Predictive models for mutations in mismatch repair genes: implication for genetic counseling in developing countries

Background: Lynch syndrome (LS) is the most common form of inherited predisposition to colorectal cancer (CRC), accounting for 2-5% of all CRC. LS is an autosomal dominant disease characterized by mutations in the mismatch repair genes mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), postmeiotic segregation increased 1 (PMS1), post-meiotic segregation increased 2 (PMS2) and mutS homolog 6 (MSH6). Mutation risk prediction models can be incorporated into clinical practice, facilitating the decision-making process and identifying individuals for molecular investigation. This is extremely important in countries with limited economic resources. This study aims to evaluate sensitivity and specificity of five predictive models for germline mutations in repair genes in a sample of individuals with suspected Lynch syndrome. Methods: Blood samples from 88 patients were analyzed through sequencing MLH1, MSH2 and MSH6 genes. The probability of detecting a mutation was calculated using the PREMM, Barnetson, MMRpro, Wijnen and Myriad models. To evaluate the sensitivity and specificity of the models, receiver operating characteristic curves were constructed. Results: Of the 88 patients included in this analysis, 31 mutations were identified: 16 were found in the MSH2 gene, 15 in the MLH1 gene and no pathogenic mutations were identified in the MSH6 gene. It was observed that the AUC for the PREMM (0.846), Barnetson (0.850), MMRpro (0.821) and Wijnen (0.807) models did not present significant statistical difference. The Myriad model presented lower AUC (0.704) than the four other models evaluated. Considering thresholds of >= 5%, the models sensitivity varied between 1 (Myriad) and 0.87 (Wijnen) and specificity ranged from 0 (Myriad) to 0.38 (Barnetson). Conclusions: The Barnetson, PREMM, MMRpro and Wijnen models present similar AUC. The AUC of the Myriad model is statistically inferior to the four other models.

Intratumoral budding as a potential parameter of tumor progression in mismatch repair-proficient and mismatch repair-deficient colorectal cancer patients

In colorectal cancer, tumor budding at the invasive front (peritumoral budding) is an established prognostic parameter and decreased in mismatch repair-deficient tumors. In contrast, the clinical relevance of tumor budding within the tumor center (intratumoral budding) is not yet known. The aim of the study was to determine the correlation of intratumoral budding with peritumoral budding and mismatch repair status and the prognostic impact of intratumoral budding using 2 independent patient cohorts. Following pancytokeratin staining of whole-tissue sections and multiple-punch tissue microarrays, 2 independent cohorts (group 1: n = 289; group 2: n = 222) with known mismatch repair status were investigated for intratumoral budding and peritumoral budding. In group 1, intratumoral budding was strongly correlated to peritumoral budding (r = 0.64; P < .001) and less frequent in mismatch repair-deficient versus mismatch repair-proficient cases (P = .177). Sensitivity and specificity for lymph node positivity were 72.7% and 72.1%. In mismatch repair-proficient cancers, high-grade intratumoral budding was associated with right-sided location (P = .024), advanced T stage (P = .001) and N stage pN (P < .001), vascular invasion (P = .041), infiltrating tumor margin (P = .003), and shorter survival time (P = .014). In mismatch repair-deficient cancers, high intratumoral budding was linked to higher tumor grade (P = .004), vascular invasion (P = .009), infiltrating tumor margin (P = .005), and more unfavorable survival time (P = .09). These associations were confirmed in group 2. High-grade intratumoral budding was a poor prognostic factor in univariate (P < .001) and multivariable analyses (P = .019) adjusting for T stage, N stage distant metastasis, and adjuvant therapy. These preliminary results on 511 patients show that intratumoral budding is an independent prognostic factor, supporting the future investigation of intratumoral budding in larger series of both preoperative and postoperative rectal and colon cancer specimens.

Prognostic impact of β-2-microglobulin expression in colorectal cancers stratified by mismatch repair status

β-2-microglobulin (B2M) is essential for antigen presentation, yet may also possess proto-oncogenic properties.

Prognostic impact of lymph node ratio outperforms positive lymph nodes and lymph nodes harvested: a time-dependent analysis in mismatch repair-proficient and –deficient colrectal cancers

Objective: We compare the prognostic strength of the lymph node ratio (LNR), positive lymph nodes (+LNs) and collected lymph nodes (LNcoll) using a time-dependent analysis in colorectal cancer patients stratified by mismatch repair (MMR) status. Method: 580 stage III-IV patients were included. Multivariable Cox regression analysis and time-dependent receiver operating characteristic (tROC) curve analysis were performed. The Area under the Curve (AUC) over time was compared for the three features. Results were validated on a second cohort of 105 stage III-IV patients. Results: The AUC for the LNR was 0.71 and outperformed + LNs and LNcoll by 10–15 % in both MMR-proficient and deficient cancers. LNR and + LNs were both significant (p<0.0001) in multivariable analysis but the effect was considerably stronger for the LNR [LNR: HR=5.18 (95 % CI: 3.5–7.6); +LNs=1.06 (95 % CI: 1.04–1.08)]. Similar results were obtained for patients with >12 LNcoll. An optimal cut off score for LNR=0.231 was validated on the second cohort (p<0.001). Conclusion: The LNR outperforms the + LNs and LNcoll even in patients with >12 LNcoll. Its clinical value is not confounded by MMR status. A cut-of score of 0.231 may best stratify patients into prognostic subgroups and could be a basis for the future prospective analysis of the LNR.

Loss of Cdx2 Expression in Primary Tumors and Lymph Node Metastases is Specific for Mismatch Repair-Deficiency in Colorectal Cancer

Background: Approximately 20% of all colorectal cancers are hypothesized to arise from the "serrated pathway" characterized by mutation in BRAF, high-level CpG Island Methylator Phenotype, and microsatellite instability/mismatch repair (MMR)-deficiency. MMR-deficient cancers show frequent losses of Cdx2, a homeodomain transcription factor. Here, we determine the predictive value of Cdx2 expression for MMR-deficiency and investigate changes in expression between primary cancers and matched lymph node metastases. Methods: Immunohistochemistry for Cdx2, Mlh1, Msh2, Msh6, and Pms2 was performed on whole tissue sections from 201 patients with primary colorectal cancer and 59 cases of matched lymph node metastases. Receiver operating characteristic curve analysis and Area under the Curve (AUC) were investigated; association of Cdx2 with clinicopathological features and patient survival was carried out. Results: Loss of Cdx2 expression was associated with higher tumor grade (p = 0.0002), advanced pT (p = 0.0166), and perineural invasion (p = 0.0228). Cdx2 loss was an unfavorable prognostic factor in univariate (p = 0.0145) and multivariate [p = 0.0427; HR (95% CI): 0.58 (0.34-0.98)] analysis. The accuracy (AUC) for discriminating MMR-proficient and - deficient cancers was 87% [OR (95% CI): 0.96 (0.95-0.98); p < 0.0001]. Specificity and negative predictive value for MMR-deficiency was 99.1 and 96.3%. One hundred and seventy-four patients had MMR-proficient cancers, of which 60 (34.5%) showed Cdx2 loss. Cdx2 loss in metastases was related to MMR-deficiency (p < 0.0001). There was no difference in expression between primary tumors and matched metastases. Conclusion: Loss of Cdx2 is a sensitive and specific predictor of MMR-deficiency, but is not limited to these tumors, suggesting that events "upstream" of the development of microsatellite instability may impact Cdx2 expression.

Possible role of Cdx2 in the serrated pathway of colorectal cancer characterized by BRAF mutation, high-level CpG Island methylator phenotype and mismatch repair-deficiency

Colorectal cancer is a heterogeneous disease at the histomorphological, clinical and molecular level. Approximately 20% of cases may progress through the "serrated" pathway characterized by BRAF mutation and high-level CpG Island Methylator Phenotype (CIMP). A large subgroup are additionally microsatellite instable (MSI) and demonstrate significant loss of tumor suppressor Cdx2. The aim of this study is to determine the specificity of Cdx2 protein expression and CpG promoter hypermethylation for BRAF(V600E) and high-level CIMP in colorectal cancer. Cdx2, Mlh1, Msh2, Msh6, and Pms2 were analyzed by immunohistochemistry using a multi-punch tissue microarray (TMA; n = 220 patients). KRAS and BRAF(V600E) mutation analysis, CDX2 methylation and CIMP were investigated. Loss of Cdx2 was correlated with larger tumor size (P = 0.0154), right-sided location (P = 0.0014), higher tumor grade (P < 0.0001), more advanced pT (P = 0.0234) and lymphatic invasion (P = 0.0351). Specificity was 100% for mismatch repair (MMR)-deficiency (P < 0.0001), 92.2% (P < 0.0001) for BRAF(V600E) and 91.8% for CIMP-high. Combined analysis of BRAF(V600E) /CIMP identified Cdx2 loss as sensitive (80%) and specific (91.5%) for mutation/high status. These results were validated on eight well-established colorectal cancer cell lines. CDX2 methylation correlated with BRAF(V600E) (P = 0.0184) and with Cdx2 protein loss (P = 0.0028). These results seem to indicate that Cdx2 may play a role in the serrated pathway to colorectal cancer as underlined by strong relationships with BRAF(V600E) , CIMP-high and MMR-deficiency. Whether this protein can only be used as a "surrogate" marker, or is functionally involved in the progression of these tumors remains to be elucidated.

Metabolomics Reveals Aging-associated Attenuation of Noninvasive Radiation Biomarkers in Mice: Potential Role of Polyamine Catabolism and Incoherent DNA Damage-repair

Development of methods for rapid screening and stratification of subjects after exposure is an integral part of countermeasures against radiation. The potential demographic and exposure history-related heterogeneity of exposed populations warrants robust biomarkers that withstand and reflect such differences. In this study, the effect of aging and repeated exposure on the metabolic response to sublethal irradiation was examined in mice using UPLC-ESI-QTOF mass spectrometry. Aging attenuated postexposure elevation in excretions of DNA damage biomarkers as well as N(1)-acetylspermidine. Although N(1)-acetylspermidine and 2'-deoxyuridine elevation was highly correlated in all age groups, xanthine and N(1)-acetylspermidine elevation was poorly correlated in older mice. These results may reflect the established decline in DNA damage-repair efficiency associated with aging and indicate a novel role for polyamine metabolism in the process. Although repeated irradiation at long intervals did not affect the elevation of N(1)-acetylspermidine, 2'-deoxyuridine, and xanthine, it did significantly attenuate the elevation of 2'-deoxycytidine and thymidine compared to a single exposure. However, these biomarkers were found to identify exposed subjects with accuracy ranging from 82% (xanthosine) to 98% (2'-deoxyuridine), irrespective of their age and exposure history. This indicates that metabolic biomarkers can act as robust noninvasive signatures of sublethal radiation exposure.

Mismatch Repair Deficient Tumors Lacking Known Sporadic Causes: Are They All Due to Lynch Syndrome?

BACKGROUND: Mismatch repair deficient (MMRD) colorectal (CRC) or endometrial (EC) cancers in the absence of MLH1 promoter hypermethylation and BRAF mutations are suggestive of Lynch syndrome (LS). Positive germline genetic test results confirm LS. It is unclear if individuals with MMRD tumors but no identified germline mutation or sporadic cause (MMRD+/germline-) have LS. HYPOTHESIS: Since LS is hereditary, individuals with LS should have a stronger family history of LS-related cancers than individuals with sporadic tumors. We hypothesized that MMRD+/germline- CRC and/or EC patients would have less suggestive family histories than LS CRC and/or EC patients. METHODS: 253 individuals with an MMRD CRC or EC who underwent genetic counseling at one institution were included in analysis in 1 of 4 groups: LS, MMRD+/germline-, MMRD+/VUS, sporadic MSI-H (MMRD tumor with MLH1 promoter hypermethylation or BRAF mutation). Family histories were analyzed utilizing MMRpro and PREMM1,2,6. Kruskal-Wallis tests were used to compare family history scores. Logistic regression was used to determine what factors were predictive of LS. RESULTS: MMRD+/germline- individuals had significantly lower median family history scores (PREMM1,2,6=7.3, MMRpro=8.1) than LS individuals (PREMM1,2,6=26.1, MMRpro=89.8, p CONCLUSION: MMRD+/germline- individuals have less suggestive family histories of LS than individuals with LS, but more suggestive family histories than sporadic MSI-H individuals. CRC and/or EC patients with abnormal tumor studies are more likely to have a germline LS mutation if they have a family history suggestive of hereditary cancer. These results imply that the MMRD+/germline- group may not all have LS. This finding highlights the need to determine other somatic, epigenetic or germline causes of MMRD tumors so that these patients and their families can be accurately counseled regarding screening and management.

DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSα/hMutSβ ratio and reduces the efficiency of base–base mismatch repair

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSα (hMSH2⋅hMSH6) and hMutSβ (hMSH2⋅hMSH3). In HL-60 extracts the hMutSα to hMutSβ ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base–base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSα and hMutSβ display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion–deletion heterology, but only hMutSα restores base–base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.

The role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae

In most organisms, the mismatch repair (MMR) system plays an important role in substantially lowering mutation rates and blocking recombination between nonidentical sequences. In Saccharomyces cerevisiae, the products of three genes homologous to Escherichia coli mutS—MSH2, MSH3, and MSH6—function in MMR by recognizing mispaired bases. To determine the effect of MMR on single-base pair mismatches, we have measured reversion rates of specific point mutations in the CYC1 gene in both wild-type and MMR-deficient strains. The reversion rates of all of the point mutations are similar in wild-type cells. However, we find that in the absence of MSH2 or MSH6, but not MSH3, reversion rates of some mutations are increased by up to 60,000-fold, whereas reversion rates of other mutations are essentially unchanged. When cells are grown anaerobically, the reversion rates in MMR-deficient strains are decreased by as much as a factor of 60. We suggest that the high reversion rates observed in these MMR-deficient strains are caused by misincorporations opposite oxidatively damaged bases and that MMR normally prevents these mutations. We further suggest that recognition of mispairs opposite damaged bases may be a more important role for MMR in yeast than correction of errors opposite normal bases.