The DNA damage response (DDR) pathway coordinates the identification, signaling, and repair of DNA damage caused by endogenous or exogenous factors and regulates cell-cycle progression with DNA repair to minimize DNA damage being permanently passed through cell division. Severe DNA damage that cannot be repaired may trigger apoptosis; as such, the DDR pathway is of crucial importance as a cancer target. Poly (ADP-ribose) polymerase (PARP) is the best-known element of the DDR, and several PARP inhibitors have been licensed. However, there are approximately 450 proteins involved in DDR, and a number of these other targets are being investigated in the laboratory and clinic. We review the most recent evidence for the clinical effect of PARP inhibition in breast and ovarian cancer and explore expansion into the first-line setting and into other tumor types. We critique the evidence for patient selection techniques and summarize what is known about mechanisms of PARP inhibitor resistance. We then discuss what is known about the preclinical rationale for targeting other members of the DDR pathway and the associated tumor cell genetics that may confer sensitivity to these agents. Examples include DNA damage sensors (MLH1), damage signaling molecules (ataxia-telangiectasia mutated; ataxia-telangiectasia mutated-related and Rad3-related; CHK1/2; DNA-dependent protein kinase, catalytic subunit; WEE1; CDC7), or effector proteins for repair (POLQ [also referred to as POLθ], RAD51, poly [ADP-ribose] glycohydrolase). Early-phase clinical trials targeting some of these molecules, either as a single agent or in combination, are discussed. Finally, we outline the challenges that must be addressed to maximize the therapeutic opportunity that targeting DDR provides.

Ovarian malignancies occurring in the setting of hereditary nonpolyposis colorectal carcinoma syndrome typically present in young women, often as the first or "sentinel" cancer, but the frequency of microsatellite instability (MSI) and mismatch repair (MMR) defects in ovarian surface epithelial malignancies in women <or=50 years of age is neither well known nor well tested. Fifty-two ovarian surface epithelial carcinomas, including 4 with synchronous endometrial carcinomas, were identified in patients 50 years of age or younger and evaluated for evidence of MSI and MMR protein deficiency. Each case was tested for MSI by multiplex polymerase chain reaction amplification of the National Cancer Institute reference panel (BAT25, BAT26, D2S123, D5S346, and D17S250) and deficiency of MMR protein expression by immunohistochemistry (<em>MLH1</em>, MSH2, MSH6, and PMS2). MMR protein expression and MSI (in a subset of cases) were also evaluated in 50 unselected ovarian serous tumors of low malignant potential , a tumor common in younger women. Defects in MMR were detected in 5 of 52 (10%) ovarian carcinomas by at least 1 testing method, including 2 of 4 (50%) ovarian cancers presenting with synchronous endometrial cancer. Three of the 5 (60%) ovarian carcinomas were clear cell carcinomas (17% of all pure clear cell carcinomas) and the remaining 2 were high-grade carcinomas with endometrioid and mixed histology. Loss of MSH2 and MSH6 was detected in all of the affected clear cell carcinomas and a synchronous endometrial cancer with endometrioid histology. Loss of 1 or more MMR proteins was initially noted in 10/50 ovarian serous tumors of low malignant potential on tissue microarray, but further testing on full tissue sections showed intact protein expression and microsatellite stability in all informative cases. This study demonstrates a 10% rate of MMR-deficient ovarian cancer in women <or=50 years of age. MMR-deficient ovarian cancer is frequently associated with loss of expression of MSH2 and MSH6 proteins and clear cell histology. The occurrence of MMR inactivation in a significant proportion of ovarian clear cell carcinomas (17% in this study) suggests that this tumor may warrant targeted testing in women <or=50 years of age.

BACKGROUND

Microsatellite-unstable colorectal cancers (CRC) that are due to deficient DNA mismatch repair (dMMR) represent approximately 15% of all CRCs in the United States. These microsatellite-unstable CRCs represent a heterogenous group of diseases with distinct oncogenesis pathways. There are overlapping clinicopathologic features between some of these groups, but many important differences are present. Therefore, determination of the etiology for the dMMR is vital for proper patient management and follow-up.

UNASSIGNED

Epigenetic inactivation of MLH1 MMR gene (sporadic microsatellite-unstable CRC) and germline mutation in an MMR gene (Lynch syndrome, LS) are the two most common mechanisms in the pathogenesis of microsatellite instability in CRC. However, in a subset of dMMR CRC cases that are identified by screening tests, no known LS-associated genetic alterations are appreciated by current genetic analysis. When the etiology for dMMR is unclear, it leads to patient anxiety and creates challenges for clinical management.

CONCLUSIONS

It is critical to distinguish LS patients from other patients with tumors due to dMMR, so that the proper screening protocol can be employed for the patients and their families, with the goal to save lives while avoiding unnecessary anxiety and costs. This review summarizes the major pathogenesis pathways of dMMR CRCs, their clinicopathologic features, and practical screening suggestions. In addition, we include frequently asked questions for MMR immunohistochemistry interpretation.

BACKGROUND

The ability to maintain DNA integrity is a critical cellular function. DNA repair is conducted by distinct pathways of genes, many of which are thought to be altered in colorectal cancer. However, there has been little characterization of these pathways in colorectal cancer.

METHODS

By using the TaqMan real-time quantitative PCR, RNA expression profiling of 20 DNA repair pathway genes was done in matched tumor and normal tissues from 52 patients with Dukes' C colorectal cancer.

RESULTS

The relative mRNA expression level across the 20 DNA repair pathway genes varied considerably, and the individual variability was also quite large, with an 85.4 median fold change in the tumor tissue genes and a 127.2 median fold change in the normal tissue genes. Tumor-normal differential expression was found in 13 of 20 DNA repair pathway genes (only XPA had a lower RNA level in the tumor samples; the other 12 genes had significantly higher tumor levels, all P<0.01). Coordinated expression of ERCC6, HMG1, MSH2, and POLB (RS>or=0.60) was observed in the tumor tissues (all P<0.001). Apoptosis index was not correlated with expression of the 20 DNA repair pathway genes. MLH1 and XRCC1 RNA expression was correlated with microsatellite instability status (P=0.045 and 0.020, respectively). An inverse correlation was found between tumor MLH1 RNA expression and MLH1 DNA methylation (P=0.003).

CONCLUSIONS

Our study provides an initial characterization of the DNA repair pathways for understanding the cellular DNA damage/repair system in human colorectal cancer.

Transcriptional inactivation of tumor-suppressor genes by promoter CpG island methylation is thought to be an important mechanism in human carcinogenesis. The CpG island methylator phenotype (CIMP) with extensive promoter methylation appears to be a distinct epigenetic subtype of colorectal carcinoma. Most previous studies on CpG island methylation in colorectal carcinoma used methylation-specific PCR, which may detect low levels of DNA methylation with little or no biological significance. In contrast, quantitative DNA methylation assays have been shown to provide useful information beyond that which can be achieved with methylation-specific PCR. Synchronous neoplasias provide a unique model for investigators to examine molecular alterations in multistep tumorigenesis within one individual. However, no study to date has quantified DNA methylation of CIMP-specific promoters in synchronous colorectal neoplasias. Utilizing real-time PCR (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G (calcium channel, voltage-dependent, T type alpha-1G subunit), CDKN2A (p16/INK4A), CRABP1 (cellular retinoic acid binding protein-1), MLH1 and NEUROG1 (neurogenin 1)] and MGMT in six synchronous carcinoma pairs (12 carcinomas) and eight synchronous carcinoma and adenoma pairs (16 tumors). We found that while some synchronous tumor pairs showed discordant promoter methylation patterns, other tumor pairs showed similar, but not exactly identical, patterns of promoter methylation. All but two pairs showed concordant patterns of CIMP status (CIMP positive vs CIMP negative) (P = 0.05 in cancer pairs). BRAF mutations were present in only CIMP-positive tumors. A high degree of microsatellite instability (MSI-H) was observed in both CIMP-positive and CIMP-negative tumors. KRAS mutations were not concordant in any synchronous neoplasia pair. In conclusion, epigenetic alterations at CIMP-specific promoter CpG islands in synchronous colorectal neoplasias likely have both random and nonrandom components.

OBJECTIVE

We have performed a case-control study among prostate cancer patients treated with three-dimensional conformational radiotherapy (3D-CRT) in order to investigate the association between single nucleotide polymorphisms (SNPs), treatment and patient features with gastrointestinal and genitourinary acute toxicity.

METHODS

A total of 698 patients were screened for 14 SNPs located in the ATM, ERCC2, LIG4, MLH1 and XRCC3 genes. Gastrointestinal and genitourinary toxicities were recorded prospectively using the Common Terminology Criteria for Adverse Events v3.0.

RESULTS

The XRCC3 SNP rs1799794 (G/G OR=5.65; 95% CI: 1.95-16.38; G/A OR=2.75; 95% CI: 1.25-6.05; uncorrected p-value=2.8×10(-03); corrected p-value=0.03; FDR q-value=0.06) as well as the mean dose received by the rectum (OR=1.06; 95% CI: 1.02-1.1; uncorrected p-value=2.49×10(-03); corrected p-value=0.03; FDR q-value=0.06) were significantly associated with gastrointestinal toxicity after correction for multiple testing. Those patients who undergone previous prostatectomy were less prone to develop genitourinary toxicity (OR=0.38; 95% CI: 0.18-0.71; uncorrected p-value=4.95×10(-03); corrected p-value=0.03; FDR q-value=0.08). Our study excludes the possibility of a >2-fold risk increase in genitourinary acute toxicity being due to rs1801516 ATM SNP, the rs1805386 and rs1805388 LIG4 markers, as well as all the SNPs evaluated in the ERCC2, MLH1 and XRCC3 genes.

CONCLUSIONS

The XRCC3 rs1799794 SNP and the mean dose received by the rectum are associated with the development of gastrointestinal toxicity after 3D-CRT.

To investigate the relationship between fenofibrate (FF) and oxidative stress, enzymatic, histopathological, and molecular biological analyses were performed in the liver of male F344 rats fed 2 doses of FF (Experiment 1; 0 and 6000 ppm) for 3 weeks and 3 doses (Experiment 2; 0, 3000, and 6000 ppm) for 9 weeks. FF treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver. However, it decreased those of superoxide dismutase in the liver in both experiments. Increased 8-hydroxy-2'-deoxyguanosine levels in liver DNA and lipofuscin accumulation were observed in the treated rats of Experiment 2. In vitro measurement of reactive oxygen species (ROS) in rat liver microsomes revealed a dose-dependent increase due to FF treatment. Microarray (only Experiment 1) or real-time reverse transcription-polymerase chain reaction analyses revealed that the expression levels of metabolism and DNA repair-related genes such as Aco, Cyp4a1, Cat, Yc2, Gpx2, Apex1, Xrcc5, Mgmt, Mlh1, Gadd45a, and Nbn were increased in FF-treated rats. These results provide evidence of a direct or indirect relationship between oxidative stress and FF treatment. In addition, increases in the expression levels of cell cycle-related genes such as Chek1, Cdc25a, and Ccdn1; increases in the expression levels of cell proliferation-related genes such as Hdgfrp3 and Vegfb; and fluctuations in the expression levels of apoptosis-related genes such as Casp11 and Trp53inp1 were observed in these rats. This suggests that cell proliferation induction, apoptosis suppression, and DNA damage due to oxidative stresses are probably involved in the mechanism of hepatocarcinogenesis due to FF in rats.

OBJECTIVE

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that strongly inhibits epithelial cell growth. Disabling of TGF-beta signaling is thought to be involved in development of a variety of tumors in which abnormal expression or function of TGF-beta receptor plays critical roles. In the present study, we examined aberrant expression and mutation of the gene TGF-beta receptor type II (TbetaRII) in endometrial cancers of endometrioid subtype.

RESULTS

Real-time PCR analysis using surgical tissue specimens of 27 endometrial cancers and 24 normal endometria revealed that endometrial cancers had significantly decreased levels of TbetaRII mRNA expression (mean level 2.44 +/- 2.65), compared to normal endometria (mean level 7.23 +/- 6.07) (P < 0.001). Methylation status of TbetaRII promoter containing 30 CpGs was examined by bisulfite sequencing analysis, and 98% (51/52) of the patients were found to have unmethylated TbetaRII promoter, indicating that promoter hypermethylation is not the major cause of decreased expression of TbetaRII in endometrial cancers. Mutational analysis revealed that 15.1% (8/53) of endometrial cancers had frameshift mutations at polyadenine repeats in exon 3 of the TbetaRII gene. Notably, these mutations were preferentially accumulated in patients with MSI-H phenotype (7/19:37%) (P < 0.001) or with those with methylated MLH1 promoters (6/16:38%) (P < 0.01). Thus, it appears that the TbetaRII gene is a target of mismatch repair deficiency.

CONCLUSIONS

Taken together, we found that the decreased expression of TbetaRII as well as frameshift mutation of TbetaRII via mismatch repair deficiency frequently occurs in this tumor type, possibly causing loss of receptor function and unresponsiveness of TGF-beta signaling that may lead to endometrial carcinogenesis.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant condition due to heterozygous germline mutations in DNA mismatch repair genes, in particular MLH1, MSH2 and MSH6. Recently, a syndrome was recognized in which children develop haematological malignancies, solid tumours and signs of neurofibromatosis type 1 due to bi-allelic MMR gene mutations in MLH1, MSH2 and PMS2. Here we describe the child of healthy consanguineous parents who had café-au-lait spots, oligodendroglioma, and rectal cancer. The patient was homozygous for the MSH6 mutation c.3386_3388delGTG in exon 5 which has a predicted pathogenic effect. Germline NF1 gene mutation testing was negative. The rectal tumour showed microsatellite instability and absence of MSH6 staining, whereas the brain tumour was MSI stable and showed normal immunohistochemical expression of MSH6. Apparently, not only MLH1, MSH2 and PMS2, but also MSH6 is involved in the syndrome of childhood cancer and signs of neurofibromatosis type 1.

Heterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inherited cancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancer syndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes. MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/or gastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-based mutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection by the analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered by the presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based on direct cDNA sequencing of RT-PCR products, we investigated two families with children suspected to suffer from MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients of the first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family. Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL "hybrid" allele carrier, that RNA-based PMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogene coamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMR deficiency and in HNPCC patients with PMS2 defects.

Although breast cancer is the second most common cause of brain metastasis with a notable increase of incidence, genes that mediate breast cancer brain metastasis (BCBM) are not fully understood. To study the molecular nature of brain metastasis, we performed gene expression profiling of brain metastasis and matched primary breast cancer (BC). We used the Ion AmpliSeq Cancer Panel v2 covering 2,855 mutations from 50 cancer genes to analyze 18 primary BC and 42 BCBM including 15 matched pairs. The most common BCBM subtypes were triple-negative (42.9%) and basal-like (36.6%). In a total of 42 BCBM samples, 32 (76.2%) harbored at least one mutation (median 1, range 0-7 mutations). Frequently detected somatic mutations included TP53 (59.5%), MLH1 (14.3%), PIK3CA (14.3%), and KIT (7.1%). We compared BCBM with patient-matched primary BC specimens. There were no significant differences in mutation profiles between the two groups. Notably, gene expression in BCBM such as TP53, PIK3CA, KIT, MLH1, and RB1 also seemed to be present in primary breast cancers. The TP53 mutation frequency was higher in BCBM than in primary BC (59.5% vs 38.9%, respectively). In conclusion, we found actionable gene alterations in BCBM that were maintained in primary BC. Further studies with functional testing and a delineation of the role of these genes in specific steps of the metastatic process should lead to a better understanding of the biology of metastasis and its susceptibility to treatment.

OBJECTIVE

The specificity of the autoantibody response in different autoimmune diseases makes autoantibodies useful for diagnostic purposes. It also focuses attention on tissue- and event-specific circumstances that may select unique molecules for an autoimmune response in specific diseases. Defining additional phenotype-specific autoantibodies may identify such circumstances. This study was undertaken to investigate the disease specificity of PMS1, an autoantigen previously identified in some sera from patients with myositis.

METHODS

We used immunoprecipitation analysis to determine the frequency of autoantibodies to PMS1 in sera from patients with myositis, systemic lupus erythematosus, or scleroderma and from healthy controls. Additional antigens recognized by PMS1-positive sera were further characterized in terms of their susceptibility to cleavage by apoptotic proteases.

RESULTS

PMS1, a DNA mismatch repair enzyme, was identified as a myositis-specific autoantigen. Autoantibodies to PMS1 were found in 4 of 53 patients with autoimmune myositis (7.5%), but in no sera from 94 patients with other systemic autoimmune diseases (P = 0.016). Additional mismatch repair enzymes (PMS2, MLH1) were targeted, apparently independently. Sera recognizing PMS1 also recognized several other proteins involved in DNA repair and remodeling, including poly(ADP-ribose) polymerase, DNA-dependent protein kinase, and Mi-2. All of these autoantigens were efficiently cleaved by granzyme B, generating unique fragments not observed during other forms of cell death.

CONCLUSIONS

PMS1 autoantibodies are myositis specific. The striking correlation between an immune response to a group of granzyme B substrates (functioning in DNA repair and remodeling) and the myositis phenotype strongly implies that tissue- and event-specific biochemical events play a role in selecting these molecules for an autoimmune response. Understanding the role of granzyme B cleavage in this response is an important priority.

An association of Lynch syndrome (LS) with breast cancer has been long suspected; however, there have been insufficient data to address this question for each of the LS genes individually.

We conducted a retrospective review of personal and family history in 423 women with pathogenic or likely pathogenic germ-line variants in MLH1 (N = 65), MSH2 (N = 94), MSH6 (N = 140), or PMS2 (N = 124) identified via clinical multigene hereditary cancer testing. Standard incidence ratios (SIRs) of breast cancer were calculated by comparing breast cancer frequencies in our study population with those in the general population (Surveillance, Epidemiology, and End Results 18 data).

When evaluating by gene, the age-standardized breast cancer risks for MSH6 (SIR = 2.11; 95% confidence interval (CI), 1.56-2.86) and PMS2 (SIR = 2.92; 95% CI, 2.17-3.92) were associated with a statistically significant risk for breast cancer whereas no association was observed for MLH1 (SIR = 0.87; 95% CI, 0.42-1.83) or MSH2 (SIR = 1.22; 95% CI, 0.72-2.06).

Our data demonstrate that two LS genes, MSH6 and PMS2, are associated with an increased risk for breast cancer and should be considered when ordering genetic testing for individuals who have a personal and/or family history of breast cancer.

There is evidence that insertion of viral DNA into a mammalian genome can lead to alterations of methylation patterns. The aim of the present study was to examine the presence of DNA sequences of five human DNA viruses (assessed by PCR): JC polyoma virus (JCV), human adenovirus (AdV), Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV/HHV8) and human papillomavirus (HPV) in a cohort of 186 sporadic colorectal cancers (CRCs) and related these data with the methylation status of six CpG island methylator phenotype (CIMP)-specific genes (MLH1, CACNA1G, NEUROG1, IGF2, SOCS1, RUNX3) and seven cancer-related genes markers (p16, MINT1, MINT2, MINT31, EN1, SCTR and INHBB) assessed by methylation-specific PCR in 186 and 134 CRC cases, respectively. The AdV, KSHV and HPV were detected in four (2%), two (1%) and zero CRC cases, respectively, and thus were excluded from further analyses. Although 19% and 9% of the CRCs were positive for EBV and JCV, respectively, no associations between virus presence and CpG island methylation were found after correction for multiple testing. Our results demonstrate that the presence of DNA sequences of JCV and EBV in CRC is unrelated to the methylation of the 13 cancer-related CpG islands and CIMP.

OBJECTIVE

Aberrant expression of SOX4 in endometrial cancer has been identified and partially was contributed to hypermethylation of miR-129-2. Other miRNAs are suspected to influence SOX 4 as well. The current study seeks to identify other hypermethylated miRNAs that regulate SOX4 in endometrial carcinomas.

METHODS

Methylation levels of miRNA promoter regions were measured by combined bisulfite restriction analysis (COBRA) and pyrosequencing assays. Gene expression was determined by RT-qPCR. Methylation level of a miRNA locus was corrected with clinicopathologic factors for 252 gynecological specimens.

RESULTS

In silico analysis identified 13 miRNA loci bound on the 3'-UTR of SOX4. Using COBRA assays, increased methylation of miR-203, miR-219-2, miR-596, and miR-618 was detected in endometrial cancer cells relative to those seen in a normal cell line and in normal endometrium. Transfection of a miR-203 mimic decreased SOX4 gene expression. Hypermethylation of miR-203 was detected in 52% of type I endometrioid endometrial carcinomas (n=131) but was not seen in any of 10 uninvolved normal endometria (P<0.001). Methylation status of miR-203 was significantly associated with microsatellite instability and MLH1 methylation in endometrial tumors (P<0.001). Furthermore, hypermethylation of miR-203 was found in endometrioid and clear endometrial subtype tumors, but not in cervical squamous cell and ovarian carcinomas.

CONCLUSIONS

Hypermethylation of miR-203 is a frequent event in endometrial carcinomas and is strongly associated with microsatellite instability and MLH1 methylation status. Thus, miR-203 methylation level might represent a marker for patients with endometrioid and clear endometrial sub-cancers.

Endometrial cancer is associated with numeric and structural chromosomal abnormalities, microsatellite instability (MSI), and alterations that activate oncogenes and inactivate tumor suppressor genes. The aim of this study was to characterize a set of endometrial cancers using multiple molecular genetic and immunohistochemical techniques. Ninety-six cases were examined for genomic alterations by MSI, MLH1 promoter hypermethylation, p53 and mismatch repair protein expression (MLH1, MSH2, MSH6, PMS2), and PTEN, PIK3CA, KRAS, and BRAF mutation analysis. At least 1 alteration was identified in 48 of 87 (55%) specimens tested for PTEN, making it the most common abnormality in this study. A PIK3CA alteration was observed in 16 (17%) specimens. Twenty-nine of 94 (31%) MSI tested tumors exhibited an MSI-H phenotype. Of the 29 MSI-H cases, 24 (83%) were positive for methylation of the MLH1 promoter region. Twenty-three (82%) of the 28 MSI-H cases with immunohistochemistry results showed loss of expression of MLH1/PMS2 (n=19), MSH2/MSH6 (n=2), or MSH6 only (n=2). Of the 19 MSI-H cases with loss of MLH1/PMS2 on immunohistochemistry, 18 were positive, and 1 was equivocal for MLH1 promoter hypermethylation. Twelve of 94 cases (13%) analyzed for KRAS mutations were found to have a mutation. No BRAF V600E mutations were indentified. This study provides a comprehensive molecular genetic analysis of commonly analyzed targets in a large cohort of endometrial cancers.

Promoter hypermethylation is responsible for gene inactivation during carcinogenesis. It has been proposed that there is some degree of specificity in the set of genes that become altered by this mechanism in distinct tumor types. To understand whether promoter hypermethylation may differentiate the site of origin, 49 lung adenocarcinomas from 31 lung primaries and 18 metastases from colorectal primaries, respectively, were tested for the presence of this alteration in the APC, CDH1, DAPK, GSTP1, MLH1, MGMT, P14, P16, RARbeta2, RASSF1, sFRP1 and WIF-1 genes. A distinct profile was apparent for the 2 groups of lung tumors and the frequencies of promoter hypermethylation at sFRP1 and WIF-1, 2 genes involved in Wnt signaling, and at CDH1 were significantly higher in colorectal metastases than in lung primaries, whereas methylation of the APC promoter was significantly more common in lung primary adenocarcinomas. Some tumors showed concomitant APC, sFRP1 and WIF-1 gene inactivation, indicating that multiple DNA methylation events must have occurred to definitively down-regulate the signaling through Wnt. However, promoter hypermethylation at the APC and CDH1 genes tended to be mutually exclusive (Fisher's exact test, p = 0.006), suggesting a similar role in carcinogenesis. In conclusion, we propose that inactivation by promoter hypermethylation at the APC, CDH1, sFRP1 and WIF-1 genes may contribute to the discrimination of lung primary adenocarcinomas from colorectal metastasis to the lung, and report the simultaneous presence of methylation at the promoters of multiple genes involved in the Wnt signaling. This may have biological consequences for carcinogenesis.

Guanine rich nucleic acid sequences can form G-quadruplex (G4) structures that interfere with DNA replication, repair and RNA transcription. The human FANCJ helicase contributes to maintaining genomic integrity by promoting DNA replication through G4-forming DNA regions. Here, we combined single-molecule and ensemble biochemical analysis to show that FANCJ possesses a G4-specific recognition site. Through this interaction, FANCJ targets G4-containing DNA where its helicase and G4-binding activities enable repeated rounds of stepwise G4-unfolding and refolding. In contrast to other G4-remodeling enzymes, FANCJ partially stabilizes the G-quadruplex. This would preserve the substrate for the REV1 translesion DNA synthesis polymerase to incorporate cytosine across from a replication-stalling G-quadruplex. The residues responsible for G-quadruplex recognition also participate in interaction with MLH1 mismatch-repair protein, suggesting that the FANCJ activity supporting replication and its participation in DNA interstrand crosslink repair and/or heteroduplex rejection are mutually exclusive. Our findings not only describe the mechanism by which FANCJ recognizes G-quadruplexes and mediates their stepwise unfolding, but also explain how FANCJ chooses between supporting DNA repair versus promoting DNA replication through G-rich sequences.

OBJECTIVE

To correlate survival and surgical-pathologic factors with DNA mismatch repair status in patients with endometrial cancer.

METHODS

Specimens from 336 patients with endometrial cancer were used to create a tissue microarray. Immunohistochemistry with antibodies against the mismatch repair genes MLH1, MSH2, MSH6, and PMS2 were used to stain the tissue microarray. Clinical, pathologic, and survival data were collected and correlated with the immunohistochemistry results.

RESULTS

Mismatch repair deficiency was seen in 29% (84 of 294) of cases. Correlation was noted between lack of expression of MLH1 and an increased risk for lymphvascular space involvement (32% versus 21%, P=.05) and cervical involvement (26% versus 14%, P=.02). Lack of expression of either MLH1 or MSH2 was associated with thinner patients (85% had a body mass index less than 40 versus 73% of patients with normal expression, P=.02), as well as with the absence of a history of previous primary malignancy (0 verus 13 cases [4%], P=.023). The estimated disease-free survival is 88%; despite a small number of recurrences, there was a nonsignificant improvement in disease-free survival in tumors with an intact mismatch repair system (P=.1). Significantly improved disease-free survival was seen in patients with normal MLH1 and MSH2 expression compared with those with abnormal expression (92% versus 81%, P=.035).

CONCLUSIONS

Defects in DNA mismatch repair in endometrial cancer is correlated with negative prognostic factors and worse progression-free survival (without a difference in overall survival) compared with tumors with an intact mismatch repair system.

METHODS

II-3.

The identification of biomarkers associated with response to therapeutic agents is central to optimizing patient outcomes. Expression of the immune checkpoint proteins PD-1/L1, and DNA mismatch repair deficiency (dMMR) status may be predictive response biomarkers for immunotherapies, but their overlap requires further study. We prospectively conducted PD-L1 and MMR immunohistochemistry (IHC) on 430 consecutive patients with advanced gastrointestinal (GI) cancers, genitourinary (GU) cancers or rare cancers between June 2012 and March 2016. Overall 393/430 (91.4%) patients were evaluable for PD-L1 expression by IHC. The frequency of tumor PD-L1 positivity (PD-L1+) was 16.5% (65/393). Among anatomic tumor sites PD-L1+ was 28.6% in melanoma, 22.2% in GC, 20.9% in CRC, 12.5% in BTC, 7.1% in GU cancer, 6.7% in HCC, 0% in pancreatic cancer and 0% in sarcoma. Among the 394 evaluable for MLH1/MSH2 expression cases, 18 patients (4.5%) had dMMR tumors. The dMMR was most common in GC (7.1%) followed by 6.7% in HCC, 4.4% in CRC, and 2.7% in sarcoma. Of the 365 patients evaluable for both PD-L1 and MLH1/MSH2 expression, there was a significant association between the PD-L1 expression and MLH1/MSH2 loss (P = 0.01), but not with overall survival within tumor types. PD-L1 status and dMMR are overlapping putative response biomarkers in immunoncology. Clinical trials with biomarker enrichment restricted to PD-L1+ or dMMR may be inadequate to capture the subset of patients who may benefit from immune mediated therapies. More robust immunotherapy biomarkers and careful clinical trial design are warranted.

Gastroenteropancreatic (GEP) neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are heterogeneous neoplasms characterized by poor outcome. Microsatellite instability (MSI) has recently been found in colorectal NECs showing a better prognosis than expected. However, the frequency of MSI in a large series of GEP-NEC/MANECs is still unknown. In this work, we investigated the incidence of MSI in GEP-NEC/MANECs and characterized their clinicopathologic and molecular features. MSI analysis and immunohistochemistry for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) were performed in 89 GEP-NEC/MANECs (six esophageal, 77 gastrointestinal, three pancreatic, and three of the gallbladder). Methylation of 34 genes was studied by methylation-specific multiplex ligation probe amplification. Mutation analysis of BRAF and KRAS was assessed by PCR-pyrosequencing analysis. MSI was observed in 11 NEC/MANECs (12.4%): seven intestinal and four gastric. All but two MSI-cases showed MLH1 methylation and loss of MLH1 protein. The remaining two MSI-cancers showed lack of MSH2 or PMS2 immunohistochemical expression. MSI-NEC/MANECs showed higher methylation levels than microsatellite stable NEC/MANECs (40.6% vs 20.2% methylated genes respectively, P<0.001). BRAF mutation was detected in six out of 88 cases (7%) and KRAS mutation was identified in 15 cases (17%). BRAF mutation was associated with MSI (P<0.0008), while KRAS status did not correlate with any clinicopathologic or molecular feature. Vascular invasion (P=0.0003) and MSI (P=0.0084) were identified as the only independent prognostic factors in multivariate analysis. We conclude that MSI identifies a subset of gastric and intestinal NEC/MANECs with distinct biology and better prognosis. MSI-NEC/MANECs resemble MSI-gastrointestinal adenocarcinomas for frequency, molecular profile and pathogenetic mechanisms.

The aim of this study was to determine the frequency of microsatellite instability (MSI(+)) in tumors from a population-based series of young colorectal cancer patients and its correlation with the loss of expression of mismatch repair (MMR) proteins. The BAT-26 mononucleotide repeat was used to screen for MSI(+) in all colorectal cancers diagnosed in Western Australia throughout a 5-year period in patients <60 years of age. MSI(+) was found in 75 of 1003 (7.5%) cases, of which six contained a concomitant mutation in BRAF and were therefore excluded from further investigations as possible hereditary nonpolyposis colorectal cancer. Immunohistochemistry was used to evaluate expression of the four major MMR proteins (MLH1, MSH2, MSH6, and PMS2) in the remaining 69 MSI(+) tumors. Complete loss of MLH1 and PMS2 expression or of MSH2 and MSH6 expression was found in 35 (51%) and 17 (25%) cases, respectively, whereas other patterns of complete loss were observed in eight cases (12%). Eight tumors (12%) were initially recorded as showing normal expression, but on review seven were reclassified as having abnormal staining because of heterogeneous patterns of MMR loss. Three of these seven cases had previously been found to have germline mutations. Because of possible misinterpretation of heterogeneous immunohistochemistry staining for MMR protein loss, MSI testing is recommended as the initial screen for population-based detection of hereditary nonpolyposis colorectal cancer.

The clinicopathologic significance of mismatch repair (MMR) defects in endometrioid endometrial cancer (EEC) has not been definitively established. We undertook tumor typing to classify MMR defects to determine if MMR status is prognostic or predictive.

Primary EECs from NRG/GOG0210 patients were assessed for microsatellite instability (MSI), MLH1 methylation, and MMR protein expression. Each tumor was assigned to one of four MMR classes: normal, epigenetic defect, probable mutation (MMR defect not attributable to MLH1 methylation), or MSI-low. The relationships between MMR classes and clinicopathologic variables were assessed using contingency table tests and Cox proportional hazard models.

A total of 1,024 tumors were assigned to MMR classes. Epigenetic and probable mutations in MMR were significantly associated with higher grade and more frequent lymphovascular space invasion. Epigenetic defects were more common in patients with higher International Federation of Gynecology and Obstetrics stage. Overall, there were no differences in outcomes. Progression-free survival was, however, worse for women whose tumors had epigenetic MMR defects compared with the MMR normal group (hazard ratio, 1.37; P < .05; 95% CI, 1.00 to 1.86). An exploratory analysis of interaction between MMR status and adjuvant therapy showed a trend toward improved progression-free survival for probable MMR mutation cases.

MMR defects in EECs are associated with a number of well-established poor prognostic indicators. Women with tumors that had MMR defects were likely to have higher-grade cancers and more frequent lymphovascular space invasion. Surprisingly, outcomes in these patients were similar to patients with MMR normal tumors, suggesting that MMR defects may counteract the effects of negative prognostic factors. Altered immune surveillance of MMR-deficient tumors, and other host/tumor interactions, is likely to determine outcomes for patients with MMR-deficient tumors.

Lynch syndrome, caused by germline mutations in the mismatch repair genes, is associated with increased cancer risk. Here using a large whole-genome sequencing data bank, cancer registry and colorectal tumour bank we determine the prevalence of Lynch syndrome, associated cancer risks and pathogenicity of several variants in the Icelandic population. We use colorectal cancer samples from 1,182 patients diagnosed between 2000-2009. One-hundred and thirty-two (11.2%) tumours are mismatch repair deficient per immunohistochemistry. Twenty-one (1.8%) have Lynch syndrome while 106 (9.0%) have somatic hypermethylation or mutations in the mismatch repair genes. The population prevalence of Lynch syndrome is 0.442%. We discover a translocation disrupting MLH1 and three mutations in MSH6 and PMS2 that increase endometrial, colorectal, brain and ovarian cancer risk. We find thirteen mismatch repair variants of uncertain significance that are not associated with cancer risk. We find that founder mutations in MSH6 and PMS2 prevail in Iceland unlike most other populations.