Tim C. Diss

IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours.

Somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in gliomas and acute myeloid leukaemia (AML). Since patients with multiple enchondromas have occasionally been reported to have these conditions, we hypothesized that the same mutations would occur in cartilaginous neoplasms. Approximately 1200 mesenchymal tumours, including 220 cartilaginous tumours, 222 osteosarcomas and another ∼750 bone and soft tissue tumours, were screened for IDH1 R132 mutations, using Sequenom® mass spectrometry. Cartilaginous tumours and chondroblastic osteosarcomas, wild‐type for IDH1 R132, were analysed for IDH2 (R172, R140) mutations. Validation was performed by capillary sequencing and restriction enzyme digestion. Heterozygous somatic IDH1/IDH2 mutations, which result in the production of a potential oncometabolite, 2‐hydroxyglutarate, were only detected in central and periosteal cartilaginous tumours, and were found in at least 56% of these, ∼40% of which were represented by R132C. IDH1 R132H mutations were confirmed by immunoreactivity for this mutant allele. The ratio of IDH1:IDH2 mutation was 10.6 : 1. No IDH2 R140 mutations were detected. Mutations were detected in enchondromas through to conventional central and dedifferentiated chondrosarcomas, in patients with both solitary and multiple neoplasms. No germline mutations were detected. No mutations were detected in peripheral chondrosarcomas and osteochondromas. In conclusion, IDH1 and IDH2 mutations represent the first common genetic abnormalities to be identified in conventional central and periosteal cartilaginous tumours. As in gliomas and AML, the mutations appear to occur early in tumourigenesis. We speculate that a mosaic pattern of IDH‐mutation‐bearing cells explains the reports of diverse tumours (gliomas, AML, multiple cartilaginous neoplasms, haemangiomas) occurring in the same patient. Copyright

Resistance of t(11;18) positive gastric mucosa-associated lymphoid tissue lymphoma to Helicobacter pylori eradication therapy

20-30% of gastric mucosa-associated lymphoid tissue (MALT) lymphoma associated with Helicobacter pylori do not regress after antibiotic therapy. Regression can be assessed only by extended follow-up. To assess whether t(11;18, q21;q21), which results in a chimeric transcript between the AP12 and MLT genes, predicts lymphoma resistance to antibiotic therapy, we screened for the fusion transcript with RT-PCR in ten responsive and 12 non-responsive gastric MALT lymphomas. The AP12-MLT transcript was detected in nine (75%) of 12 patients non-responsive to antibiotic therapy but not in responsive patients. Most H pylori-associated gastric MALT lymphomas that do not respond to antibiotic therapy are associated with t(11;18, q21;q21).

Follicular colonization in B-cell lymphoma of mucosa-associated lymphoid tissue.

The formation of neoplastic B-cell follicles is universally accepted as diagnostic of a follicle centre cell (FCC) lymphoma. Low-grade B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) are characterized by a diffuse infiltrate of cells of uncertain lineage known as “centrocyte-like” cells because of their resemblance to centrocytes (small cleaved cells). Some MALT lymphomas, however, contain numerous follicles and may even have a predominantly follicular appearance. These follicles may be reactive or show immunoglobulin (Ig) light-chain restriction, indicating their neoplastic nature. We have proposed that these neoplastic follicles are not composed of follicle centre cells but result from colonization of reactive follicles by CCL cells. In this study, the immunophenotype and genotype of 10 primary gastrointestinal lymphomas with a follicular component have been determined. One case exhibited the morphological, immunophenotypic, and genotypic features of FCC lymphoma (Ig light-chain restriction, CD10+, KB61 (CDw32)-, Jh, and bcl-2 gene rearrangement). Neoplastic follicles in the remaining nine cases, which showed the features of MALT lymphoma, were of a different phenotype (Ig light-chain restriction, CD10-, KB61(CDw32)+), and these lymphomas showed Jh but not bct-2 gene rearrangement. Taken in conjunction with the morphological features, these findings suggest that in these cases the neoplastic follicles formed as the result of colonization of previously reactive follicles by neoplastic CCL cells. Thus, not all lymphomas containing neoplastic follicles are of FCC origin. Follicular colonization, as seen in lowgrade MALT lymphomas, is likely to be a recapitulation of an as yet undescribed normal immunological phenomenon that may involve marginal zone B cells.

In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low-grade serous tumours

Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS/RAF/MEK/ERK/MAP‐kinase pathway. Activating mutations in BRAF have recently been identified in melanomas, colorectal cancers, and thyroid and ovarian tumours. In the present study, an extensive characterization of BRAF and KRAS mutations has been performed in 264 epithelial and non‐epithelial ovarian neoplasms. The epithelial tumours ranged from adenomas and borderline neoplasms to invasive carcinomas including serous, mucinous, clear cell, and endometrioid lesions. It is shown that BRAF mutations in ovarian tumours occur exclusively in low‐grade serous neoplasms (33 of 91, 36%); these included serous borderline tumours (typical and micropapillary variants), an invasive micropapillary carcinoma and a psammocarcinoma. KRAS mutations were identified in 26 of 91 (29.5%) low‐grade serous tumours, 7 of 49 (12%) high‐grade serous carcinomas, 2 of 6 mucinous adenomas, 22 of 28 mucinous borderline tumours, and 10 of 18 mucinous carcinomas. Of note, two serous borderline tumours were found to harbour both BRAF and KRAS mutations. The finding that at least 60% of serous borderline tumours harbour mutations in two members of the ERK‐MAP‐kinase pathway (BRAF 36%, KRAS 30%) compared with 12% of high‐grade serous carcinomas (BRAF 0%, KRAS 12%) indicates that the majority of serous borderline tumours do not progress to serous carcinomas. Furthermore, no BRAF mutations were detected in the other 173 ovarian tumours in this study. Copyright

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets: Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

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.

EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations

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.

The polymerase chain reaction in the demonstration of monoclonality in T cell lymphomas.

AIMS--To evaluate polymerase chain reaction (PCR) amplification of T cell receptor (TCR) beta and gamma chain genes as a means of demonstrating monoclonality in T cell lymphomas using histological samples; to compare the performance of PCR with Southern blot analysis. METHODS--TCR-beta, TCR-gamma and immunoglobulin heavy chain (IGH) genes were analysed using PCR in 55 cases of T cell lymphoma (28 frozen tissue and 27 paraffin wax embedded samples), diagnosed using morphological and immunohistochemical criteria. The 28 frozen samples were subjected to Southern blot analysis using TCR-beta, TCR-gamma and IGH gene probes. Twenty five B cell lymphomas and 21 non-neoplastic lymphoid tissue samples were used as controls. RESULTS--Using TCR-beta PCR, monoclonality was detected in 24 (44%) of 55 T cell lymphomas compared with 43 (78%) of 55 using TCR-gamma PCR and in 82% with both techniques. Five (9%) of 55 T cell lymphomas were IGH PCR positive. None of the non-neoplastic lymphoid control samples were PCR positive. All B cell lymphomas showed a polyclonal pattern with TCR-beta PCR while a single B cell lymphoma was positive using TCR-gamma primers. With TCR-beta PCR, a monoclonal result was seen in 12 (43%) of 28 frozen samples of T cell lymphoma, compared with 23 (82%) of 28 using Southern blot analysis. With TCR-gamma PCR, 19 (68%) of 28 frozen tissue samples were positive, compared with 26 (93%) of 28 using Southern blot analysis. A single case showed IGH rearrangement by Southern blot analysis. CONCLUSION--TCR-gamma PCR should be the method of choice for analysis of clonality in paraffin wax embedded sections of lymphoproliferative lesions, as TCR-beta PCR has a high false negative rate. Southern blot analysis remains the most successful technique when sufficient fresh tissue samples and resources are available.

Sources of DNA for detecting B cell monoclonality using PCR.

AIMS--To evaluate the polymerase chain reaction (PCR) demonstration of clonal immunoglobulin heavy chain gene rearrangements using routinely prepared, unstained, and stained formalin fixed, paraffin wax embedded tissue samples. METHODS--Extracts from (a) fresh frozen tissue samples, (b) unstained, and (c) haematoxylin and eosin stained formalin fixed, paraffin wax embedded 5 microns tissue sections from 42 cases of low grade B cell lymphoma, all shown to be monoclonal by Southern blot analysis, were analysed using PCR. Two regions of the variable segment of the immunoglobulin heavy chain gene were amplified (framework 2 to joining region [Fr2/JH] and framework 3 to joining region [Fr3/JH]). Twelve samples of reactive lymphoid tissue were studied as controls. Products from each case were directly compared on polyacrylamide gels. RESULTS--Using both primer combinations, monoclonality was detected in 38 of 42 (90%) cases using fresh material, 37 of 42 (88%) using unstained paraffin wax embedded samples, and in 35 of 42 (83%) cases using haematoxylin and eosin stained sections. No false positive results attributable to fixation, processing, or staining were identified, although the efficiency of amplification using the Fr2/JH primers was significantly reduced. CONCLUSIONS--PCR determination of B cell clonality using paraffin wax embedded material is sufficiently sensitive and reliable for use as a routine diagnostic adjunct to conventional morphological and immunocytochemical assessment of lymphoproliferative disease.

Detection of Helicobacter pylori in gastric biopsy and resection specimens.

AIMS: To compare the sensitivity of detecting Helicobacter pylori in gastric biopsy and resection specimens using tinctorial and silver impregnation stains, immunohistochemistry and the polymerase chain reaction (PCR). METHODS: Formalin fixed, paraffin wax embedded tissue from 33 gastric biopsy specimens (26 showing chronic gastritis and seven showing low grade mucosa associated lymphoid tissue (MALT) lymphoma) together with blocks of uninvolved mucosa from gastrectomy specimens for MALT lymphoma (five cases) were studied. Consecutive sections were stained using haematoxylin and eosin, Giemsa, the Warthin-Starry silver stain, and a polyclonal antibody directed against H pylori using an immunoperoxidase technique following heat induced antigen retrieval. PCR analysis of DNA extracted from a further section was carried out using primers which amplified a 411 base pair fragment of the urease A gene. RESULTS: H pylori was detected in 14 (37%) sections stained with haematoxylin and eosin, 21 (55%) with Giemsa, 23 (61%) with Warthin-Starry, and 25 (66%) stained with the antibody. Seventeen (45%) cases were positive on PCR. Immunohistochemistry was positive in all cases in which H pylori was detected by other methods. CONCLUSION: Immunohistochemistry using an immunoperoxidase technique following heat induced antigen retrieval for detecting H pylori in gastric biopsy and resection specimens is highly sensitive and easy to use.

True histiocytic lymphoma - A morphologic, immunohistochemical, and molecular genetic study of 13 cases

We describe the morphologic, immunohistologic, and genotypic characteristics of 13 cases of true histiocytic lymphomas. Six cases presented with primary gastrointestinal involvement, five with lymphadenopathy, the other sites involved being the bone marrow and the skin. The neoplastic cells displayed large abundant eosinophilic cytoplasm, occasionally vacuolated with folded or bizarre-shaped nuclei with prominent nucleoli. Mitotic figures were numerous. Multinucleated cells were common. The pattern of growth was usually diffuse and noncohesive. Spindle cell sarcoma-like areas were evident in five cases, with a prominent foam cell component in four cases. All cases expressed histiocyte-associated markers (CD68, lysozyme, alpha-1-antitrypsin), CD45 or CD45RO, and were negative for CD1a, epithelial, and B- and T-cell lineage-specific markers. Reactivity for S-100 was observed in a variable proportion of cells in 11 cases. The proliferation fraction varied from 3 to 88%. Genotypic analysis for T-cell receptor or immunoglobulin gene rearrangement demonstrated a germline configuration in all cases. We demonstrate that true histiocytic lymphoma is a rare distinctive pathologic entity that may be defined by immunohistochemical criteria and that recognition among histiocytic disorders is important for clinical and prognosis reasons.