid tissue (MALT) lymphoma. Clonal B‐cell populations are present in both reactive and neoplastic MALT tissue, thus limiting their usefulness in the evaluation of gastric lymphoid infiltrates in endoscopic biopsy specimens. The aim of this study was to identify the presence of clonal B‐cell populations in Hp‐gastritis with MALT and to assess their usefulness in distinguishing reactive from malignant infiltrates. Routinely fixed paraffin‐embedded blocks from 20 patients with Hp‐gastritis with lymphoid hyperplasia were analysed for B‐cell clonality by a semi‐nested polymerase chain reaction (PCR) using FRIII/LJH and FRIII/VLJH primers for amplification of the VDJ region of the immunoglobulin heavy chain gene. The histopathological findings were evaluated according to a previously published scoring system. Immunohistochemistry was performed by the labelled streptavidin–biotin technique using the following primary antibodies: CD45, CD45RO, CD3, CD20, and cytokeratin. The histopathological findings were diagnostic of Hp‐chronic active gastritis (grade 2, n=17; grade 3, n=3). Scattered intraepithelial B‐cells were present in all cases and non‐destructive lymphoepithelial lesions in one grade 3 case. Amplifiable DNA was obtained from all samples. Clonal bands were observed in ten (7/17 grade 2 and 3/3 grade 3 lesions) and polyclonal smears in ten cases (all grade 2). The clonal bands were often (n=6) associated with a background polyclonal smear and were not reproducible from deeper sections (n=10) or another paraffin block (n=1), while the clonal bands in control low‐grade MALT lymphomas were not associated with a background smear and were reproducible from deeper sections. None of the patients has developed lymphoma to date (follow‐up 21–44 months). In conclusion, B‐cell clonal bands are common in H. pylori‐gastritis with lymphoid hyperplasia. The irreproducibility of these bands is a useful feature in favouring a reactive process. Copyright

Association between mutations of the follicle-stimulating-hormone receptor and repeated twinning.

Follicle-stimulating hormone (FSH) has a role in folliculogenesis and spontaneous twinning. Using the candidate gene approach, we searched for mutations in the gene encoding the FSH receptor in a woman who had given birth to two sets of dizygotic twins without fertility treatment. We identified two linked mutations (Thr307Ala and Asn680Ser) that were closely associated with this phenotype. We suggest that expression of both mutations increases the sensitivity of the receptor to FSH.

G125A single-nucleotide polymorphism in the human BAX promoter affects gene expression

Earlier we had reported a guanine to adenosine substitution at position 125 (G125A) in the BAX promoter, and its association with higher stage of the disease and failure to achieve complete response to treatment in chronic lymphocytic leukemia (CLL) patients. The aim of this study was to test the hypothesis that G125A leads to a reduction in the transcription of the BAX gene and that this is a direct cause of altered BAX mRNA and protein expression. In lymphocytes of CLL patients, BAX mRNA expression was determined by RNase protection assay and Bax protein was detected by immunoblotting. The presence of G125A in the BAX promoter was associated with lower BAX mRNA (P=0.004) and protein (P=0.024) levels. In transient expression assays using wild-type and mutant BAX promoter sequences linked to Luciferase as a reporter, the G125A polymorphism reduced expression of the BAX promoter by 2.6-fold. These studies suggest a mechanism for the biological effect of the G125A.