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PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society., (© 2011 Blackwell Publishing Ltd.)

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Hepatosplenic gamma-delta T-cell lymphoma is a rare, usually fatal lymphoma and available literature on management is sparse. Allografting is probably the only curative option. We describe a further case with a dramatic, though transient response to Fludarabine and Alemtuzumab combination, following a failure of conventional chemotherapy. Given the dreadful prognosis with conventional chemotherapy, it is a regimen worth pursuing as a disease reduction strategy prior to allograft where appropriate.

The spectrum of clinical presentation of haematological disease is wide. We highlight two features of this principle: a rare cause of a 'haematological' presentation and a possible haematological cause of a disease not normally considered as such. A case of systemic pyoderma gangrenosum presented with splenomegaly in the absence of a rash. A clonal gamma- and beta-T-cell receptor rearrangement was demonstrated. Such clones may be a general phenomenon involved in the pathogenesis of this condition.

The p16INK4A tumour suppressor gene (p16) encodes for a cyclin-dependant kinase inhibitor which plays a role in the phosphorylation of the retinoblastoma protein (pRb) during regulation of the G1-S phase of the cell cycle. Loss of heterozygosity at 9p21, the chromosomal location of the p16 gene, has been reported in a broad range of tumours and this is usually indicative of the presence of a tumour suppressor gene, the second allele being frequently inactivated by mutation or deletion. The p16 gene, however, is often found not be mutated or deleted and it has been suggested that hypermethylation of the CpG islands of the gene may be an alternative mechanism of gene inactivation. We sought to determine the levels of p16 abnormality in a series of epithelial ovarian carcinomas in an attempt to clarify the presently conflicting evidence of whether or not hypermethylation of the p16 gene plays a role in ovarian carcinogenesis. We analysed 57 primary ovarian tumours and their corresponding blood DNA using SSCP analysis, sequencing and the methylation specific PCR (MSP) technique. We found low levels of mutation (6.7% of malignant tumours) and no evidence of methylation in any of our samples, suggesting that neither mutation or hypermethylation of the CpG islands of the p16 gene play an important role in ovarian carcinogenesis.

The objective of this study was to compare the frequency of two common p21WAF1/Cip1 gene polymorphisms in ovarian cancer patients with that in age-matched controls, from a population originating from Eastern Scotland. Both polymorphisms were found significantly less frequently in both the constitutive and tumour tissue DNA of ovarian cancer patients (3/65; 4.6%), than in that from geographically and age-matched controls (25/186; 13.4%) (p=0.0495, chi2). Furthermore, we found no p21WAF1/Cip1 gene mutations in any of the tumours, reflected by a relatively low degree of loss of heterozygosity (LOH) at the chromosomal region where the gene maps, providing further evidence that the p21WAF1/Cip1 gene is not mutated in ovarian cancer. The data suggest however, that there may potentially be a protective function for the two p21WAF1/Cip1 gene polymorphisms in the population under study.

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

In breast cancers and sarcomas, a variant polymorphism in the cell cycle inhibitor P21CIP1/WAF1 is under-represented in those individuals whose tumours contain mutated TP53. The aim of this study was to determine whether this variant polymorphism was also under-represented in those with ovarian carcinoma and TP53 mutations. We studied lymphocyte DNA from 104 women with ovarian carcinoma, 15 with borderline tumours and 16 with benign tumours, using a previously-reported PCR-RFLP technique. 96 of the ovarian carcinoma cases had been previously examined for mutations in TP53 and/or for overexpression of the TP53 protein. The variant allele was seen in 11 out of 104 women (10.6%) with ovarian carcinoma. There was no significant difference in the distribution of the variant allele in the women whose tumours had (7/47) or did not have (4/49) TP53 mutations (P = 0.523). It does not appear that the presence of this variant allele of P21CIP1/WAF1 has any aetiological role in ovarian carcinomas. Studies in other tumours support this finding.

Using the single-strand conformational polymorphism technique, we have screened 66 malignant ovarian tumors for p53 mutation in exons 5 to 8. Thirty-four of the tumors demonstrated a single-strand conformational polymorphism band shift in this region of the gene, including 6 in exon 5, 7 in exon 6, 12 in exon 7, and 10 in exon 8 (one of the tumors showed a shift for exons 7 and 8). All of the single-strand conformational polymorphism shifts have been further characterized by DNA sequencing, and 31 of 35 have been shown to represent genuine DNA alterations. These include 27 point mutations (23 missense, 2 nonsense, and 2 silent mutations), 3 deletions (a 2-base pair deletion introducing, by frameshift, a stop codon further downstream; a 3-base pair deletion; and an unusual 6-base pair deletion made up of separate 2-base pair and 4-base pair deletions), and a 4-base pair insertion (introducing a stop codon downstream). In total, 29 of the 66 (44%) carcinomas analyzed had mutations affecting the primary sequence of the p53 protein. p53 mutation was found in tumors of all International Federation of Gynecologists and Obstetricians stages, suggesting that it might be an earlier genetic event in the progression of epithelial ovarian tumors than previously thought. A significantly greater number of p53 mutations were seen in high-grade serous carcinomas than in those of endometrioid and mucinous types (0.02 > P > 0.01). Analysis of the distribution of point mutations showed no preference for any particular mutation type.

Genes on chromosomes 17q and 18q have been shown to code for putative tumor suppressors. By a combination of allele-loss studies on sporadic ovarian carcinomas and linkage analysis on a breast/ovarian cancer family, we have investigated the involvement of such genes in these diseases. Allele loss occurred in sporadic tumors from both chromosome 17p, in 18/26 (69%) cases, and chromosome 17q, in 15/22 (68%) cases. In the three familial tumors studied, allele loss also occurred on chromosome 17 (in 2/3 cases for 17p markers and in 2/2 cases for a 17q allele). Allele loss on chromosome 18q, at the DCC (deleted in colorectal carcinomas) locus, was not as common (6/16 cases [38%]) in sporadic ovarian tumors but had occurred in all three familial tumors. The results of linkage analysis on the breast/ovarian cancer family suggested linkage between the disease locus and 17q markers, with a maximum lod score of 1.507 obtained with Mfd188 (D17S579) polymorphism at 5% recombination. The maximum lod score for DCC was 0.323 at 0.1% recombination. In this family our results are consistent with a predisposing gene for breast/ovarian cancer being located at chromosome 17q21.