BACKGROUND: Homologous recombination plays a vital role in the occurrence and drug resistance of gastric cancer. This study aimed to screen new gastric cancer diagnostic biomarkers in the homologous recombination pathway and then used radiomic features to construct a prediction model of biomarker expression to guide the selection of chemotherapy regimens.
METHODS: Gastric cancer transcriptome data were downloaded from The Cancer Genome Atlas database. Machine learning methods were used to screen for diagnostic biomarkers of gastric cancer and validate them experimentally. Computed Tomography image data of gastric cancer patients and corresponding clinical data were downloaded from The Cancer Imaging Archive and our imaging centre, and then the Computed Tomography images were subjected to feature extraction, and biomarker expression prediction models were constructed to analyze the correlation between the biomarker radiomics scores and clinicopathological features.
RESULTS: We screened RAD51D and XRCC2 in the homologous recombination pathway as biomarkers for gastric cancer diagnosis by machine learning, and the expression of RAD51D and XRCC2 was significantly positively correlated with pathological T stage, N stage, and TNM stage. Homologous recombination pathway blockade inhibits gastric cancer cell proliferation, promotes apoptosis, and reduces the sensitivity of gastric cancer cells to chemotherapeutic drugs. Our predictive RAD51D and XRCC2 expression models were constructed using radiomics features, and all the models had high accuracy. In the external validation cohort, the predictive models still had decent accuracy. Moreover, the radiomics scores of RAD51D and XRCC2 were also significantly positively correlated with the pathologic T, N, and TNM stages.
CONCLUSIONS: The gastric cancer diagnostic biomarkers RAD51D and XRCC2 that we screened can, to a certain extent, reflect the expression status of genes through radiomic characteristics, which is of certain significance in guiding the selection of chemotherapy regimens for gastric cancer patients.

BACKGROUND: RAD51C and RAD51D are crucial in homologous recombination (HR) DNA repair. The prevalence of the RAD51C and RAD51D mutations in breast cancer varies across ethnic groups. Associations of RAD51C and RAD51D germline pathogenic variants (GPVs) with breast and ovarian cancer predisposition have been recently reported and are of interest.
METHODS: We performed multi-gene panel sequencing to study the prevalence of RAD51C and RAD51D germline mutations among 3728 patients with hereditary breast and/or ovarian cancer (HBOC).
RESULTS: We identified 18 pathogenic RAD51C and RAD51D mutation carriers, with a mutation frequency of 0.13% (5/3728) and 0.35% (13/3728), respectively. The most common recurrent mutation was RAD51D c.270_271dupTA; p.(Lys91Ilefs*13), with a mutation frequency of 0.30% (11/3728), which was also commonly identified in Asians. Only four out of six cases (66.7%) of this common mutation tested positive for homologous recombination deficiency (HRD).
CONCLUSIONS: Taking the family studies in our registry and tumor molecular pathology together, we concluded that this relatively common RAD51D variant showed incomplete penetrance in our local Chinese community. Personalized genetic counseling emphasizing family history for families with this variant, as suggested at the UK Cancer Genetics Group (UKCGG) Consensus meeting, would also be appropriate in Chinese families.

UNASSIGNED: RAD51D has been reported as a breast cancer (BC) and ovarian cancer (OC) predisposition gene, particularly among Caucasian populations. We studied the prevalence of RAD51D variants in Pakistani BC/OC patients.
UNASSIGNED: In total, 371 young or familial BC/OC patients were thoroughly analyzed for RAD51D sequence variants using denaturing high-performance liquid chromatography pursued by DNA sequencing of differentially eluted amplicons. We also assessed the pathogenic effects of novel variants using in-silico algorithms. All detected RAD51D variants were investigated in 400 unaffected controls.
UNASSIGNED: No pathogenic RAD51D variant was detected. However, we identified nine unique heterozygous variants. Of these, two missense variants (p.Pro10Leu and p.Ile311Asn) and one intronic variant (c.481-26_23delGTTC) were classified as in silico-predicted variants of uncertain significance, with a frequency of 0.8% (3/371). The p.Pro10Leu variant was detected in a 28-year-old female BC patient of Punjabi ethnic background, whose mother and maternal cousin had BCs at ages 53 and 40, respectively. This variant was also detected in 1/400 (0.25%) healthy controls, where the control subject\'s daughter had acute lymphoblastic leukemia. The p.Ile311Asn variant was identified in a female BC patient at age 29 of Punjabi ethnicity and in 1/400 (0.25%) healthy controls, where the control subject\'s daughter had Hodgkin\'s disease at age 14. A novel intronic variant, c.481-26_-23delGTTC, was found in a 30-year-old Punjabi female BC patient but not in 400 healthy controls.
UNASSIGNED: No pathogenic RAD51D variant was identified in the current study. Our study data suggested a negligible association of RAD51D variants with BC/OC risk in Pakistani women.

RAD51D mutations have been implicated in the transformation of normal fallopian tube epithelial (FTE) cells into high-grade serous ovarian cancer (HGSOC), one of the most prevalent and aggressive gynecologic malignancies. Currently, no suitable model exists to elucidate the role of RAD51D in disease initiation and progression. Here, we established organoids from primary human FTE and introduced TP53 as well as RAD51D knockdown to enable the exploration of their mutational impact on FTE lesion generation. We observed that TP53 deletion rescued the adverse effects of RAD51D deletion on the proliferation, stemness, senescence, and apoptosis of FTE organoids. RAD51D deletion impaired the homologous recombination (HR) function and induced G2/M phase arrest, whereas concurrent TP53 deletion mitigated G0/G1 phase arrest and boosted DNA replication when combined with RAD51D mutation. The co-deletion of TP53 and RAD51D downregulated cilia assembly, development, and motility, but upregulated multiple HGSOC-associated pathways, including the IL-17 signaling pathway. IL-17A treatment significantly improved cell viability. TP53 and RAD51D co-deleted organoids exhibited heightened sensitivity to platinum, poly-ADP ribose polymerase inhibitors (PARPi), and cell cycle-related medication. In summary, our research highlighted the use of FTE organoids with RAD51D mutations as an invaluable in vitro platform for the early detection of carcinogenesis, mechanistic exploration, and drug screening.

Many breast cancer (BC) predisposition genes encode proteins involved in DNA damage repair (DDR). Identification of germline pathogenic va-riants (PV) in DDR genes raises the question whether their presence can influence the treatment outcomes and potential radiation-induced toxicity in their carriers treated by adjuvant radiotherapy, which has not yet been answered conclusively. We retrospectively examined records of 213 BC patients treated by adjuvant radiotherapy, including 39 (18.3 %) BRCA1/2 PV carriers, 25 carriers (11.7 %) of PV in other breast cancer-predisposing genes, and 149 (70 %) non-carriers. Our goal was to examine 5-year disease-free survival (5y DFS) rates among the study groups and determine the impact of radiotherapy-induced lymphopoenia (RIL) on this outcome. While we found no significant difference in 5y DFS between non-carriers and carriers of BRCA mutations (86.4 % vs 78.4 % P = 0.24) or between non-carriers and other studied mutations (86.4 % vs 93.3 %; P = 0.27), respectively, we observed that the entire group of PV carriers had a significantly lower proportion of patients without RIL (P = 0.04) than the non-carriers. In contrast, subsequent analyses indicated a non-significant trend toward an increased 5y DFS in PV carriers with RIL. Our single-centre study indicated that the presence of PV in BC patients has an insignificant impact on DFS but can reduce the risk of RIL associated with adjuvant radiotherapy. It remains unclear whether this may result from the paradoxical activation of anti-tumour immunity in PV carriers with higher lymphocyte consumption resulting from higher immune effectiveness.

Ovarian cancer is the leading cause of gynecologic cancer-related death, and PARP inhibitors (PARPis) are becoming a promising treatment option, as demonstrated by recent clinical trials. After PARPi exposure, somatic reversion mutations in the homologous recombination genes may be a mechanism of PARPi resistance in ovarian carcinoma. We present an ovarian cancer case of a 61-year-old woman, who underwent routine tumor reduction surgery followed by platinum and PARPis. She demonstrated a good response to PARPis for 15 months before recurrence and secondary tumor reduction surgery. However, post-surgery platinum and PARPi treatment only kept the disease stable for 5 months. A potential molecular mechanism for PARPi resistance was investigated using next-generation sequencing, immunohistochemical (IHC) staining, and other functional assays. A germline RAD51D loss-of-function mutation was found in the reported case (LRG_516t1:c.270_271dup p1:p.(Lys91fs*13)). Subsequently, a secondary mutation (LRG_516t1:c.271_282 del) was identified in the same locus of the germline duplication in the post-progression biopsies and ctDNA. The IHC staining supported low expression of RAD51D in the initial tumor tissue, but the expression was restored after the correction of the open reading frame by the secondary mutation. The in vitro results supported that the loss-of-function mutation of RAD51D was the basis for the initial response to the platinum and PARPi therapy, while the newly acquired reversion mutation could be attributed to the observed PARPi resistance. An acquired mutation can reverse a loss-of-function change in RAD51D and can result in PARPi resistance in a hereditary ovarian cancer patient. Liquid biopsy could be considered for longitudinal monitoring in ovarian patients under PARPi-based therapy, which can identify acquired resistant mutations earlier and facilitate precision management.

For many individuals harboring a variant of uncertain functional significance (VUS) in a homologous recombination (HR) gene, their risk of developing breast and ovarian cancer is unknown. Integral to the process of HR are BRCA1 and regulators of the central HR protein, RAD51, including BRCA2, PALB2, RAD51C and RAD51D. Due to advancements in sequencing technology and the continued expansion of cancer screening panels, the number of VUS identified in these genes has risen significantly. Standard practices for variant classification utilize different types of predictive, population, phenotypic, allelic and functional evidence. While variant analysis is improving, there remains a struggle to keep up with demand. Understanding the effects of an HR variant can aid in preventative care and is critical for developing an effective cancer treatment plan. In this review, we discuss current perspectives in the classification of variants in the breast and ovarian cancer genes BRCA1, BRCA2, PALB2, RAD51C and RAD51D.

Breast cancer (BC) is the most common cancer and the leading cause of cancer death in women worldwide. Since the discovery of the highly penetrant susceptibility genes BRCA1 and BRCA2, many other predisposition genes that confer a moderate risk of BC have been identified. Advances in multigene panel testing have allowed the simultaneous sequencing of BRCA1/2 with these genes in a cost-effective way. Germline DNA from 521 cases with BC fulfilling diagnostic criteria for hereditary BC were screened with multigene NGS testing. Pathogenic (PVs) and likely pathogenic (LPVs) variants in moderate penetrance genes were identified in 15 out of 521 patients (2.9%), including 2 missense, 7 non-sense, 1 indel, and 3 splice variants, as well as two different exon deletions, as follows: ATM (n = 4), CHEK2 (n = 5), PALB2 (n = 2), RAD51C (n = 1), and RAD51D (n = 3). Moreover, the segregation analysis of PVs and LPVs into first-degree relatives allowed the detection of CHEK2 variant carriers diagnosed with in situ melanoma and clear cell renal cell carcinoma (ccRCC), respectively. Extended testing beyond BRCA1/2 identified PVs and LPVs in a further 2.9% of BC patients. In conclusion, panel testing yields more accurate genetic information for appropriate counselling, risk management, and preventive options than assessing BRCA1/2 alone.

Meiotic crossovers ensure accurate chromosome segregation and increase genetic diversity. RAD51C and RAD51D play an early role in facilitating RAD51 during homologous recombination. However, their later function in meiosis is largely unknown in plants. Here, through targeted disruption of RAD51C and RAD51D, we generated three new mutants and revealed their later meiotic role in crossover maturation. The rad51c-3 and rad51d-4 mutants showed a mixture of bivalents and univalents and no chromosomal entanglements, whereas rad51d-5 exhibited an intermediate phenotype with reduced chromosomal entanglements and increased bivalent formation compared with knockout alleles. Comparisons of RAD51 loadings and chromosomal entanglements in these single mutants, rad51c-3 rad51d-4, rad51c-3 dmc1a dmc1b, and rad51d-4 dmc1a dmc1b suggest that the retained level of RAD51 in mutants is required for uncovering their function in crossover formation. Reductions in chiasma frequency and later HEI10 foci in these mutants support that crossover maturation requires RAD51C and RAD51D. Moreover, interaction between RAD51D and MSH5 indicates that RAD51 paralogs may cooperate with MSH5 to ensure accurate Holliday junction processing into crossover products. This finding of the role of RAD51 paralogs in crossover control may be conserved from mammals to plants and advances our current understanding of these proteins.

Plants cope with various recurring stress conditions that often induce DNA damage, ultimately affecting plant genome integrity, growth, and productivity. The CROWDED NUCLEI (CRWN) family comprises lamin-like proteins with multiple functions, such as regulating gene expression, genome organization, and DNA damage repair in Arabidopsis (Arabidopsis thaliana). However, the mechanisms and consequences of CRWNs in DNA damage repair are largely unknown. Here, we reveal that CRWNs maintain genome stability by forming repairing nuclear bodies at DNA double-strand breaks. We demonstrate that CRWN1 and CRWN2 physically associate with the DNA damage repair proteins RAD51D and SUPPRESSOR OF NPR1-1 Inducible 1 (SNI1) and act in the same genetic pathway to mediate this process. Moreover, CRWN1 and CRWN2 partially localize at γ-H2AX foci upon DNA damage. Notably, CRWN1 and CRWN2 undergo liquid-liquid phase separation to form highly dynamic droplet-like structures with RAD51D and SNI1 to promote the DNA damage response (DDR). Collectively, our data shed light on the function of plant lamin-like proteins in the DDR and maintenance of genome stability.