Sequential azacitidine and carboplatin induces immune activation in platinum-resistant high-grade serous ovarian cancer cell lines and primes for checkpoint inhibitor immunotherapy.

BACKGROUND: Platinum chemoresistance results in high-grade serous ovarian cancer (HGSOC) disease recurrence. Recent treatment advances using checkpoint inhibitor immunotherapy has not benefited platinum-resistant HGSOC. In ovarian cancer, DNA methyltransferase inhibitors (DNMTi) block methylation and allow expression of silenced genes, primarily affecting immune reactivation pathways. We aimed to determine the epigenome and transcriptome response to sequential treatment with DNMTi and carboplatin in HGSOC. METHODS: In vitro studies with azacitidine or carboplatin alone and in sequential combination. Response was determined by cell growth, death and apoptosis. Genome-wide DNA methylation levels and transcript expression were compared between untreated and azacitidine and carboplatin sequential treatment. RESULTS: Sequential azacitidine and carboplatin significantly slowed cell growth in 50% of cell lines compared to carboplatin alone. The combination resulted in significantly higher cell death in 25% of cell lines, and significantly higher cell apoptosis in 37.5% of cell lines, than carboplatin alone. Pathway analysis of upregulated transcripts showed that the majority of changes were in immune-related pathways, including those regulating response to checkpoint inhibitors. CONCLUSIONS: Sequential azacitidine and carboplatin treatment slows cell growth, and demethylate and upregulate pathways involved in immune response, suggesting that this combination may be used to increase HGSOC response to immune checkpoint inhibitors in platinum-resistant patients who have exhausted all currently-approved avenues of treatment.

The presence of the intron 3 16 bp duplication polymorphism of p53 (rs17878362) in breast cancer is associated with a low Δ40p53:p53 ratio and better outcome.

Breast cancer is the most common female cancer, but it has relatively low rates of p53 mutations, suggesting other mechanisms are responsible for p53 inactivation. We have shown that the p53 isoform, Δ40p53, is highly expressed in breast cancer, where it may contribute to p53 inactivation. Δ40p53 can be produced by alternative splicing of p53 in intron 2 and this is regulated by the formation of G-quadruplex structures in p53 intron 3, from which the nucleotides forming these structures overlap with a common polymorphism, rs17878362. rs17878362 alters p53 splicing to decrease fully spliced p53 messenger RNA (mRNA) in vitro following ionizing radiation and this in turn alters Δ40p53:p53. Hence, the presence of rs17878362 may be important in regulating Δ40p53:p53 in breast cancer. This study aimed to determine if rs17878362 was associated with altered Δ40p53 and p53 expression and outcome in breast cancer. We sequenced p53 in breast tumours from 139 patients and compared this with Δ40p53 and p53 mRNA expression. We found that the ratio of Δ40p53:p53 was significantly lower in tumours homozygous for the polymorphic A2 allele compared with those who were wild-type (A1/A1). Furthermore, there was a lower proportion of breast cancers carrying the A2 allele from patients who subsequently developed metastasis compared with those that did not. Finally, we show that patients whose tumours carried the polymorphic A2 allele had significantly better disease-free survival. These results show that rs17878362 is associated with a low Δ40p53:p53 ratio in breast cancer and that this is associated with better outcome.

Reevaluation of RINT1 as a breast cancer predisposition gene.

Rad50 interactor 1 (RINT1) has recently been reported as an intermediate-penetrance (odds ratio 3.24) breast cancer susceptibility gene, as well as a risk factor for Lynch syndrome. The coding regions and exon-intron boundaries of RINT1 were sequenced in 2024 familial breast cancer cases previously tested negative for BRCA1, BRCA2, and PALB2 mutations and 1886 population-matched cancer-free controls using HaloPlex Targeted Enrichment Assays. Only one RINT1 protein-truncating variant was detected in a control. No excess was observed in the total number of rare variants (truncating and missense) (28, 1.38 %, vs. 27, 1.43 %. P > 0.999) or in the number of variants predicted to be pathogenic by various in silico tools (Condel, Polyphen2, SIFT, and CADD) in the cases compared to the controls. In addition, there was no difference in the incidence of classic Lynch syndrome cancers in RINT1 rare variant-carrying families compared to RINT1 wild-type families. This study had 90 % power to detect an odds ratio of at least 2.06, and the results do not provide any support for RINT1 being a moderate-penetrance breast cancer susceptibility gene, although larger studies will be required to exclude more modest effects. This study emphasizes the need for caution before designating a cancer predisposition role for any gene based on very rare truncating variants and in silico-predicted missense variants.

When is a mutation not a mutation: the case of the c.594-2A>C splice variant in a woman harbouring another BRCA1 mutation in trans.

Since the identification of BRCA1 there has only ever been described two bi-allelic mutation carriers, one of whom was subsequently shown to be a mono-allelic carrier. The second patient diagnosed with two BRCA1 mutations appears to be accurate but there remain some questions about the missense variant identified in that patient. In this report we have identified a woman who is a bi-allelic mutation carrier of BRCA1 and provide an explanation as to why this patient has a phenotype very similar to that of any mono-allelic mutation carrier. The splice variant identified in this patient appears to be associated with the up-regulation of a BRCA1 splice variant that rescues the lethality of being a double mutant. The consequences of the findings of this report may have implications for mutation interpretation and that could serve as a model for not only BRCA1 but also for other autosomal dominant disorders that are considered as being embryonically lethal.

Prevalence of PALB2 mutations in Australian familial breast cancer cases and controls.

INTRODUCTION: PALB2 is emerging as a high-penetrance breast cancer predisposition gene in the order of BRCA1 and BRCA2. However, large studies that have evaluated the full gene rather than just the most common variants in both cases and controls are required before all truncating variants can be included in familial breast cancer variant testing. METHODS: In this study we analyse almost 2000 breast cancer cases sourced from individuals referred to familial cancer clinics, thus representing typical cases presenting in clinical practice. These cases were compared to a similar number of population-based cancer-free controls. RESULTS: We identified a significant excess of truncating variants in cases (1.3 %) versus controls (0.2 %), including six novel variants (p = 0.0001; odds ratio (OR) 6.58, 95 % confidence interval (CI) 2.3-18.9). Three of the four control individuals carrying truncating variants had at least one relative with breast cancer. There was no excess of missense variants in cases overall, but the common c.1676A > G variant (rs152451) was significantly enriched in cases and may represent a low-penetrance polymorphism (p = 0.002; OR 1.24 (95 % CI 1.09-1.47). CONCLUSIONS: Our findings support truncating variants in PALB2 as high-penetrance breast cancer susceptibility alleles, and suggest that a common missense variant may also lead to a low level of increased breast cancer risk.

Prevalence of BRCA1 and BRCA2 germline mutations in patients with triple-negative breast cancer.

Triple-negative breast cancers (TNBC) lack expression of oestrogen, progesterone and HER2 receptors. The gene expression profiles of TNBCs are similar to those of breast tumours in women with BRCA1 mutations. Reports to date indicate that up to 20 % of TNBC patients harbour germline BRCA mutations; however, the prevalence of BRCA mutations in TNBC patients varies widely between countries and from study to study. We studied 774 women with triple-negative breast cancer, diagnosed on average at age 58.0 years. Samples of genomic DNA were provided by the Australian Breast Cancer Tissue Bank (ABCTB) (439 patients) and by the Department of Genetics and Pathology of the Pomeranian Medical University (335 patients). The entire coding regions and the exon-intron boundaries of BRCA1 and BRCA2 were amplified and sequenced by next-generation sequencing. We identified a BRCA1 or BRCA2 mutation in 74 of 774 (9.6 %) triple-negative patients. The mutation prevalence was 9.3 % in Australia and was 9.9 % in Poland. In both countries, the mean age of diagnoses of BRCA1 mutation carriers was significantly lower than that of non-carriers, while the age of onset of BRCA2 mutation carriers was similar to that of non-carriers. In the Australian cohort, 59 % of the mutation-positive patients did not have a family history of breast or ovarian cancer, and would not have qualified for genetic testing. The triple-negative phenotype should be added as a criterion to genetic screening guidelines.

The relative mRNA expression of p53 isoforms in breast cancer is associated with clinical features and outcome.

Mutation of p53 is a common feature of cancer. Breast cancer is the most common malignancy that develops in women; however, somatic mutation of p53 is rare, suggesting that p53 becomes inactivated by other mechanisms. p53 is expressed as smaller isoforms, some of which inhibit wild-type p53. There are no studies that have examined the relative expression of all isoforms in this disease. We have analysed the relative messenger RNA expression of the p53 isoforms, Δ40, Δ133, ß and γ in a panel of 6 breast cancer cell lines, 148 breast cancers specimens and 31 matched normal adjacent tissues by semi-quantitative real-time reverse transcription-PCR and analysed their relationship to clinical features and outcome. We have identified several important clinical associations, particularly with Δ40p53, which was expressed at levels that were ~50-fold higher than the least expressed isoform p53γ. Δ40p53 was significantly upregulated in tumour tissue when compared with the normal breast and was significantly associated with an aggressive breast cancer subtype-triple negative. Additionally, p53ß expression was significantly negatively associated with tumour size and positively associated with disease-free survival, where high levels of p53ß were protective, particularly in patients with a mutation in p53, suggesting p53ß may counteract the damage inflicted by mutant p53. In conclusion, the relative expression of p53 isoforms is related to clinical features of breast cancer and outcome. These results have implications for the stratification of breast cancer based on p53 function and may provide an alternate explanation for deregulated p53 signalling in breast cancer.

Low prevalence of germline PALB2 mutations in Australian triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a tumour classification that is defined by oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 receptor negativity. TNBCs share a similar gene expression profile to BRCA-mutated tumours, have been shown to carry a high proportion of BRCA mutations and have a more adverse prognosis compared to other types of breast tumours. PALB2 has been shown to be a moderate-penetrance breast cancer susceptibility gene and is involved in the same DNA damage repair pathway as BRCA1 and BRCA2; this raises the possibility that germline PALB2 mutations may be involved in the pathogenesis of TNBCs. In our study, we sequenced the coding regions of PALB2 (including intron/exon boundaries) in genomic DNA from 347 patients diagnosed with TNBC to determine the prevalence of deleterious mutations in this population. Two novel truncating mutations (c.758dup and c.2390del) and one previously detected truncating mutation (c.3113+5G>C) were found. In addition, five variants predicted to be protein-affecting were also identified. Our study shows that the prevalence of PALB2 germline mutations in individuals with TNBC is ∼1%, similar to the prevalence of PALB2 germline mutation of 1% in familial non-BRCA1/2 breast cancer cohorts.

Fusobacterium nucleatum: An Overview of Evidence, Demi-Decadal Trends, and Its Role in Adverse Pregnancy Outcomes and Various Gynecological Diseases, including Cancers.

Gynecological and obstetric infectious diseases are crucial to women's health. There is growing evidence that links the presence of Fusobacterium nucleatum (F. nucleatum), an anaerobic oral commensal and potential periodontal pathogen, to the development and progression of various human diseases, including cancers. While the role of this opportunistic oral pathogen has been extensively studied in colorectal cancer in recent years, research on its epidemiological evidence and mechanistic link to gynecological diseases (GDs) is still ongoing. Thus, the present review, which is the first of its kind, aims to undertake a comprehensive and critical reappraisal of F. nucleatum, including the genetics and mechanistic role in promoting adverse pregnancy outcomes (APOs) and various GDs, including cancers. Additionally, this review discusses new conceptual advances that link the immunomodulatory role of F. nucleatum to the development and progression of breast, ovarian, endometrial, and cervical carcinomas through the activation of various direct and indirect signaling pathways. However, further studies are needed to explore and elucidate the highly dynamic process of host-F. nucleatum interactions and discover new pathways, which will pave the way for the development of better preventive and therapeutic strategies against this pathobiont.

ERCC2 mutations alter the genomic distribution pattern of somatic mutations and are independently prognostic in bladder cancer.

Excision repair cross-complementation group 2 (ERCC2) encodes the DNA helicase xeroderma pigmentosum group D, which functions in transcription and nucleotide excision repair. Point mutations in ERCC2 are putative drivers in around 10% of bladder cancers (BLCAs) and a potential positive biomarker for cisplatin therapy response. Nevertheless, the prognostic significance directly attributed to ERCC2 mutations and its pathogenic role in genome instability remain poorly understood. We first demonstrated that mutant ERCC2 is an independent predictor of prognosis in BLCA. We then examined its impact on the somatic mutational landscape using a cohort of ERCC2 wild-type (n = 343) and mutant (n = 39) BLCA whole genomes. The genome-wide distribution of somatic mutations is significantly altered in ERCC2 mutants, including T[C>T]N enrichment, altered replication time correlations, and CTCF-cohesin binding site mutation hotspots. We leverage these alterations to develop a machine learning model for predicting pathogenic ERCC2 mutations, which may be useful to inform treatment of patients with BLCA.

Epigenetic Mechanisms and Therapeutic Targets in Chemoresistant High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype, and the overall survival rate has not improved in the last three decades. Currently, most patients develop recurrent disease within 3 years and succumb to the disease within 5 years. This is an important area of research, as the major obstacle to the treatment of HGSOC is the development of resistance to platinum chemotherapy. The cause of chemoresistance is still largely unknown and may be due to epigenetics modifications that are driving HGSOC metastasis and treatment resistance. The identification of epigenetic changes in chemoresistant HGSOC enables the development of epigenetic modulating drugs that may be used to improve outcomes. Several epigenetic modulating drugs have displayed promise as drug targets for HGSOC, such as demethylating agents azacitidine and decitabine. Others, such as histone deacetylase inhibitors and miRNA-targeting therapies, demonstrated promising preclinical results but resulted in off-target side effects in clinical trials. This article reviews the epigenetic modifications identified in chemoresistant HGSOC and clinical trials utilizing epigenetic therapies in HGSOC.

Investigation of monogenic causes of familial breast cancer: data from the BEACCON case-control study.

Breast cancer (BC) has a significant heritable component but the genetic contribution remains unresolved in the majority of high-risk BC families. This study aims to investigate the monogenic causes underlying the familial aggregation of BC beyond BRCA1 and BRCA2, including the identification of new predisposing genes. A total of 11,511 non-BRCA familial BC cases and population-matched cancer-free female controls in the BEACCON study were investigated in two sequencing phases: 1303 candidate genes in up to 3892 cases and controls, followed by validation of 145 shortlisted genes in an additional 7619 subjects. The coding regions and exon-intron boundaries of all candidate genes and 14 previously proposed BC genes were sequenced using custom designed sequencing panels. Pedigree and pathology data were analysed to identify genotype-specific associations. The contribution of ATM, PALB2 and CHEK2 to BC predisposition was confirmed, but not RAD50 and NBN. An overall excess of loss-of-function (LoF) (OR 1.27, p = 9.05 × 10-9) and missense (OR 1.27, p = 3.96 × 10-73) variants was observed in the cases for the 145 candidate genes. Leading candidates harbored LoF variants with observed ORs of 2-4 and individually accounted for no more than 0.79% of the cases. New genes proposed by this study include NTHL1, WRN, PARP2, CTH and CDK9. The new candidate BC predisposition genes identified in BEACCON indicate that much of the remaining genetic causes of high-risk BC families are due to genes in which pathogenic variants are both very rare and convey only low to moderate risk.

Global DNA methylation and cognitive and behavioral outcomes at 4 years of age: A cross-sectional study.

BACKGROUND: Accumulating evidence suggests that breastfeeding exclusivity and duration are positively associated with child cognition. This study investigated whether DNA methylation, an epigenetic mechanism modified by nutrient intake, may contribute to the link between breastfeeding and child cognition. The aim was to quantify the relationship between global DNA methylation and cognition and behavior at 4 years of age. METHODS: Child behavior and cognition were measured at age 4 years using the Wechsler Preschool and Primary Scale of Intelligence, third version (WPPSI-III), and Child Behavior Checklist (CBC). Global DNA methylation (%5-methylcytosines (%5mC)) was measured in buccal cells at age 4 years, using an enzyme-linked immunosorbent assay (ELISA) commercial kit. Linear regression models were used to quantify the statistical relationships. RESULTS: Data were collected from 73 children recruited from the Women and Their Children's Health (WATCH) study. No statistically significant associations were found between global DNA methylation levels and child cognition or behavior (p > .05), though the estimates of effect were consistently negative. Global DNA methylation levels in males were significantly higher than in females (median %5mC: 1.82 vs. 1.03, males and females, respectively, (p < .05)). CONCLUSION: No association was found between global DNA methylation and child cognition and behavior; however given the small sample, this study should be pooled with other cohorts in future meta-analyses.

BCL-2 family isoforms in apoptosis and cancer.

The BCl-2 family has long been identified for its role in apoptosis. Following the initial discovery of BCL-2 in the context of B-cell lymphoma in the 1980s, a number of homologous proteins have since been identified. The members of the Bcl-2 family are designated as such due to their BCL-2 homology (BH) domains and involvement in apoptosis regulation. The BH domains facilitate the family members' interactions with each other and can indicate pro- or anti-apoptotic function. Traditionally, these proteins are categorised into one of the three subfamilies; anti-apoptotic, BH3-only (pro-apoptotic), and pore-forming or 'executioner' (pro-apoptotic) proteins. Each of the BH3-only or anti-apoptotic proteins has a distinct pattern of activation, localisation and response to cell death or survival stimuli. All of these can vary across cell or stress types, or developmental stage, and this can cause the delineation of the roles of BCL-2 family members. Added to this complexity is the presence of relatively uncharacterised isoforms of many of the BCL-2 family members. There is a gap in our knowledge regarding the function of BCL-2 family isoforms. BH domain status is not always predictive or indicative of protein function, and several other important sequences, which can contribute to apoptotic activity have been identified. While therapeutic strategies targeting the BCL-2 family are constantly under development, it is imperative that we understand the molecules, which we are attempting to target. This review, discusses our current knowledge of anti-apoptotic BCL-2 family isoforms. With significant improvements in the potential for splicing therapies, it is important that we begin to understand the distinctions of the BCL-2 family, not limited to just the mechanisms of apoptosis control, but in their roles outside of apoptosis.

Methyl-Donor and Cofactor Nutrient Intakes in the First 2-3 Years and Global DNA Methylation at Age 4: A Prospective Cohort Study.

BACKGROUND: During the early postnatal period, the impact of nutrition on DNA methylation has not been well studied in humans. The aim was to quantify the relationship between one-carbon metabolism nutrient intake during the first three years of life and global DNA methylation levels at four years. DESIGN: Childhood dietary intake was assessed using infant feeding questionnaires, food frequency questionnaires, 4-day weighed food records and 24-h food records. The dietary records were used to estimate the intake of methionine, folate, vitamins B2, B6 and B12 and choline. The accumulative nutrient intake specific rank from three months to three years of age was used for analysis. Global DNA methylation (%5-methyl cytosines (%5-mC)) was measured in buccal cells at four years of age, using an enzyme-linked immunosorbent assay (ELISA) commercial kit. Linear regression models were used to quantify the statistical relationships. RESULTS: Data were collected from 73 children recruited from the Women and their Children's Health (WATCH) study. No association was found between one-carbon metabolism nutrient intake and global DNA methylation levels (P > 0.05). Global DNA methylation levels in males were significantly higher than in females (median %5-mC: 1.82 vs. 1.03, males and females respectively, (P < 0.05)). CONCLUSION: No association was found between the intake of one-carbon metabolism nutrients during the early postnatal period and global DNA methylation levels at age four years. Higher global DNA methylation levels in males warrants further investigation.

DNA methylation profile of triple negative breast cancer-specific genes comparing lymph node positive patients to lymph node negative patients.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no targeted treatment available. Our previous study identified 38 TNBC-specific genes with altered expression comparing tumour to normal samples. This study aimed to establish whether DNA methylation contributed to these expression changes in the same cohort as well as disease progression from primary breast tumour to lymph node metastasis associated with changes in the epigenome. We obtained DNA from 23 primary TNBC samples, 12 matched lymph node metastases, and 11 matched normal adjacent tissues and assayed for differential methylation profiles using Illumina HumanMethylation450 BeadChips. The results were validated in an independent cohort of 70 primary TNBC samples. The expression of 16/38 TNBC-specific genes was associated with alteration in DNA methylation. Novel methylation changes between primary tumours and lymph node metastases, as well as those associated with survival were identified. Altered methylation of 18 genes associated with lymph node metastasis were identified and validated. This study reveals the important role DNA methylation plays in altered gene expression of TNBC-specific genes and lymph node metastases. The novel insights into progression of TNBC to secondary disease may provide potential prognostic indicators for this hard-to-treat breast cancer subtype.

Panel Testing for Familial Breast Cancer: Calibrating the Tension Between Research and Clinical Care.

PURPOSE: Gene panel sequencing is revolutionizing germline risk assessment for hereditary breast cancer. Despite scant evidence supporting the role of many of these genes in breast cancer predisposition, results are often reported to families as the definitive explanation for their family history. We assessed the frequency of mutations in 18 genes included in hereditary breast cancer panels among index cases from families with breast cancer and matched population controls. PATIENTS AND METHODS: Cases (n = 2,000) were predominantly breast cancer-affected women referred to specialized Familial Cancer Centers on the basis of a strong family history of breast cancer and BRCA1 and BRCA2 wild type. Controls (n = 1,997) were cancer-free women from the LifePool study. Sequencing data were filtered for known pathogenic or novel loss-of-function mutations. RESULTS: Excluding 19 mutations identified in BRCA1 and BRCA2 among the cases and controls, a total of 78 cases (3.9%) and 33 controls (1.6%) were found to carry potentially actionable mutations. A significant excess of mutations was only observed for PALB2 (26 cases, four controls) and TP53 (five cases, zero controls), whereas no mutations were identified in STK11. Among the remaining genes, loss-of-function mutations were rare, with similar frequency between cases and controls. CONCLUSION: The frequency of mutations in most breast cancer panel genes among individuals selected for possible hereditary breast cancer is low and, in many cases, similar or even lower than that observed among cancer-free population controls. Although multigene panels can significantly aid in cancer risk management and expedite clinical translation of new genes, they equally have the potential to provide clinical misinformation and harm at the individual level if the data are not interpreted cautiously.

Novel genes associated with lymph node metastasis in triple negative breast cancer.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and no targeted treatments. TNBC patients are more likely to develop metastases and relapse than patients with other breast cancer subtypes. We aimed to identify TNBC-specific genes and genes associated with lymph node metastasis, one of the first signs of metastatic spread. A total of 33 TNBCs were used; 17 of which had matched normal adjacent tissues available, and 15 with matched lymph node metastases. Gene expression microarray analysis was used to reveal genes that were differentially expressed between these groups. We identified and validated 66 genes that are significantly altered when comparing tumours to normal adjacent samples. Further, we identified 83 genes that are associated with lymph node metastasis and correlated these with miRNA-expression. Pathway analysis revealed their involvement in DNA repair, recombination and cell death, chromosomal instability and other known cancer-related pathways. Finally, four genes were identified that were specific for TNBC, of which one was associated with overall survival. This study has identified novel genes involved in LN metastases in TNBC and genes that are TNBC specific that may be used as treatment targets or prognostic indicators in the future.

Reevaluation of the BRCA2 truncating allele c.9976A > T (p.Lys3326Ter) in a familial breast cancer context.

The breast cancer predisposition gene, BRCA2, has a large number of genetic variants of unknown effect. The variant rs11571833, an A > T transversion in the final exon of the gene that leads to the creation of a stop codon 93 amino acids early (K3326*), is reported as a neutral polymorphism but there is some evidence to suggest an association with an increased risk of breast cancer. We assessed whether this variant was enriched in a cohort of breast cancer cases ascertained through familial cancer clinics compared to population-based non-cancer controls using a targeted sequencing approach. We identified the variant in 66/2634 (2.5%) cases and 33/1996 (1.65%) controls, indicating an enrichment in the breast cancer cases (p = 0.047, OR 1.53, 95% CI 1.00-2.34). This data is consistent with recent iCOGs data suggesting that this variant is not neutral with respect to breast cancer risk. rs11571833 may need to be included in SNP panels for evaluating breast cancer risk.