Mutation spectra of the BRCA1/2 genes in human breast and ovarian cancer and germline.

Annotating genomic sequence alterations is sometimes a difficult decision, particularly in missense variants with uncertain pathogenic significance and also in those presumed as germline pathogenic variants. We here suggest that mutation spectrum may also be useful for judging them. From the public databases, 982 BRCA1/1861 BRCA2 germline missense variants and 294 BRCA1/420 BRCA2 somatic missense variants were obtained. We then compared their mutation spectra, i.e., the frequencies of two transition- and four transversion-type mutations, in each category. Intriguingly, in BRCA1 variants, A:T to C:G transversion, which was relatively frequent in the germline, was extremely rare in somatic, particularly breast cancer, cells (p = .03). Conversely, A:T to T:A transversion was most infrequent in the germline, but not rare in somatic cells. Thus, BRCA1 variants with A:T to T:A transversion may be suspected as somatic, and those with A:T to C:G as being in the germline. These tendencies of mutation spectrum may also suggest the biological and chemical origins of the base alterations. On the other hand, unfortunately, variants of uncertain significance (VUS) were not distinguishable by mutation spectrum. Our findings warrant further and more detailed studies.

Activation of recombinational repair in Ewing sarcoma cells carrying EWS-FLI1 fusion gene by chromosome translocation.

Chromosome translocation (TL) is an important mode of genomic changes underlying human tumorigenesis, the detailed mechanisms of which are, however, still not well understood. The two major modalities of DNA double strand break repair, i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ), have been hypothesized. In a typical TL+ human neoplasm, Ewing sarcoma, which is frequently associated with t(11;22) TL encoding the EWS-FLI1 fusion gene, NHEJ has been regarded as a model to explain the disease-specific TL. Using comprehensive microarray approaches, we observed that expression of the HR genes, particularly of RAD51, is upregulated in TL+ Ewing sarcoma cell lines, WE-68 and SK-N-MC, as in the other TL+ tumor cell lines and one defective in DNA mismatch repair (MMR). The upregulated RAD51 expression indeed lead to frequent focus formation, which may suggest an activation of the HR pathway in these cells. Furthermore, sister chromatid exchange was frequently observed in the TL+ and MMR-defective cells. Intriguingly, ionizing irradiation revealed that the decrease of 53BP1 foci was significantly retarded in the Ewing sarcoma cell lines, suggesting that the NHEJ pathway may be less active in the cells. These observations may support an HR involvement, at least in part, to explain TL in Ewing sarcoma.

Serum immunoglobulin G as a discriminator of infection in follicular lymphoma patients undergoing chemotherapy with bendamustine in combination with rituximab.

OBJECTIVES: Chemotherapy, including bendamustine, usually causes lymphocytopaenia and hypogammaglobulinaemia as side effects in patients with haematological malignancies. Therefore, the possibility has been considered that these immunological adverse events induced by bendamustine may lead to infectious diseases. However, lymphocytopaenia and/or hypogammaglobulinaemia have not yet been shown to have a statistically significant association with infection in cancer patients who receive bendamustine. METHODS: We retrospectively studied 27 patients with relapsed or refractory indolent follicular lymphoma who were treated with bendamustine and rituximab (BR). In order to elucidate relationships between immune-related laboratory parameters (i.e. peripheral blood leukocyte, neutrophil, lymphocyte and immunoglobulin G [IgG]) and infectious events, receiver operating characteristic (ROC) curve and multivariate logistic regression analyses were performed. RESULTS: Infectious diseases occurred in 11 patients (11/27, 41%), including 3 (3/27, 11%) with severe diseases. The area under the ROC curve (AUC) showed that the lowest IgG level during and after BR discriminated infectious events (cut-off value, 603 mg/dL) with 81.8% sensitivity and 68.8% specificity (AUC, 0.76; 95% CI, 0.52-0.90). Furthermore, a multivariate regression analysis revealed that the minimal serum IgG value during and after BR therapy was the only variable that was significantly associated with infection (odds ratio, 8.29; 95% CI, 1.19-57.62; p value, 0.03). CONCLUSION: Serum IgG ≤603 mg/dL during and after BR therapy was independently associated with an increased risk of infection. The monitoring of serum IgG during chemotherapy may help to predict the development of infection in blood cancer patients undergoing chemotherapy with bendamustine in combination with rituximab.

Fatal Hepatitis C after Chemotherapy in a Patient with Malignant Lymphoma: Possible Reactivation of Seronegative Occult Hepatitis C Virus Infection Due to Chemotherapy.

A 79-year-old man with lymphoma who tested negative for anti-hepatitis C virus (HCV) antibody received rituximab-containing chemotherapy. Liver dysfunction of unknown cause had persisted since the second cycle of chemotherapy. Ten months after treatment, he rapidly developed massive ascites and atrophy of the liver, and we detected HCV RNA in his serum using real time polymerase chain reaction. Furthermore, medical interviews showed that the patient had no episodes for acute HCV infection, but he did have a history of unspecified liver dysfunction. These findings support the possibility of the reactivation of seronegative occult HCV infection due to chemotherapy in a cancer patient.

Efficacy and Safety of the Modified EPOCH Regimen (Etoposide, Vincristine, Doxorubicin, Carboplatin, and Prednisolone) for Adult T-cell Leukemia/Lymphoma: A Multicenter Retrospective Study.

BACKGROUND: We retrospectively analyzed patients with untreated aggressive adult T-cell leukemia/lymphoma who received the modified EPOCH (mEPOCH) regimen. PATIENTS AND METHODS: Patients received up to 6 mEPOCH cycles. Etoposide (50 mg/m2/day), doxorubicin (10 mg/m2/day), and vincristine (0.4 mg/m2/day) were each given as a continuous 96-hour infusion on days 1 to 4. Prednisolone (40 mg/m2/day) was given intravenously or orally on days 1 to 4 and then tapered and stopped on day 7, and carboplatin (dose calculated for each patient individually using Calvert's formula according to a target under the curve of 3 mg/mL/min) was given as a 2-hour intravenous infusion on day 6. RESULTS: In 103 patients, overall response rate and complete response rate were 58% and 25%, respectively. With a median follow-up of 8.9 months, the median survival time was 9.8 months (95% confidence interval, 7.2-13.9 months). The median progression-free survival (PFS) was 4.2 months (95% confidence interval, 3.4-5.7 months). Patients who completed ≥ 4 cycles experienced significantly better overall survival and PFS compared with those who completed < 4 cycles. Twenty-eight patients underwent allogeneic hematopoietic stem cell transplantation after mEPOCH and demonstrated significantly prolonged overall survival and PFS compared with those who did not undergo transplantation. CONCLUSION: The mEPOCH regimen is effective with tolerable adverse effects and may be an alternative treatment option for adult T-cell leukemia/lymphoma.

Clinicopathological analysis of sinonasal malignant lymphoma in an HTLV-1 endemic area in Japan -special focus on primary sinonasal diffuse large B-cell lymphoma.

The present study investigated histological subtypes of lymphoma in patients newly diagnosed with malignant lymphoma in the human T-cell leukemia virus type 1 (HTLV-1) endemic area of Japan, and further analyzed the clinicopathological features and clinical outcomes of patients with primary sinonasal lymphoma. We retrospectively examined 151 patients aged 18-90 years in Fukuoka, Japan. Subtypes of lymphoma were determined according to the WHO classification. Among the 151 patients, 104 were diagnosed with malignant lymphoma, including 96 at the time of initial diagnosis. Ninety-two of the 96 lymphomas (96%) were non-Hodgkin lymphoma. Mature B-cell neoplasms comprised 78% (n = 75). Primary lymphoma of the sinonasal cavity was found in six patients (6%). The histological subtype of sinonasal lymphoma was diffuse large B-cell lymphoma (DLBCL) in all six tumors. Furthermore, overall survival was significantly different among three distinct DLBCL patient groups, including primary sinonasal lymphoma patients (p = 0.0016; 3-year overall survival: sinonasal DLBCL group, 53%; DLBCL of the CNS group, 0%; other DLBCL group, 83%). Our study suggests that primary DLBCL of the sinonasal tract is a distinct disease entity of DLBCL.

A retrospective analysis of haplo-identical HLA-mismatch hematopoietic transplantation without posttransplantation cyclophosphamide for GVHD prophylaxis in patients with adult T-cell leukemia-lymphoma.

Currently, allogeneic hematopoietic stem cell transplantation (allo-HCT) is the only available curative modality for patients with adult T-cell leukemia-lymphoma (ATL). When used in conjunction with posttransplantation cyclophosphamide (PTCY) for graft-versus-host disease prophylaxis, allo-HCT from an HLA haplo-identical donor yields promising outcomes for many diseases other than ATL. However, appropriate comparisons with other donor sources, especially cord blood and conventional HLA haplo-identical donors, are needed to validate the safety and efficacy of this modality. In this study, we retrospectively evaluated the outcome of allo-HCT without PTCY in patients with ATL registered in the Japan Society for Hematopoietic Cell Transplantation TRUMP database between 1985 and 2015. During that period, 46 patients received allo-HCT without PTCY and survivors were followed for a median of 2316.5 days (range: 220-3884 days). Although the estimated 1- and 5-year overall survival rates of the entire cohort were 34.5% and 17.7%, respectively, the cumulative 1- and 5-year non-ATL mortality rates of 41.3% and 55.8%, respectively, were high. The results of our study will serve as a platform for discussions of the safety and efficacy of haplo-HCT for future clinical trials in patients with ATL.

A specific mode of microsatellite instability is a crucial biomarker in adult T-cell leukaemia/lymphoma patients.

PURPOSE: Microsatellite instability (MSI) has been a long-standing biomarker candidate for drug resistance in tumour cells. Despite numerous clinical studies, the data in the literature are not conclusive. The complexity of the MSI phenomenon in some malignancies may, at least partly, account for the discrepancy. In addition, methodological problems are also pointed out in the assay techniques. We previously established a unique fluorescent technique in which the major methodological problems in conventional assays are overcome. Application of this technique has revealed two distinct modes of microsatellite alterations, i.e. Type A and Type B. More importantly, we demonstrated that Type A MSI is the direct consequence of defective DNA mismatch repair (MMR) that causes cellular resistance against antineoplastic agents. METHOD: We first applied this technique to adult T-cell leukaemia/lymphoma (ATLL). RESULTS: The MSI phenomenon was indeed observed in ATLLs (4/20, 20%). Intriguingly, the observed microsatellite alterations were invariably Type A, which implies that the tumours were MMR-defective. Indeed, clinical outcomes of patients with these MSI+ tumours were significantly worse. Furthermore, multivariate analysis revealed that Type A MSI is an independent prognostic factor. CONCLUSION: These observations strongly suggest the possibility of Type A MSI as a prognostic and potentially predictive biomarker in ATLL.

Modal variety of microsatellite instability in human endometrial carcinomas.

PURPOSE: Microsatellite instability (MSI) in human endometrial cancer (EC) was analysed using a unique fluorescent technique. MSI is associated with various human neoplasms. However, the reported frequency of MSI differs widely in each malignancy. Methodological difficulties have in fact been pointed out in its assay techniques. METHODS: We previously established a sensitive fluorescent technique in which the major methodological problems are overcome. Application of this technique has revealed two distinct modes of microsatellite alterations, i.e. Type A and Type B. In the present study, we have applied this technique to 94 ECs. RESULTS: Significant microsatellite alterations were observed in 38 (40.4%) tumours of the panel. The two modes, Type A and Type B, were indeed observed in this malignancy. More importantly, we found that the modes more closely correlated with the molecular and clinicopathological backgrounds of the tumours than the established and widely used MSI grades, MSI-H and MSI-L. Type B MSI widely correlated with family history of hereditary non-polyposis colorectal cancer-associated cancers, whereas MSI-H only did with that of colorectal cancer. Furthermore, mutation in the KRAS oncogene, which has been regarded as generally infrequent in microsatellite-unstable tumours, was clearly associated with Type A MSI. CONCLUSIONS: Our observations may suggest a biological relevance and a potential utility of the modal classification of MSI and, furthermore, added complexities to genomic instability underlying tumourigenesis in human endometrium.

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer.

Genomic sequences encoding the 3' exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms.

Simulation-based analyses reveal stable microsatellite sequences in human pancreatic cancer.

Genomic analysis using tissue samples is an essential approach in cancer genetics. However, technical and biological limits exist in this approach. Microsatellite instability (MSI) is frequently observed in human tumors. MSI assays are now prevalent and regarded as commonplace. However, several technical problems have been left unsolved in the conventional assay technique. Indeed, the reported frequencies of MSI differ widely in each malignancy. An example is pancreatic cancer. Using a unique fluorescent technique, we found that MSI is extremely infrequent in this malignancy, despite the relatively high frequencies in some reports. In a series of simulations, we have demonstrated that the extremely low frequency was derived neither from less sensitive assays nor from a scarcity of cancer cells in tissue samples. Furthermore, analyzing laser-capture microdissection (LCM)-processed cell populations of a microsatellite-unstable colorectal cancer cell line, HCT116, we have shown that MSI can be detected only when comparing two cell populations that have grown independently to a sufficiently large size. When MSI is not detected in analyses using tissue samples, LCM is not advisable. We therefore did not extend our study to LCM of tissue specimens. We conclude that microsatellite sequence alterations are not detectable in human pancreatic cancer.

Exclusive KRAS mutation in microsatellite-unstable human colorectal carcinomas with sequence alterations in the DNA mismatch repair gene, MLH1.

Microsatellite instability (MSI) is regarded as reflecting defective DNA mismatch repair (MMR). MMR defects lead to an increase in point mutations, as well as repeat instability, on the genome. However, despite the highly unstable microsatellites, base substitutions in representative oncogenes or tumor suppressors are extremely infrequent in MSI-positive tumors. Recently, the heterogeneity in MSI-positive colorectal tumors is pointed out, and the 'hereditary' and 'sporadic settings' are proposed. Particularly in the former, base substitution mutations in KRAS are regarded as relatively frequent. We sequenced the KRAS gene in a panel of 76 human colorectal carcinomas in which the MSI status has been determined. KRAS mutations were detected in 22 tumors (28.9%). Intriguingly, all of the KRAS-mutant MSI-H (high) tumors harbored sequence alterations in an essential MMR gene, MLH1, which implies that KRAS mutation more frequently and almost exclusively occurs in MMR gene-mutant MSI-H tumors. Furthermore, in contrast with the prevailing viewpoint, some of these tumors are derived from sporadic colorectal cancer patients. The tight connection between MMR gene mutation and KRAS mutation may suggest previously unrecognized complexities in the relationship between MSI and the mutator phenotype derived from defective MMR.

Frequent microsatellite instability in non-Hodgkin lymphomas irresponsive to chemotherapy.

Microsatellite instability (MSI) in haematopoietic malignancies has been controversial. Particularly in non-Hodgkin lymphoma, the data published to date lack unity. Using a unique fluorescent technique, we found MSI in eight (14%) tumours in a panel of 59 carefully selected non-Hodgkin lymphoma patients. Our fluorescent technique also reveals two qualitatively distinct modes of MSI, i.e. Type A and Type B. Based on our previous studies using DNA mismatch repair (MMR) gene-knock out animals, we have concluded that Type A MSI is a direct consequence of defective MMR. MSI observed in non-Hodgkin lymphomas was uniformly Type A, which implies that MMR deficiency occurs in this malignancy. Intriguingly, in non-Hodgkin lymphoma patients treated by CHOP/VEPA-based therapies, response to chemotherapy was significantly worse in those with microsatellite-unstable tumours (p=0.027). As a consequence, the patient outcomes at 1 year after treatment were significantly less favourable in this population (p=0.046), although the survival difference was not statistically confirmed in a longer term. These findings suggest that in some non-Hodgkin lymphomas MMR deficiency may lead to drug resistance in tumour cells and, consequently, to poor patient outcomes. In non-Hodgkin lymphoma, MSI may be a potential biomarker that predicts the tumour response against chemotherapy.

Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability.

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of < or =6 bp. Type B changes are more drastic and involve modifications of > or =8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Frequent loss of heterozygosity but rare microsatellite instability in oesophageal cancer in Japanese and Chinese patients.

Reported frequencies for microsatellite instability (MSI) in oesophageal cancer differ widely in the literature, perhaps due to the high incidence of loss of heterozygosity (LOH) in this cancer. Using high-resolution fluorescent microsatellite analysis (HRFMA), we analysed microsatellite alterations in detail in 50 Japanese and 50 Chinese patients with squamous cell carcinoma in the oesophagus. In HRFMA, several devices have been developed to improve the detection characteristics, reproducibility of polymerase chain reaction and the migration accuracy of electrophoresis. All the alterations observed were separable into MSI, LOH and alterations ambiguous for both. MSI was rare in these panels of oesophageal carcinomas. The frequencies of MSI in the Japanese and Chinese subjects were 8 and 4%, respectively. All the alterations were mild (within 2 base pairs) and were observed in a limited number of markers. More drastic types of MSI, such as those typical in colorectal cancer, were not observed. On the other hand, the incidence of LOH was high, reaching 50% for the Japanese and 70% for the Chinese subjects. In many of these cases, LOH was observed in multiple microsatellite markers. The frequency of LOH in each marker was not apparently biased. Although in many cases MSI and LOH were clearly distinguished with use of the sensitive and quantitative fluorescent assay, theoretically indistinguishable patterns were noted in some cases. In conclusion, MSI is rare and LOH predominates in squamous cell carcinoma in the oesophagus.