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BackgroundVaginal brachytherapy is currently recommended as adjuvant treatment in patients with high-intermediate risk endometrial cancer to maximize local control and has only mild side effects and no or limited impact on quality of life. However, there is still considerable overtreatment and also some undertreatment, which may be reduced by tailoring adjuvant treatment to the patients’ risk of recurrence based on molecular tumor characteristics.Primary objectivesTo compare the rates of vaginal recurrence in women with high-intermediate risk endometrial cancer, treated after surgery with molecular-integrated risk profile-based recommendations for either observation, vaginal brachytherapy or external pelvic beam radiotherapy or with standard adjuvant vaginal brachytherapyStudy hypothesisAdjuvant treatment based on a molecular-integrated risk profile provides similar local control and recurrence-free survival as current standard adjuvant brachytherapy in patients with high-intermediate risk endometrial cancer, while sparing many patients the morbidity of adjuvant treatment and reducing healthcare costs.Trial designA multicenter, international phase III randomized trial (2:1) of molecular-integrated risk profile-based adjuvant treatment (experimental arm) or adjuvant vaginal brachytherapy (standard arm).Major inclusion/exclusion criteriaWomen aged 18 years and over with a histological diagnosis of high-intermediate risk endometrioid endometrial cancer after total abdominal or laparoscopic hysterectomy and bilateral salpingo-oophorectomy. High-intermediate risk factors are defined as: (i) International Federation of Gynecology and Obstetrics stage IA (with invasion) and grade 3; (ii) stage IB grade 1 or 2 with age ≥60 and/or lymph-vascular space invasion; (iii) stage IB, grade 3 without lymph-vascular space invasion; or (iv) stage II (microscopic and grade 1).EndpointsThe primary endpoint is vaginal recurrence. Secondary endpoints are recurrence-free and overall survival; pelvic and distant recurrence; 5-year vaginal control (including treatment for relapse); adverse events and patient-reported symptoms and quality of life; and endometrial cancer-related healthcare costs.Sample size500 eligible and evaluable patients.Estimated dates for completing accrual and presenting resultsEstimated date for completing accrual will be late 2021. Estimated date for presentation of (first) results is expected in 2023.Trial registrationThe trial is registered at clinicaltrials.gov (NCT03469674) and ISRCTN (11659025).

Myotonic dystrophy type 1 (DM1) is associated with one of the most highly unstable CTG*CAG repeat expansions. The formation of further repeat expansions in transgenic mice carrying expanded CTG*CAG tracts requires the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSbeta complex. It has been proposed that binding of MutSbeta to CAG hairpins blocks its ATPase activity compromising hairpin repair, thereby causing expansions. This would suggest that binding, but not ATP hydrolysis, by MutSbeta is critical for trinucleotide expansions. However, it is unknown if the MSH2 ATPase activity is dispensible for instability. To get insight into the mechanism by which MSH2 generates trinucleotide expansions, we crossed DM1 transgenic mice carrying a highly unstable >(CTG)(300) repeat tract with mice carrying the G674A mutation in the MSH2 ATPase domain. This mutation impairs MSH2 ATPase activity and ablates base-base MMR, but does not affect the ability of MSH2 (associated with MSH6) to bind DNA mismatches. We found that the ATPase domain mutation of MSH2 strongly affects the formation of CTG expansions and leads instead to transmitted contractions, similar to a Msh2-null or Msh3-null deficiency. While a decrease in MSH2 protein level was observed in tissues from Msh2(G674) mice, the dramatic reduction of expansions suggests that the expansion-biased trinucleotide repeat instability requires a functional MSH2 ATPase domain and probably a functional MMR system.

Eukaryotic MutSβ is a heterodimer composed of Msh2 and Msh3 that recognizes insertion-deletion loops (IDLs) and 3′ overhangs during mismatch repair. Now crystal structures of MutSβ in complex with DNA, containing IDLs of varying lengths, reveal that this complex interacts with its substrate differently than MutSα and bacterial MutS do. DNA mismatch repair corrects replication errors, thus reducing mutation rates and microsatellite instability. Genetic defects in this pathway cause Lynch syndrome and various cancers in humans. Binding of a mispaired or unpaired base by bacterial MutS and eukaryotic MutSα is well characterized. We report here crystal structures of human MutSβ in complex with DNA containing insertion-deletion loops (IDL) of two, three, four or six unpaired nucleotides. In contrast to eukaryotic MutSα and bacterial MutS, which bind the base of a mismatched nucleotide, MutSβ binds three phosphates in an IDL. DNA is severely bent at the IDL; unpaired bases are flipped out into the major groove and partially exposed to solvent. A normal downstream base pair can become unpaired; a single unpaired base can thereby be converted to an IDL of two nucleotides and recognized by MutSβ. The C-terminal dimerization domains form an integral part of the MutS structure and coordinate asymmetrical ATP hydrolysis by Msh2 and Msh3 with mismatch binding to signal for repair.

ObjectiveAlthough early-detected cervical cancer is associated with good survival, the prognosis for late-stage disease is poor and treatment options are sparse. Mismatch repair deficiency (MMR-D) has surfaced as a predictor of prognosis and response to immune checkpoint inhibitor(s) in several cancer types, but its value in cervical cancer remains unclear. This study aimed to define the prevalence of MMR-D in cervical cancer and assess the prognostic value of MMR protein expression.MethodsExpression of the MMR proteins MLH-1, PMS-2, MSH-2, and MSH-6 was investigated by immunohistochemical staining in a prospectively collected cervical cancer cohort (n=508) with corresponding clinicopathological and follow-up data. Sections were scored as either loss or intact expression to define MMR-D, and by a staining index, based on staining intensity and area, evaluating the prognostic potential. RNA and whole exome sequencing data were available for 72 and 75 of the patients and were used for gene set enrichment and mutational analyses, respectively.ResultsFive (1%) tumors were MMR-deficient, three of which were of neuroendocrine histology. MMR status did not predict survival (HR 1.93, p=0.17). MSH-2 low (n=48) was associated with poor survival (HR 1.94, p=0.02), also when adjusting for tumor stage, tumor type, and patient age (HR 2.06, p=0.013). MSH-2 low tumors had higher tumor mutational burden (p=0.003) and higher frequency of (frameshift) mutations in the double-strand break repair gene RAD50 (p<0.01).ConclusionMMR-D is rare in cervical cancer, yet low MSH-2 expression is an independent predictor of poor survival.

Although DNA double-strand break (DSB) repair is mediated by numerous proteins accumulated at DSB sites, how DNA repair proteins are assembled into damaged chromatin has not been fully elucidated. Here we show that a member of the tripartite motif protein family, TRIM29, is a histone-binding protein responsible for DNA damage response (DDR). We found that TRIM29 interacts with BRCA1-associated surveillance complex, cohesion, DNA-PKcs and components of TIP60 complex. The dynamics of the TRIM29-containing complex on H2AX nucleosomes is coordinated by a cross-talk between histone modifications. TRIM29 binds to modified histone H3 and H4 tails in the context of nucleosomes. Furthermore, chromatin binding of TRIM29 is required for the phosphorylation of H2AX and cell viability in response to ionizing radiation. Our results suggest that TRIM29 functions as a scaffold protein to assemble DNA repair proteins into chromatin followed by efficient activation of DDR. Many proteins are assembled into damaged chromatins to repair DNA double-strand breaks (DSBs). Here, the authors show that TRIM29 binds to modified histone H3 and H4 tails following DSB formation and functions as a scaffold protein to assemble DNA repair proteins into chromatin.

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

Although early-detected cervical cancer is associated with good survival, the prognosis for late-stage disease is poor and treatment options are sparse. Mismatch repair deficiency (MMR-D) has surfaced as a predictor of prognosis and response to immune checkpoint inhibitor(s) in several cancer types, but its value in cervical cancer remains unclear. This study aimed to define the prevalence of MMR-D in cervical cancer and assess the prognostic value of MMR protein expression. Expression of the MMR proteins MLH-1, PMS-2, MSH-2, and MSH-6 was investigated by immunohistochemical staining in a prospectively collected cervical cancer cohort (n=508) with corresponding clinicopathological and follow-up data. Sections were scored as either loss or intact expression to define MMR-D, and by a staining index, based on staining intensity and area, evaluating the prognostic potential. RNA and whole exome sequencing data were available for 72 and 75 of the patients and were used for gene set enrichment and mutational analyses, respectively. Five (1%) tumors were MMR-deficient, three of which were of neuroendocrine histology. MMR status did not predict survival (HR 1.93, p=0.17). MSH-2 low (n=48) was associated with poor survival (HR 1.94, p=0.02), also when adjusting for tumor stage, tumor type, and patient age (HR 2.06, p=0.013). MSH-2 low tumors had higher tumor mutational burden (p=0.003) and higher frequency of (frameshift) mutations in the double-strand break repair gene RAD50 (p<0.01). MMR-D is rare in cervical cancer, yet low MSH-2 expression is an independent predictor of poor survival.

Trinucleotide repeat expansion disorders (TRED) are caused by genomic expansions of trinucleotide repeats, such as CTG and CAG. These expanded repeats are unstable in germline and somatic cells, with potential consequences for disease severity. Previous studies have demonstrated the involvement of DNA repair proteins in repeat instability, although the key factors affecting large repeat expansion and contraction are unclear. Here we investigated these factors in a human cell model harboring 800 CTG•CAG repeats by individually knocking down various DNA repair proteins using short interfering RNA. Knockdown of MSH2 and MSH3, which form the MutSβ heterodimer and function in mismatch repair, suppressed large repeat expansions, whereas knockdown of MSH6, which forms the MutSα heterodimer with MSH2, promoted large expansions exceeding 200 repeats by compensatory increases in MSH3 and the MutSβ complex. Knockdown of topoisomerase 1 (TOP1) and TDP1, which are involved in single-strand break repair, enhanced large repeat contractions. Furthermore, knockdown of senataxin, an RNA/DNA helicase which affects DNA:RNA hybrid formation and transcription-coupled nucleotide excision repair, exacerbated repeat instability in both directions. These results indicate that DNA repair factors, such as MutSβ play important roles in large repeat expansion and contraction and can be an excellent therapeutic target for TRED.

Microsatellite instability, a well-established driver pathway in colorectal carcinogenesis, can develop in both sporadic and hereditary conditions via different molecular alterations in the DNA mismatch repair (MMR) genes. MMR protein immunohistochemistry (IHC) is currently widely used for the detection of MMR deficiency in solid tumors. The IHC test, however, can show varied staining patterns, posing challenges in the interpretation of the staining results in some cases. Here we report a case of an 80-year-old female with a colonic adenocarcinoma that exhibited an unusual “null” IHC staining pattern with complete loss of all four MMR proteins (MLH1, MSH2, MSH6, and PMS2). This led to subsequent MLH1 methylation testing and next generation sequencing which demonstrated that the loss of all MMR proteins was associated with concurrent promoter hypermethylation of MLH1 and double somatic truncating mutations in MSH2. These molecular findings, in conjunction with the patient’s age being 80 years and the fact that the patient had no personal or family cancer history, indicated that the MMR deficiency was highly likely sporadic in nature. Thus, the stringent Lynch syndrome type surveillance programs were not recommended to the patient and her family members. This case illustrates a rare but important scenario where a null IHC phenotype signifies complex underlying molecular alternations that bear clinical management implications, highlighting the need for recognition and awareness of such unusual IHC staining patterns.

Background: This study aimed to explore the effects of microRNA-21-5p (miR-21-5p) on the radiation sensitivity of non-small cell lung cancer (NSCLC) and the involvement of human MutS homolog 2 (hMSH2) One hundred fourteen NSCLC patients at stage II or III who received surgery and postoperative radiotherapy were enrolled in this study. Methods: The patients were assigned into radiation-sensitive and -insensitive groups. NSCLC A549 cells were transfected to generate control, Negative control (NC), miR-21-5p inhibitor, miR-21-5p mimic, small interfering hMSH2 (sihMSH2), miR-21-5p inhibitor + sihMSH2 and hMSH2 overexpression groups. Immunohistochemistry was performed to detect the hMSH2 expression in transfected and irradiated cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to evaluate A549 miR-21-5p and hMSH2 expression in transfected and irradiated cells. A colony formation assay was adopted for cell survival analysis. The relationship between miR-21-5p and hMSH2 was verified by a luciferase reporter assay. Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and apoptosis was assessed by flow cytometry. NSCLC nude mouse models were established, and tumor volumes and tumor weights were recorded. Results: The radiation-sensitive group of patients exhibited lower miR-21-5p but higher hMSH2 expression than the insensitive group. For irradiated A549 cells, lower cell survival, higher apoptosis, increased miR-21-5p expression and decreased hMSH2 expression were observed at 6 and 8 Gy than at 0, 2 and 4 Gy; compared to 6 Gy, cell survival and hMSH2 expression were decreased and apoptosis and miR-21-5p expression were increased at 8 Gy. Additionally, miR-21-5p was found to target hMSH2. Compared with the control group, the cell survival rate was lower and the apoptosis rate higher in the miR-21-5p inhibitor group, whereas the opposite was observed for the miR-21-5p mimic and sihMSH2 groups. For the mouse model, decreased tumor volume and tumor weight and higher hMSH2 expression were found in the miR-21-5p inhibitor, radiation, hMSH2 overexpression, miR-21-5p inhibitor + radiation and hMSH2 overexpression + radiation groups compared with the control group. In addition, tumor volume and tumor weight were decreased and hMSH2 expression increased in the miR-21-5p inhibitor + radiation and hMSH2 overexpression + radiation groups compared with the radiation alone group. Conclusion: These findings indicate that inhibition of miR-21 can promote the radiation sensitivity of NSCLC by targeting hMSH2.