DNA Mismatch Repair Gene Variant Classification: Evaluating the Utility of Somatic Mutations and Mismatch Repair Deficient Colonic Crypts and Endometrial Glands.

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6xMLH1, 9xMSH2, 6xMSH6, 4xPMS2), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an MSH2 exon 1-6 duplication provides further support for an upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care.

Telehealth Delivery of Evidence-Based Intervention Within Older Adult Populations: A Scoping Review.

Telehealth services enable individuals to participate in meaningful occupations; however, the evidence on this topic has not been synthesized in older adult populations. This scoping review synthesized the evidence on interventions delivered through telehealth (and the mode of delivery) in occupational therapy for older adult populations. A search for studies on occupational therapy, older adults, and telehealth was performed in six research databases, and identified 536 articles. Four reviewers independently screened the titles and abstracts; and reviewed eligible full text. Ten articles were extracted into a table and summarized through narrative format. The studies focused on performance-based intervention (60%), cognition (10%), health (10%), occupation (10%), and the environment (10%) in older adult populations (N = 1 - 208); including those with Alzheimer's disease, chronic pain, cancer, and stroke. The interventions were delivered through electronic audio-visual platforms (e.g., zoom) (80%) and teleconference platforms (e.g., phone calls) (20%).

Identifying primary and secondary MLH1 epimutation carriers displaying low-level constitutional MLH1 methylation using droplet digital PCR and genome-wide DNA methylation profiling of colorectal cancers.

BACKGROUND: MLH1 epimutation is characterised by constitutional monoallelic MLH1 promoter hypermethylation, which can cause colorectal cancer (CRC). Tumour molecular profiles of MLH1 epimutation CRCs were used to classify germline MLH1 promoter variants of uncertain significance and MLH1 methylated early-onset CRCs (EOCRCs). Genome-wide DNA methylation and somatic mutational profiles of tumours from two germline MLH1: c.-11C > T and one MLH1: c.-[28A > G; 7C > T] carriers and three MLH1 methylated EOCRCs (< 45 years) were compared with 38 reference CRCs. Methylation-sensitive droplet digital PCR (ddPCR) was used to detect mosaic MLH1 methylation in blood, normal mucosa and buccal DNA. RESULTS: Genome-wide methylation-based Consensus Clustering identified four clusters where the tumour methylation profiles of germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs clustered with the constitutional MLH1 epimutation CRCs but not with the sporadic MLH1 methylated CRCs. Furthermore, monoallelic MLH1 methylation and APC promoter hypermethylation in tumour were observed in both MLH1 epimutation and germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs. Mosaic constitutional MLH1 methylation in MLH1: c.-11C > T carriers and 1 of 3 MLH1 methylated EOCRCs was identified by methylation-sensitive ddPCR. CONCLUSIONS: Mosaic MLH1 epimutation underlies the CRC aetiology in MLH1: c.-11C > T germline carriers and a subset of MLH1 methylated EOCRCs. Tumour profiling and ultra-sensitive ddPCR methylation testing can be used to identify mosaic MLH1 epimutation carriers.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n=135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n=137; 80xCRCs, 33xECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

A metabolomics investigation into the effects of HIV protease inhibitors on HPV16 E6 expressing cervical carcinoma cells.

Recently, it has been reported that anti-viral drugs, such as indinavir and lopinavir (originally targeted for HIV), also inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells. In order to understand more about the mode-of-action(s) of these drugs the metabolome of HPV16 E6 expressing cervical carcinoma cell lines was investigated using mass spectrometry (MS)-based metabolic profiling. The metabolite profiling of C33A parent and E6-transfected cells exposed to these two anti-viral drugs was performed by ultra performance liquid chromatography (UPLC)-MS and gas chromatography (GC)-time of flight (TOF)-MS. Using a combination of univariate and multivariate analyses, these metabolic profiles were investigated for analytical and biological reproducibility and to discover key metabolite differences elicited during anti-viral drug challenge. This approach revealed both distinct and common effects of these two drugs on the metabolome of two different cell lines. Finally, intracellular drug levels were quantified, which suggested in the case of lopinavir that increased activity of membrane transporters may contribute to the drug sensitivity of HPV infected cells.

Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells.

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 µM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.

Identification of a de novo BRCA1 mutation in a woman with early onset bilateral breast cancer.

De novo mutations are rarely reported in BRCA1 and BRCA2. We report a proven BRCA1 de novo mutation in a woman diagnosed with young onset bilateral breast cancer with a limited family history.

Increased colorectal cancer incidence in obligate carriers of heterozygous mutations in MUTYH.

BACKGROUND & AIMS: MUTYH-associated polyposis (MAP) is an autosomal recessive disorder caused by mutations in the MUTYH gene. Patients with MAP are at extremely high risk of colorectal cancer, but the risks of colorectal and other cancers in heterozygous carriers of a single MUTYH mutation are uncertain. We performed a retrospective study of cancer incidence and causes of death among obligate MUTYH heterozygote individuals. METHODS: MAP index cases were identified from polyposis registers in Germany, The Netherlands, and the United Kingdom. Cancer incidence, cancer mortality, and all-cause mortality data were collected from 347 parents of unrelated MAP index cases and the spouses of 3 index cases who were also found to be heterozygous for single MUTYH mutations. These data were compared with appropriate national sex-, age-, and period-specific population data to obtain standardized mortality ratios (SMR) and standardized incidence ratios (SIR). RESULTS: There was a 2-fold increase in the incidence of colorectal cancer among parents of MAP cases, compared with the general population (SIR, 2.12; 95% confidence interval [CI]: 1.30-3.28). Their colorectal cancer mortality was not increased significantly (SMR, 1.02; 95% CI: 0.41-2.10) nor was overall cancer risk (SIR, 0.92; 95% CI: 0.70-1.18), cancer mortality (SMR, 1.12; 95% CI: 0.83-1.48), or overall mortality (SMR, 0.94; 95% CI: 0.80-1.08). CONCLUSIONS: The risk of colorectal cancer in heterozygous carriers of single MUTYH mutations who are relatives of patients with MAP is comparable with that of first-degree relatives of patients with sporadic colorectal cancer. Screening measures should be based on this modest increase in risk.

1H MRS identifies specific metabolite profiles associated with MYCN-amplified and non-amplified tumour subtypes of neuroblastoma cell lines.

Neuroblastoma is the most common extracranial solid malignancy in children. The disease possesses a broad range of clinical phenotypes with widely varying prognoses. Numerous studies have sought to identify the associated genetic abnormalities in the tumour, resulting in the identification of useful prognostic markers. In particular, the presence of multiple copies of the MYCN oncogene (referred to as MYCN amplification) has been found to confer a poor prognosis. However, the molecular pathways involved are as yet poorly defined. Metabolite profiles generated by in vitro (1)H MRS provide a means of investigating the downstream metabolic consequences of genetic alterations and can identify potential targets for new agents. Thirteen neuroblastoma cell lines possessing multiple genetic alterations were investigated; seven were MYCN amplified and six MYCN non-amplified. In vitro magic angle spinning (1)H MRS was performed on cell suspensions, and the spectra analysed to obtain metabolite concentration ratios relative to total choline (tCho). A principal component analysis using these concentration ratios showed that MYCN-amplified and non-amplified cell lines form separate classes according to their metabolite profiles. Phosphocholine/tCho and taurine/tCho were found to be significantly raised (p < 0.05) and glycerophosphocholine/tCho significantly reduced (p < 0.05) in the MYCN-amplified compared with the MYCN non-amplified cell lines (two-tailed t test). (1)H MRS of the SH-EP1 cell line and an isogenic cell line transfected with the MYCN oncogene also showed that MYCN oncogene over-expression causes alterations in phosphocholine, glycerophosphocholine and taurine concentrations. Molecular pathways of choline and taurine metabolism are potential targets for new agents tailored to MYCN-amplified neuroblastoma.