Concordance between microsatellite instability testing and immunohistochemistry for mismatch repair proteins and efficient screening of mismatch repair deficient gastric cancer.

Microsatellite instability (MSI) testing, an established technique that has gained prominence in recent years for its predictive potential regarding the efficacy of immune checkpoint inhibitors, is used to evaluate DNA mismatch repair (MMR) deficiency (dMMR). As with other methods, the immunohistochemistry (IHC) of MMR proteins is also widely adopted. Although both techniques have been validated, their concordance rate remains unknown, particularly regarding non-colorectal cancer. Therefore, the aim of the present study was to explore and elucidate their concordance in the context of gastric cancer (GC). A total of 489 surgically resected primary GC tissues were analyzed to compare the results yielded by the MSI test and those from IHC. Of 488 GC cases, 56 (11.5%) exhibited a loss of MMR proteins, whereas 52 (10.7%) were classified as high-frequency MSI (MSI-H). The concordance rate between these two categories was 99.2%. The microsatellite markers BAT26 and MONO27 demonstrated 100% sensitivity and 99.5% specificity in detecting dMMR GC. In addition, histopathological analysis revealed that MSI-H was more prevalent in GCs exhibiting coexisting Tub2 and Por1 subtypes. However, four discordant cases were observed. All four cases were microsatellite-stable cases but exhibited loss of MLH1 protein expression with hypermethylation of the MLH1 promoter. The results of the present study highlight that while there is a strong concordance between MSI and IHC testing results for determining dMMR status, IHC testing may offer superior efficacy in detecting dMMR.

The interplay of KRAS mutational status with tumor laterality in non-metastatic colorectal cancer: An international, multi-institutional study in patients with known KRAS, BRAF, and MSI status.

BACKGROUND: Although the prognostic relevance of KRAS status in metastatic colorectal cancer (CRC) depends on tumor laterality, this relationship is largely unknown in non-metastatic CRC. METHODS: Patients who underwent resection for non-metastatic CRC between 2000 and 2018 were identified from institutional databases at six academic tertiary centers in Europe and Japan. The prognostic relevance of KRAS status in patients with right-sided (RS), left-sided (LS), and rectal cancers was assessed. RESULTS: Of the 1093 eligible patients, 378 had right-sided tumors and 715 had left-sided tumors. Among patients with RS tumors, the 5-year overall (OS) and recurrence-free survival (RFS) for patients with KRASmut versus wild-type tumors was not shown to differ significantly (82.2% vs. 83.2% and 72.1% vs. 76.7%, respectively, all p > .05). Among those with LS tumors, KRAS mutation was associated with shorter 5-year OS and RFS on both the univariable (OS: 79.4% vs. 86.1%, p = .004; RFS: 68.8% vs. 77.3%, p = .005) and multivariable analysis (OS: HR: 1.52, p = .019; RFS: HR: 1.32, p = .05). CONCLUSIONS: KRAS mutation status was independently prognostic among patients with LS tumors, but this association failed to reach statistical significance in RS and rectal tumors. These findings confirm reports in metastatic CRC and underline the possible biologic importance of tumor location.

Quantitative evaluation of MSI testing using NGS detects the imperceptible microsatellite changed caused by MSH6 deficiency.

Microsatellite instability (MSI) is an effective biomarker for diagnosing Lynch syndrome (LS) and predicting the responsiveness of cancer therapy. MSI testing is conventionally performed by capillary electrophoresis, and MSI status is judged by visual assessment of allele size change. Here, we attempted to develop a quantitative evaluation model of MSI using next-generation sequencing (NGS). Microsatellite markers were analyzed in tumor and non-tumor tissues of colorectal cancer patients by NGS after a single multiplex polymerase chain reaction amplification. The read counts corresponding to microsatellite loci lengths were calculated independently of mapping against a reference genome, and their distribution was digitized by weighted mean. Weighted mean differences between tumor and non-tumor samples with different MSI status were assessed, and cut-off values for each marker in the discovery cohort were determined. Each microsatellite maker was defined as unstable if the weighted mean difference was greater than the cut-off value. In the discovery cohort, the evaluation model demonstrated sensitivity and specificity of 100% for all markers. In the validation cohort, MSI status determined by the new model was consistent with the outcome of the conventional method in 29/30 cases (97%). The single inconsistent case was classified as low-frequency MSI by the conventional method but considered MSI-high by NGS. Genetic testing for mismatch repair genes revealed a pathogenic variant in MSH6 in the discordant case. We successfully developed a quantitative evaluation method for determining MSI status using NGS. This is a robust and sensitive method and could improve LS diagnosis.

SVA retrotransposon insertion in exon of MMR genes results in aberrant RNA splicing and causes Lynch syndrome.

Lynch syndrome is an autosomal dominant hereditary cancer syndrome in which many cancers develop, the main one being colorectal cancer. Germline pathogenic variants in one of four mismatch repair (MMR) genes are known to be causative of this disease. Accurate diagnosis using genetic testing can greatly benefit the health of those affected. Recently, owing to the improvement of sequence techniques, complicated variants affecting the functions of MMR genes were discovered. In this study, we analyzed insertions of a retrotransposon-like sequence in exon 5 of the MSH6 gene and exon 3 of the MSH2 gene found in Japanese families suspected of having Lynch syndrome. Both of these insertions induced aberrant splicing, and these variants were successfully identified by mRNA sequencing or visual observation of mapping results, although a standard DNA-seq analysis pipeline failed to detect them. The insertion sequences were ~2.5 kbp in length and were found to have the structure of an SVA retrotransposon (SVA). One SVA sequence was not present in the hg19 or hg38 reference genome, but was in a Japanese-specific reference sequence (JRGv2). Our study illustrates the difficulties of identifying SVA insertions in disease genes, and that the possibility of polymorphic insertions should be considered when analyzing mobile elements.

Development of metachronous rectal cancers in a young man with dyskeratosis congenita: a case report.

BACKGROUND: DKC1 (dyskerin pseudouridine synthase 1) is a causative gene for X-linked dyskeratosis congenita. Approximately 8% of patients with dyskeratosis congenita have malignancy, but information about the development of malignancy in patients with dyskeratosis congenita is limited. CASE PRESENTATION: A young Japanese patient with bone marrow failure developed metachronous rectal adenocarcinomas at the ages of 16 and 18 years. He had no family history of cancer. Microsatellite instability testing with rectal tumor tissue demonstrated low-level microsatellite instability. To clarify whether any cancer susceptibility genes were involved in the development of rectal cancer, RNA sequencing was performed. Cancer-related genes were assessed, and a c.361A>G (p.Ser121Gly) germline variant was detected in DKC1. The same missense variant was previously reported in two patients with dyskeratosis congenita as a pathogenic variant, but those patients did not develop malignancies. CONCLUSIONS: Our patient developed rectal cancer at an early age of onset compared with the previously reported typical onset age of patients with dyskeratosis congenita. DKC1 might be involved in predisposition to colorectal cancer in young adulthood; therefore, appropriate surveillance may be considered.

Predictive model for high-frequency microsatellite instability in colorectal cancer patients over 50 years of age.

Microsatellite instability (MSI) is an important biomarker for screening for Lynch syndrome, and also of response to immune checkpoint inhibitors. The aim of this study is to create a predictive model to determine which elderly patients with colorectal cancer (CRC) should undergo MSI and/or immunohistochemistry testing on the basis of clinicopathological data. We analyzed a test cohort of CRC patients aged ≥50 years (n = 2219) by multivariate logistic regression analyses to identify predictors of high-frequency MSI (MSI-H). The created prediction model was validated in an external cohort (n = 992). The frequency of MSI-H was 5.5% among CRC patients aged ≥ 50 years. The following five predictors of MSI-H were identified in the test cohort: female (1 point), mucinous component (2 points), tumor size ≥ 60 mm (2 points), location in proximal colon (3 points), and BRAF mutation (6 points). The area under curve (AUC) in the receiver-operating characteristic (ROC) analysis of this prediction model was 0.832 (95% confidence interval: 0.790-0.874). The sensitivity and specificity were 74.4% and 77.7%, respectively, for a cut-off score of 4 points. The receiver-operating characteristic curve of the validation cohort also showed an AUC of 0.856 (95% CI: 0.806-0.905). This prediction model is useful to select elderly CRC patients who should undergo MSI testing, and who may benefit from treatment with 5-FU-based adjuvant chemotherapy and cancer immunotherapy.

Routine genetic testing of lung cancer specimens derived from surgery, bronchoscopy and fluid aspiration by next generation sequencing.

After the development of EGFR tyrosine kinase inhibitors (TKIs), genetic testing of EGFR became required for effective treatment of lung cancer. Initially, the testing was conducted separately for each mutated region. However, many EGFR mutations have since been identified that determine the efficacy of EGFR-TKIs. Therefore, genetic testing of EGFR by next generation sequencing (NGS) may be a suitable strategy for lung cancer. Here we examined the applicability of the NGS method in regard to sensitivity, time and cost. A total of 939 specimens were obtained from 686 lung cancer patients at our hospital. DNA and RNA were simultaneously extracted from specimens derived from surgery, bronchoscopy, and fluid aspiration. Specimens included cerebrospinal fluid, pleural effusion, abdominal fluid, and pericardial effusion. From RNA, target regions (EGFR, KRAS, ALK fusion and RET fusion) were enriched by RT-PCR and sequenced with MiSeq. From DNA, PCR or PCR-RFLP conventional methods were performed. NGS and conventional methods were carried out routinely per week. Among the total 939 specimens, 38 specimens could not be examined with NGS. Among these, 34 specimens were analyzed by conventional testing with simultaneously extracted DNA. The remaining four specimens could not be tested with either method. Compared with the conventional method, the concordance rate of mutations was 99% (892/901), excluding specimens with NGS failure. The time period required from processing of specimens to results was 4 days, and the cost per sample was sufficiently low. In conclusion, the genetic testing with NGS method was useful for lung cancer treatment. The cost, sensitivity and time were able to tolerate routine examinations.

High concordance rate of KRAS/BRAF mutations and MSI-H between primary colorectal cancer and corresponding metastases.

Genetic testing is needed for the treatment of colorectal cancer (CRC), especially molecular-targeted therapy. The effects of anti-EGFR therapy and prognosis are affected by the presence of KRAS mutations. However, whether primary CRC or metastatic tissues are appropriate in the analysis is still unclear. In the present study, we assessed the concordance of KRAS/BRAF mutation status and microsatellite instability (MSI) in primary CRC and corresponding metastases. This study enrolled 457 patients with surgically resected primary and corresponding metastatic CRC (499 synchronous metastases and 57 metachronous metastases) and seven local recurrences, and KRAS/BRAF mutation and MSI status were analysed for these tumours. The concordance rates of KRAS mutation, BRAF mutation, wild-type, MSI-H and MSS between primary CRC and corresponding metastases were 93.9% (214/228), 100% (30/30), 99.3% (304/306), 87.5% (21/24) and 100% (137/137), respectively. These high concordance rates were not different between synchronous and metachronous metastases. In conclusion, a high concordance of KRAS/BRAF mutation status and MSI status was observed between primary CRC and corresponding metastases in this study. Either primary CRC or metastatic tissues can be used for testing KRAS/BRAF mutation status and MSI status.

Metastatic Pattern of Stage IV Colorectal Cancer with High-Frequency Microsatellite Instability as a Prognostic Factor.

BACKGROUND: A recent clinical trial on the immune check-point inhibitor pembrolizumab demonstrated that microsatellite instability (MSI) is a good biomarker for response to this inhibitor. However, clinicopathological features of advanced colorectal cancer (CRC) with high-frequency MSI (MSI-H) are unclear. PATIENTS AND METHODS: A total of 2,439 surgically resected CRC tissues were analyzed for MSI status, and mutational status of V-Ki-Ras2 Kirsten rat sarcoma 2 viral oncogene homolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS) and v-Raf murine sarcoma viral oncogene homolog B (BRAF). Stage IV cases were selected, and clinical and molecular features were evaluated. RESULTS: There was no significant survival difference observed between MSI-H CRC and microsatellite-stable (MSS) CRC in patients with stage IV disease (3.92 vs. 2.50 years; p=0.766). However, hematogenous and lymphogenous metastasis-dominant CRC with MSI-H demonstrated poor prognosis, whereas peritoneal metastasis-dominant CRC with MSI-H demonstrated good prognosis, (1.33 vs. 5.2 years; p=0.006). CONCLUSION: Prognosis of stage IV CRC with MSI-H depended on the metastatic pattern. These findings provide useful information for the adaptation of CRC immunotherapy.

Prognostic value of KRAS and BRAF mutations in curatively resected colorectal cancer.

AIM: To investigate the prognostic role of KRAS and BRAF mutations after adjustment for microsatellite instability (MSI) status in Japanese colorectal cancer (CRC) population. METHODS: We assessed KRAS and BRAF mutations and MSI status in 813 Japanese patients with curatively resected, stage I-III CRC and examined associations of these mutations with disease-free survival (DFS) and overall survival (OS) using uni- and multivariate Cox proportional hazards models. RESULTS: KRAS and BRAF mutations were detected in 312 (38%) of 812 and 40 (5%) of 811 tumors, respectively. KRAS mutations occurred more frequently in females than in males (P=0.02), while the presence of BRAF mutations was significantly associated with the female gender (P=0.006), proximal tumor location (P<0.001), mucinous or poorly differentiated histology (P<0.001), and MSI-high tumors (P<0.001). After adjusting for relevant variables, including MSI status, KRAS mutations were associated with poorer DFS (HR=1.35; 95%CI: 1.03-1.75) and OS (HR=1.46; 95%CI: 1.09-1.97). BRAF mutations were poor prognostic factors for DFS (HR=2.20; 95%CI: 1.19-4.06) and OS (HR=2.30; 95%CI: 1.15-4.71). Neither the BRAF by MSI interaction test nor the KRAS by MSI interaction test yielded statistically significant results for DFS and OS. CONCLUSION: KRAS and BRAF mutations are associated with inferior survival, independent of MSI status, in Japanese patients with curatively resected CRC.

Combined microsatellite instability and BRAF gene status as biomarkers for adjuvant chemotherapy in stage III colorectal cancer.

BACKGROUND: The clinical relevance of combined microsatellite instability (MSI) and BRAF status for adjuvant treatment in stage III colorectal cancer (CRC) remains elusive. METHODS: In 405 patients with curatively resected stage III CRC, the prognostic value of combined MSI and BRAF status was assessed in four groups, as follows: high-levels of microsatellite instability (MSI-H) and BRAF-wild type, MSI-H and BRAF-mutation, microsatellite stable (MSS) and BRAF-wild type, and MSS and BRAF-mutation. RESULTS: Combined MSI and BRAF status provided significant prognostic stratification of disease-free survival (DFS), and was independently associated with worse DFS. The MSI-H and BRAF-wild type group had similar outcomes to stage II CRC patients, despite no benefit from 5-FU monotherapy. Further, patients in the MSS and BRAF-wild type group with stage IIIA CRC had favorable outcomes to 5-FU monotherapy, similar to those with stage II CRC. In contrast, 5-FU monotherapy was insufficient among patients in the MSS and BRAF-wild type group with stage IIIB or IIIC CRC or patients in the MSS and BRAF-mutation group with stage III CRC. CONCLUSIONS: The combination of MSI and BRAF status serves as both a prognostic and predictive marker and may provide much-needed guidance during the planning of therapeutic strategies.

Clinicopathological characteristics and prognostic impact of colorectal cancers with NRAS mutations.

At present, molecular markers of colorectal cancer (CRC), including KRAS, NRAS and BRAF mutations, and the microsatellite status are evaluated for the development of personalized treatments. However, clinicopathological and molecular characteristics and the prognostic role of NRAS mutations remain unclear. In the present study, a total of 1,304 consecutive stage 0-IV CRC tumor samples were analyzed for KRAS (exon 2, 3 and 4), NRAS (exon 2 and 3) and BRAF (exon 15) mutations. Multivariate analysis was performed to assess the prognostic impact of NRAS mutations. KRAS, NRAS and BRAF mutations were identified in 553 (42.4%), 35 (2.7%), and 59 (4.5%) of 1,304 CRC cases, respectively. Tumors with NRAS mutations were more frequently located in the distal colorectum compared with those with KRAS or BRAF mutations. Multivariate analysis indicated that KRAS and BRAF mutations were found to be associated with poor prognosis [hazard ratio (HR)=1.44, 95% confidence interval (CI), 1.18-1.76 and HR=2.09; 95% CI, 1.33-3.28, respectively], whereas NRAS mutations were associated with a trend toward favorable prognosis (HR=0.53; 95% CI, 0.27-1.03). Characteristics and prognosis of CRC with NRAS mutations are different from those with KRAS or BRAF mutations.

Molecular and clinical characteristics of MSH6 germline variants detected in colorectal cancer patients.

The MSH6 gene is one of the mismatch repair genes involved in Lynch syndrome and its mutations account for 10-20% of Lynch syndrome. Although previous studies suggested that the difference of the geographical region affects the clinical phenotype of Lynch syndrome, there has been no report on the detailed features of Japanese Lynch syndrome patients carrying an MSH6 mutation. The aim of the present study was to investigate the clinical and molecular features of MSH6 mutation carriers in Japan. Surgically resected 1720 colorectal carcinoma specimens were screened by microsatellite instability (MSI) testing and the MSI-high cases were subjected to a germline mutation analysis of the mismatch repair genes MLH1, MSH2 and MSH6. We investigated the clinical and molecular features of the MSH6 variants, such as the family cancer history, pathological findings, immunohistochemistry, methylation status of the MLH1 promoter and BRAF mutation in the colorectal tumor. Furthermore, the impact of the missense variants on MSH6 protein was predicted by using in silico tools. We identified nine novel pathogenic mutations and eight unclassified missense variants. Among the eight missense variants, three were suspected pathogenic by in silico analysis. We also found that most colorectal cancers in the MSH6 mutation carrier were diagnosed after the age of 50 and were localized distally. Furthermore, the mean age at diagnosis of endometrial cancer in Japanese MSH6 mutation carriers (49.2 years) was earlier than previous reports from Western countries (56.5 years). These results may improve the surveillance program for Japanese MSH6 mutation carriers.

The effects of sense of coherence, demands of illness, and social support on quality of life after surgery in patients with gastrointestinal tract cancer.

PURPOSE/OBJECTIVES: To examine the relationship between quality of life (QOL) as an index of adaptation status and concepts related to self-care skills of patients who have been diagnosed with and undergone surgery for digestive system cancer: sense of coherence (SOC), social support, demands of illness, and the thought "Why me?" DESIGN: Cross-sectional survey. SETTING: General hospitals in Japan. SAMPLE: 60 patients who had been newly diagnosed with digestive system cancer and had undergone surgery. METHODS: Questionnaires were distributed to participants whose discharge date had been determined. The questionnaires were returned through the mail within two weeks of the discharge date. MAIN RESEARCH VARIABLES: QOL, SOC, social support, demands of illness, and the thought "Why me?" FINDINGS: QOL was strongly correlated with SOC and the demands of illness and was moderately correlated with social support. The only variable that was negatively correlated with SOC was the question, "Why me?" SOC and demands of illness accounted for 54% of the variance in QOL; social support was not a significant factor. CONCLUSIONS: This study suggests that SOC is positively correlated with QOL and the demands of illness are negatively correlated with QOL among study participants. IMPLICATIONS FOR NURSING: Nursing interventions focusing on SOC and illness demands may have a significant effect on QOL of patients following cancer surgery.

Experiences of Japanese patients with colorectal cancer during the first six months after surgery.

PURPOSE/OBJECTIVES: To investigate the dynamic experience of patients who recently experienced the stressful situation of being diagnosed with colorectal cancer and undergoing surgery. RESEARCH APPROACH: A qualitative and inductive inquiry. SETTING: General hospitals in Japan. PARTICIPANTS: 12 patients who had undergone colorectal cancer surgery and had been discharged in the previous six months. METHODOLOGIC APPROACH: To inductively elicit the findings from patients' real experiences, interviews and qualitative analysis were used. MAIN RESEARCH VARIABLES: Experience of patients with colorectal cancer. FINDINGS: Three domains that symbolized experiences of patients with colorectal cancer during the first six months after surgery emerged: types of burdens, steps in accepting the cancer diagnosis, and way of living a normal life. Burdens included vulnerability, lack of control, asking for assistance or support, and a "why has this happened to me?" attitude. Way of living a normal life included resisting vulnerability, constructing a daily living routine, and asking for help. CONCLUSIONS: Although patients faced many and varied burdens, they were able to take several actions to gradually accept their cancer diagnoses and resume normal lives. INTERPRETATION: Some of the patients were sincere and skillful in asking for help to live normal lives, but others found that difficult. The present findings are significant in helping nurses provide interventions to enable patients to make use of assistance or support.

Needs of ambulatory patients with cancer who visited outpatient units in Japanese hospitals.

PURPOSE/OBJECTIVES: To document the domains and properties of the self-reported needs of ambulatory patients with cancer. DESIGN: Descriptive. SETTING: Outpatient units in three general hospitals in Japan. SAMPLE: 139 ambulatory patients with cancer. METHODS: The data were collected using questionnaires. Five theoretical groups, which were composed of 30 items, were extracted empirically as domains. Alpha coefficients for each domain ranged from 0.70-0.89. Relationships between each domain and other variables and among the domains themselves were examined. MAIN RESEARCH VARIABLES: Expressed needs of ambulatory patients with cancer, their backgrounds, medical and treatment characteristics, and physical functioning. FINDINGS: All domains for patient needs, except for healthcare needs, were negatively correlated with the level of their physical function. Emotional, physical, and functional needs were positively correlated with the frequency of visiting an outpatient unit. Compared with other needs, adaptation needs were greater for patients who were employed or within three months of discharge. Among patients with one of three cancer sites (i.e., breast, stomach, and colorectal cancers), the needs for individualized care were the lowest for patients with colorectal cancer and highest for patients with breast cancer. CONCLUSIONS: From the needs that ambulatory patients with cancer expressed, five domains were derived. Those domains had relationships with other variables. IMPLICATIONS FOR NURSING: The findings shed light on a segment of ambulatory cancer nursing and may be useful when developing and testing programs needed in the future.