DNA damage response (DDR) pathway is the coordinated cellular network dealing with the identification, signaling, and repair of DNA damage. It tightly regulates cell cycle progression and promotes DNA repair to minimize DNA damage to daughter cells. Key proteins involved in DDR are frequently mutated/inactivated in human cancers and promote genomic instability, a recognized hallmark of cancer. Besides being an intrinsic property of tumors, DDR also represents a unique therapeutic opportunity. Indeed, inhibition of DDR is expected to delay repair, causing persistent unrepaired breaks, to interfere with cell cycle progression, and to sensitize cancer cells to several DNA-damaging agents, such as radiotherapy and chemotherapy. In addition, DDR defects in cancer cells have been shown to render these cells more dependent on the remaining pathways, which could be targeted very specifically (synthetic lethal approach). Research over the past two decades has led to the synthesis and testing of hundreds of small inhibitors against key DDR proteins, some of which have shown antitumor activity in human cancers. In parallel, the search for synthetic lethality interaction is broadening the use of DDR inhibitors. In this review, we discuss the state-of-art of ataxia-telangiectasia mutated, ataxia-telangiectasia-and-Rad3-related protein, checkpoint kinase 1, Wee1 and Polθ inhibitors, highlighting the results obtained in the ongoing clinical trials both in monotherapy and in combination with chemotherapy and radiotherapy.

Mismatch repair (MMR) immunohistochemical (IHC) evaluation has entered pathology routine practice as the first-line screening method to identify patients with MMR deficient (MMRd)/microsatellite instability (MSI) colorectal cancer (CRC), and its misdiagnosis may significantly impact the personalization of CRC patient care. To determine the prevalence of MMR protein intratumor heterogeneity in real-world practice, we collected a series of 8282 CRCs tested for MMR proteins in the setting of Lynch syndrome universal screening. Four heterogenous cases were also investigated for tumor infiltrating lymphocytes count, MSI status, and consensus molecular subtypes by Nanostring nCounter® Platform. Overall, 1056 (12.8%) CRCs showed a MMR altered status, with 46 cases showing a heterogeneous MMR profile (0.56% of the total, and 4.36% of all MMRd cases). To conclude, the authors make some critical remarks regarding the approach to MMR heterogeneity in clinical practice and routine diagnostics.

The immunohistochemical assessment of mismatch repair (MMR) proteins represents a pivotal screening tool for identifying Lynch syndrome (LS)-related cancers, as the loss of their expression often indicates MMR dysfunction associated with genetic or epigenetic alterations. Frequently, LS-related colorectal cancers present germline pathogenic variants in the MLH1 or MSH2 genes, which result in the simultaneous immunohistochemical loss of MLH1 and PMS2 or MSH2 and MSH6 proteins expression, respectively. Less commonly observed is the single involvement of the MSH6 or PMS2 proteins expression, indicative of the presence of germline pathogenic variants in the corresponding genes. Extremely rarely reported are the null immunohistochemistry phenotypes represented by the complete loss of expression of all MMR proteins. The molecular mechanisms contributing to the raising of this latter uncommon immunohistochemical phenotype are derived from the combination of pathogenic germline variants in MMR genes with the somatic hypermethylation of the MLH1 gene promoter. This study focuses on elucidating the molecular cascade leading to the development of the null immunohistochemical phenotype, providing valuable insights into understanding the sequential molecular events driving the LS-associated tumorigenesis, which may have pivotal implications in the clinical management of patients with LS-related cancers.

Mismatch repair (MMR) protein expression in colorectal cancer (CRC) cells is usually homogeneously retained or lost. Rare lesions may show a heterogeneous pattern of MMR protein expression. We evaluated MMR protein expression (MLH1, MSH2, MSH6, and PMS2) in 200 CRCs, identifying 3 groups with proficient MMR protein expression (MMRp), deficient MMR protein expression (MMRd), and heterogeneous MMR protein expression (MMRh). MMRh tumors were microdissected on the basis of the expression of the heterogeneous marker. DNA was extracted and subjected to targeted sequencing. RNA was purified from bulk tumors of all MMRh cases and in a control series of 15 MMRp and 10 MMRd CRCs and analyzed using the PanCancer IO 360 Panel (NanoString Technologies). Twenty-nine of the 200 cases (14.5%) were MMRd. Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, thus featuring areas with double loss at immunohistochemistry (IHC) testing (MMRh double-loss cases). Four of the 6 MMRh double-loss cases were suitable for a separate sequence variant analysis of IHC double-negative and IHC single-negative components of the tumor. In all lesions, both components exhibited a high tumor mutation burden (TMB). Nevertheless, a significant increase in TMB in the double-negative components was observed (mean TMB: negative, 70 mut/Mb vs positive, 59 mut/Mb) because of a higher number of subclonal variants compared with the other component. Comparative gene expression analyses among MMRd, MMRp, and MMRh CRCs highlighted differential gene expression patterns and an increased number of tumor-infiltrating lymphocytes in MMRh lesions, which is also characterized by a substantial population of exhausted CD8⁺ lymphocytes. We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins in a background of concomitant loss of one of the other markers.

Immunohistochemistry (IHC) and/or MSI-PCR (microsatellite instability-polymerase chain reaction) tests are performed routinely to detect mismatch repair deficiency (MMR-D). Classical MMR-D tumors present a loss of MLH1/PMS2 or MSH2/MSH6 with MSI-High. Other profiles of MMR-D tumors have been described but have been rarely studied. In this study, we established a classification of unusual MMR-D tumors and determined their frequency and clinical impact. All MMR-D tumors identified between 2007 and 2017 were selected. Any profile besides the classical MMR-D phenotype was defined as unusual. For patients with unusual MMR-D tumors, IHC, and PCR data were reviewed, the tumor mutation burden (TMB) was evaluated and clinical and genetic features were collected. Of the 4948 cases of MMR testing, 3800 had both the available IHC and MSI-PCR results and 585 of these had MMR-D. After reviewing the IHC and PCR, 21% of the cases initially identified as unusual MMR-D were reclassified, which resulted in a final identification of 89 unusual MMR-D tumors (15%). Unusual MMR-D tumors were more often associated with non-CRC than classical MMR-D tumors. Unusual MMR-D tumors were classified into four sub-groups: i) isolated loss of PMS2 or MSH6, ii) classical loss of MLH1/PMS2 or MSH2/MSH6 without MSI, iii) four MMR proteins retained with MSI and, iv) complex loss of MMR proteins, with clinical characteristics for each sub-group. TMB-high or -intermediate was shown in 96% of the cancers studied (24/25), which confirmed MMR deficiency. Genetic syndromes were identified in 44.9% (40/89) and 21.4% (106/496) of patients with unusual and classical MMR-D tumors, respectively (P < 0.001). Five patients treated with an immune checkpoint inhibitor (ICI) had a prolonged clinical benefit. Our classification of unusual MMR-D phenotype helps to identify MMR deficiency. Unusual MMR-D phenotype occurs in 15% of MMR-D tumors. A high frequency of genetic syndromes was noted in these patients who could benefit from ICI.

In mismatch repair (MMR) immunohistochemistry, four MMR proteins' staining pattern reveals which particular gene may be defective. However, in the null phenotype, four MMR proteins are lost; consequently, it will be challenging to assume the target gene by immunohistochemistry and to determine whether deficient MMR was sporadic or germline.

A 70-year-old man underwent right hemicolectomy with the diagnosis of ascending colon cancer. The postoperative histopathology revealed the diagnosis of medullary carcinoma and the loss of all four MMR expressions in immunohistochemistry. The mutation analysis using a peripheral blood sample showed no germline mutations in the four genes.

This clinical case presents an unusual colon carcinoma that showed a MMR protein immunohistochemistry null phenotype. The cause of expression loss of MMR proteins can be explained by the loss of MLH1 and MSH2 functions associated with somatic loss of function mutations, functional loss in all four MMR proteins associated with somatic loss of function mutations, or Lynch-like syndrome. Correct interpretation and accumulation of relevant cases are necessary to unveil unusual cases in the era of universal screening.

Screening all patients newly diagnosed with colorectal cancer (CRC) for possible Lynch syndrome (LS) has been recommended in the United Kingdom since the National Institute for Health and Care Excellence (NICE) released new diagnostics guidance in February 2017. We sought to validate the NICE screening pathway through a prospective regional programme throughout a 5.2-million population during a 2-year period.

Pathology departments at 14 hospital trusts in the Yorkshire and Humber region of the United Kingdom were invited to refer material from patients with newly diagnosed CRC aged 50 years or over between 1 April 2017 and 31 March 2019 for LS screening. Testing consisted of immunohistochemistry for MLH1, PMS2, MSH2 and MSH6 followed by BRAF mutation analysis ± MLH1 promoter methylation testing in cases showing MLH1 loss. A total of 3141 individual specimens were submitted for testing from 12 departments consisting of 3061 unique tumours and 2791 prospectively acquired patients with CRC. Defective mismatch repair (dMMR) was observed in 15% of cases. In cases showing MLH1 loss, 76% contained a detectable BRAF mutation and, of the remainder, 77% showed MLH1 promoter hypermethylation. Of the patients included in the final analysis, 81 (2.9%) had an indication for germline testing.

LS screening using the NICE diagnostics guidance pathway is deliverable at scale identifying significant numbers of patients with dMMR. This information is used to refer patients to regional clinical genetics services in addition to informing treatment pathways including the use of adjuvant/neoadjuvant chemotherapy and immunotherapy.

Approximately 15% of colorectal cancer (CRC) cases present with high levels of microsatellite instability (MSI-H). Bulk RNA-sequencing approaches have been employed to elucidate transcriptional differences between MSI-H and microsatellite stable (MSS) CRC tumors. These approaches are frequently confounded by the complex cellular heterogeneity of tumors. We performed single-cell deconvolution of bulk RNA-sequencing on The Cancer Genome Atlas colon adenocarcinoma (TCGA-COAD) dataset. Cell composition within each dataset was estimated using CIBERSORTx. Cell composition differences were analyzed using linear regression. Significant differences in abundance were observed for 13 of 19 cell types between MSI-H and MSS/MSI-L tumors in TCGA-COAD. This included a novel finding of increased enteroendocrine (q = 3.71E-06) and reduced colonocyte populations (q = 2.21E-03) in MSI-H versus MSS/MSI-L tumors. We were able to validate some of these differences in an independent biopsy dataset. By incorporating cell composition into our regression model, we identified 3,193 differentially expressed genes (q = 0.05), of which 556 were deemed novel. We subsequently validated many of these genes in an independent dataset of colon cancer cell lines. In summary, we show that some of the challenges associated with cellular heterogeneity can be overcome using single-cell deconvolution, and through our analysis we highlight several novel gene targets for further investigation.

Tumor mutation burden (TMB) is the total exonic mutation count per megabase of tumor DNA. Recent advances in precision medicine occasionally detect Lynch syndrome (LS) by germline sequencing for mismatch-repair (g.MMR) genes but not using TMB. The current study analyzes the utility of TMB in detecting LS.

Whole-exome sequencing (ion-semiconductor sequencing) was performed for somatic and germline DNA from 2,501 various cancer patients to detect TMB and g.MMR sequencing. MMR IHC was conducted when high TMB (≥10) was detected in LS-related cancers with an additional condition of wild-type BRAF in colorectal cancers. Target sequencing and multiplex ligation-dependent probe amplification (MLPA) were further performed for g.MMR genes in MMR-deficient cancers (TMB-based g.MMR target sequencing). We compared universal sequencing and TMB-based target sequencing in their sensitivity for detecting LS.

LS was detected in 16 (0.6%) of the 2,501 patients: 1.1% (9/826) of colorectal cancer patients, 16.2% (6/37) of endometrial cancer patients, and 14.3% (1/7) of small intestine cancer patients. TMB-based g.MMR target sequencing (81.3%) showed superior sensitivity for detecting LS than universal g.MMR sequencing (56.3%; P = 0.127) but missed 3 LS patients (1 with a low-TMB cancer, 1 with a BRAF-mutant colorectal cancer, and 1 with an MMR-proficient cancer). Ion-semiconductor sequencing could detect single-nucleotide substitutions but not large deletions. POL-mutated cancers showed extremely high TMBs (48.4-749.2).

g.MMR target sequencing, combined with TMB, somatic BRAF mutation, and MMR IHC is an effective strategy for detecting LS.

TMB can be a biomarker for detecting LS in precision medicine.

Tobacco smoking is a common risk factor for lung cancer and head and neck cancer. Molecular changes such as deregulation of miRNA expression have been linked to tobacco smoking in both types of cancer. Dysfunction of the Mismatch DNA repair (MMR) mechanism has also been associated with a poor prognosis of these cancers, while a cross-talk between specific miRNAs and MMR genes has been previously proposed. We hypothesized that exposure of lung and head and neck squamous cancer cells (NCI and FaDu, respectively) to tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is capable of altering the expression of MSH2 and MLH1, key MMR components, by promoting specific miRNA deregulation. We found that either a low (1 μM) or high (2 μM) dose of NNK induced significant upregulation of "oncomirs" miR-21 and miR-155 and downregulation of "tumor suppressor" miR-422a, as well as the reduction of MMR protein and mRNA expression, in NCI and FaDu, compared to controls. Inhibition of miR-21 restored the NNK-induced reduced MSH2 phenotype in both NCI and FaDu, indicating that miR-21 might contribute to MSH2 regulation. Finally, NNK exposure increased NCI and FaDu survival, promoting cancer cell progression. We provide novel findings that deregulated miR-21, miR-155, and miR-422a and MMR gene expression patterns may be valuable biomarkers for lung and head and neck squamous cell cancer progression in smokers.

Colorectal cancer (CRC) is associated with significant morbidity and mortality in the US and worldwide. CRC is the second most common cancer-related death in both men and women globally. Chronic inflammation has been identified as one of the major risk factors of CRC. It may drive genetic and epigenetic/epigenomic alterations, such as DNA methylation, histone modification, and non-coding RNA regulation. Current prevention modalities for CRC are limited and some treatment regimens such as use the nonsteroidal anti-inflammatory drug aspirin may have severe side effects, namely gastrointestinal ulceration and bleeding. Therefore, there is an urgent need of developing alternative strategies. Recently, increasing evidence suggests that several dietary cancer chemopreventive phytochemicals possess anti-inflammation and antioxidative stress activities, and may prevent cancers including CRC. Curcumin (CUR) is the yellow pigment that is found in the rhizomes of turmeric (Curcuma longa). Many studies have demonstrated that CUR exhibit strong anticancer, antioxidative stress, and anti-inflammatory activities by regulating signaling pathways, such as nuclear factor erythroid-2-related factor 2, nuclear factor-κB, and epigenetics/epigenomics pathways of histones modifications, and DNA methylation. In this review, we will discuss the latest evidence in epigenetics/epigenomics alterations by CUR in CRC and their potential contribution in the prevention of CRC.