Germline Tissues Research Articles

Stable introduction of Wolbachia wPip into invasive Anopheles stephensi for potential malaria control.

The spread and invasion of the urban malaria vector Anopheles stephensi has emerged as a significant threat to ongoing malaria control and elimination efforts, particularly in Africa. The successful use of the maternally inherited endosymbiotic bacterium Wolbachia for arbovirus control has inspired the exploration of similar strategies for managing malaria vectors, necessitating the establishment of a stable Wolbachia-Anopheles symbiosis. In this study, we successfully transferred Wolbachia wPip into An. stephensi, resulting in the establishment of a stable transinfected HP1 line with 100% maternal transmission efficiency. We demonstrate that wPip in the HP1 line induces nearly complete unidirectional cytoplasmic incompatibility (CI) and maintains high densities in both somatic and germline tissues. Despite a modest reduction in lifespan and female reproductive capacity, our results suggest the Wolbachia infection in the HP1 line has little impact on life history traits, body size, and male mating competitiveness, as well as the ability of its larvae to tolerate rearing temperatures up to 38°C, although wPip densities moderately decrease when larvae are exposed to a constant 33°C and diurnal cyclic temperatures of 27-36°C and 27-38°C. These findings highlight the potential of the HP1 line as a robust candidate for further development in malaria control.

Abstract C156: Examining the role of T cell responses in colorectal cancer sex disparities

Abstract Purpose: Persistent sex disparities in colorectal cancer (CRC) incidence and mortality exist and vary by race and ethnicity. The overarching goal of this proposal is to quantify differences in the CRC tumor immune microenvironment by sex in the Latino Colorectal Cancer Consortium (LC3). Methods: The LC3 is a consortium of pooled individual-level data from Latino participants diagnosed with CRC from California, Florida, and Puerto Rico. Biospecimens, epidemiologic data, and clinical outcomes were collected from each contributing study. DNA was extracted from tumor tissue and paired germline or normal tissue samples. Whole exome sequencing was performed using established protocols. Global genetic ancestry was estimated from whole exome sequence data. High-throughput T cell receptor (TCR) sequencing was used to estimate features of the T cell receptor (TCR) repertoire (e.g. abundance, clonality), and tumor-infiltrating lymphocyte (TIL) counts were obtained through pathology review. We evaluated differences in TCR abundance and TILs by sex using linear models for continuous measurements and generalized linear models for binary or count measurements. Primary analyses were unadjusted, and secondary analyses adjusted for age at diagnosis, stage at diagnosis, tumor location (right colon vs. left colorectum), and microsatellite instability (MSI) status. To examine how genetic ancestry modified the relationship between sex and T cell responses, we fit separate models with an interaction term between each genetic ancestry proportion variable (i.e. African, European, Native American) and sex, and assessed effect modification using a likelihood ratio test. Results: To date, 240 participants had TCR repertoire profiling and whole exome sequencing data generated. 52% were male, with a mean age of 58. 17% of participants had rectal tumors. With respect to Hispanic ethnic heritage or country of origin, 49% of participants were of Mexican descent, followed by 13% of Central or South American descent, and 10% were of Puerto Rican descent. Mean T cell abundance was 0.18 (standard deviation (SD): 0.16) and mean clonality was 0.05 (SD: 0.05). Further analysis is underway. Conclusions: This project leverages within-Latino genetic diversity to disentangle the contributions of sex and genetic ancestry to variability in immune function and to understand how this may further sex disparities in CRC. We anticipate that results from this analysis will provide insight on the relationship between the tumor immune microenvironment and sex disparities in CRC. Citation Format: Nicole C. Loroña, Ya-Yu Tsai, Daniel Sobieski, Eric Cockman, Stephanie L. Schmit, Jane C. Figueiredo. Examining the role of T cell responses in colorectal cancer sex disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C156.

Exploring TSGA10 Function: A Crosstalk or Controlling Mechanism in the Signaling Pathway of Carcinogenesis?

Cancer-specific antigens have been a significant area of focus in cancer treatment since their discovery in the mid-twentieth century. Cancer germline antigens are a class of antigens specifically overexpressed in germline tissues and cancer cells. Among these, TSGA10 (testis-specific gene antigen 10) is of great interest because of its crucial impact on cancer progression. Early studies explored TSGA10 expression in a variety of cancer types. More recent studies revealed that TSGA10 can suppress tumor progression by blocking cancer cell metabolism, angiogenesis, and metastasis. An open question regarding the TSGA10 is why cancer cells must express a protein that prevents their progression. To answer this question, we conducted a comprehensive review to engage the TSGA10 in the context of the current understanding of "malignant transformation". This review demonstrated that TSGA10 expression level in cancer cells depends on the cancer stage across malignant transformation. In addition, we evaluated how TSGA10 expression can prevent the "cancer hallmarks". Given this information, TSGA10 can be of great interest in developing effective targeted anti-cancer therapies.

Somatic and germline aberrations in homologous recombination repair genes among Chinese high-risk breast cancer patients by multi-gene next-generation sequencing.

Recently, genes involved in homologous recombination repair (HRR) pathway have been extensively studied. However, the landscapes of HRR gene mutations remain poorly defined in Chinese high-risk breast cancer (BC) patients. Our study aims to identify the status of germline and somatic HRR gene mutations and their association with clinicopathological features in these patients. A total of 100 high-risk BC patients from our institution who underwent paired peripheral blood germline and BC tissues somatic 26 genes next-generation sequencing (NGS) from January 2018 to July 2023 were enrolled for retrospective analysis. Out of 100 high-risk BC patients, 55 (55%) had at least one germline or somatic mutation in HRR genes. Among them, 22% carried germline pathogenic variants (19 BRCA1/2 and 3 non-BRCA genes), 9% harbored somatic pathogenic mutations (3 BRCA1/2 and 6 non-BRCA genes). Among high-risk factors, family history and early onset BC showed a correlation with HRR gene mutations (p < 0.05). BRCA1 germline and HRR gene somatic mutations showed a correlation with TNBC, but BRCA2 germline mutations were associated with Luminal B/HER2-negative BC (p < 0.05). Patients with HRR gene somatic pathogenic variant more likely had a lympho-vascular invasion and distant metastasis (p < 0.05). The prevalence of HRR gene germline and somatic mutations were higher in Chinese BC patients with high risk factors. We strongly recommend that these high-risk BC patients receive comprehensive gene mutation testing, especially HRR genes, which are not only related to genetic consultation for BC patients and provide a theoretical basis for necessary prevention and individualized treatment.

The germline coordinates mitokine signaling

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C.elegans, neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the Wnt ligand EGL-20, which activate the mitochondrial unfolded protein response (UPRMT) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-periphery UPRMT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, Wnt and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPRMT activation. We also find that the germline tissue itself is essential for UPRMT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

Advancing cancer MRD monitoring through tumor whole-genome informed, duplex ctDNA sequencing.

e15044 Background: Circulating tumor DNA (ctDNA), a cell-free DNA (cfDNA) present in the plasma of cancer patients, originates from cancer cells and serves as a crucial biomarker during cancer treatment. Despite its importance, the current limit of detection (LoD) of conventional ctDNA assays is suboptimal (0.01%; 10-4) for detecting ctDNA from microscopic residual and recurrent cancer tissues. To address this, we integrated two cutting-edge techniques: whole-genome sequencing (WGS) of primary cancer tissue (a mutation capture step) and duplex DNA sequencing-based cfDNA sequencing (mutation recapture steps). Methods: Thirteen colorectal cancer patients participated in this study. Primary tumor tissues and matched normal blood tissues were collected for WGS in the mutation capture step, while plasma samples for cfDNA sequencing in the mutation recapture steps were obtained just prior to colorectal cancer surgery and 1-month post-surgery follow-up. Tumor DNA and germline DNA were analyzed using CancerVision, a CLIA-certified clinical-grade cancer WGS platform, in the mutation capture step. In the mutation recapture steps, somatically acquired mutations from the previous step were tracked in cfDNA samples using the Concatenating Original Duplex for Error Correction (CODEC) technique, a duplex DNA sequencing technique that examines both Watson and Crick strands of double-stranded DNA molecules to discern true mutations from sequencing noise. Results: The median WGS depth of tumor and germline tissues was 40x and 20x, respectively. The mean depth of cfDNA WGS with CODEC was 25x, with a median duplex sequencing rate of 70%. Overall, 4,828-301,434 somatic base substitutions (SBSs) per sample were discovered in the capture step. Four of the thirteen tumors exhibited hypermutator characteristics with microsatellite instability features. The CODEC technique demonstrated excellent capability in tracing cancer-specific mutations in cfDNA sequencing with a technical sequencing error rate of 4 x 10-7, defining the maximum technical LoD, which is 250-fold lower errors than conventional sequencing. In our sample cohort, tumor fractions in cfDNA sequencing were robustly estimated from 0.001% (10-5), below the LoD of conventional MRD methods. Conclusions: Our integration of tumor-WGS-informed duplex cfDNA sequencing methods significantly enhances the sensitivity of sequencing-based MRD assays, achieving a technical LoD limit of 10-7 for detecting ctDNA in cancer patients. These approaches represent a breakthrough in monitoring MRD in real-world cancer patients.

Abstract PO2-13-10: Predicting outcomes in locally advanced breast cancer through profiling post-neoadjuvant tissue-based minimal residual disease

Abstract Background: Neoadjuvant chemotherapy (NAC) for locally advanced breast cancer can down-stage disease prior to surgery, monitor chemo-sensitivity, and provide key prognostic information via pathologic response that guides adjuvant treatment. Greater residual disease as assessed by the residual cancer burden (RCB) index is associated with inferior outcomes but imperfectly predicts recurrence, particularly in triple negative breast cancer (TNBC) where there is heterogeneity of outcomes across RCB scores. As an alternative to RCB and PCR, blood-based minimal residual disease (MRD) from circulating tumor DNA (ctDNA) has been explored. However, poor sensitivity limits ctDNA, which is typically negative post-NAC when treatment decisions are made in patients who later recur. Methods: To address this lack of sensitivity, we developed tissue-based MRD (t-MRD) to assess molecular disease burden in post-NAC breast and lymph node resection tissue. We performed whole exome sequencing on diagnostic tumor and matched germline tissues from five pilot patients with early-stage TNBC with a range of responses to NAC. For each patient, we designed a personalized assay to track tumor-specific SNVs, combined with limited panel of genes recurrently mutated in breast cancer, to enable MRD detection. We then applied this assay to DNA from individual pathology blocks from the tumor resection via ultra-deep targeted hybrid capture. We assessed each sample for the presence of MRD using a Monte Carlo statistical framework to integrate all tumor mutations as previously described (Newman et al, Nature Biotechnology 2016). We aggregated results across all pathology blocks from each case to define each patient’s t-MRD profile and assessed the performance of t-MRD versus gold standard pathologic assessment. Results: We tracked a mean of 136 MRD reporters per case across 119 post-NAC blocks derived from five patients. Twenty-five samples had pathologic residual disease and 28 had detectable t-MRD. There was strong concordance between t-MRD mean allele frequency (AF) and tumor cellularity as assessed by pathology, defined as percent cellularity within the region containing viable tumor (Spearman r = 0.9052, p&amp;lt; 0.0001), and excellent classification of true positive pathology samples by genomic sequencing (AUC = 0.9675, p &amp;lt; 0.0001). Samples (n = 6) where t-MRD but not pathologic residual disease was detected had a lower mean AF than those where there was also histologic residual disease (p=0.0001). Among the five studied cases, we confirmed a molecular complete response with no t-MRD detected in the RCB-0 case (0/44 t-MRD positive blocks). In the two RCB-III cases, we observed more diffuse t-MRD than that assessed by pathology (n = 4 additional blocks with t-MRD but not pathologic disease detected), and in the RCB-II case, we detected minimal t-MRD in only a single block where pathology did not detect residual disease. Significance: By applying blood-based MRD assessment tools to tumor tissue, we developed t-MRD to profile molecular residual disease at the landmark post-NAC timepoint when disease burden is low and adjuvant treatment decisions are made. We demonstrated high concordance with gold standard pathologic assessment, with increased sensitivity and minimal false positive signal. Further studies assessing the additional prognostic value of t-MRD beyond stage-based prognostication and post-NAC RCB scores are ongoing. Citation Format: Julia Ransohoff, Mia Carleton, Alisha Birk, Gregory Bean, Melinda Telli, Ash Alizadeh, James Ford, George Duran, Michael Khodadoust, David Kurtz. Predicting outcomes in locally advanced breast cancer through profiling post-neoadjuvant tissue-based minimal residual disease [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-10.

Combinations of the azaquinazoline anti-Wolbachia agent, AWZ1066S, with benzimidazole anthelmintics synergise to mediate sub-seven-day sterilising and curative efficacies in experimental models of filariasis.

Lymphatic filariasis and onchocerciasis are two major neglected tropical diseases that are responsible for causing severe disability in 50 million people worldwide, whilst veterinary filariasis (heartworm) is a potentially lethal parasitic infection of companion animals. There is an urgent need for safe, short-course curative (macrofilaricidal) drugs to eliminate these debilitating parasite infections. We investigated combination treatments of the novel anti-Wolbachia azaquinazoline small molecule, AWZ1066S, with benzimidazole drugs (albendazole or oxfendazole) in up to four different rodent filariasis infection models: Brugia malayi-CB.17 SCID mice, B. malayi-Mongolian gerbils, B. pahangi-Mongolian gerbils, and Litomosoides sigmodontis-Mongolian gerbils. Combination treatments synergised to elicit threshold (>90%) Wolbachia depletion from female worms in 5 days of treatment, using 2-fold lower dose-exposures of AWZ1066S than monotherapy. Short-course lowered dose AWZ1066S-albendazole combination treatments also delivered partial adulticidal activities and/or long-lasting inhibition of embryogenesis, resulting in complete transmission blockade in B. pahangi and L. sigmodontis gerbil models. We determined that short-course AWZ1066S-albendazole co-treatment significantly augmented the depletion of Wolbachia populations within both germline and hypodermal tissues of B. malayi female worms and in hypodermal tissues in male worms, indicating that anti-Wolbachia synergy is not limited to targeting female embryonic tissues. Our data provides pre-clinical proof-of-concept that sub-seven-day combinations of rapid-acting novel anti-Wolbachia agents with benzimidazole anthelmintics are a promising curative and transmission-blocking drug treatment strategy for filarial diseases of medical and veterinary importance.

Aging disrupts spatiotemporal regulation of germline stem cells and niche integrity.

A major factor driving stem cell decline is stem cell niche aging, but its molecular mechanism remains elusive. We use the Caenorhabditis elegans distal tip cell (DTC), the mesenchymal niche that employs Notch signaling to regulate germline stem cells (GSCs), as an in vivo niche aging model and delineate the molecular details of the DTC/niche aging process. Here, we demonstrate that a drastic decrease in C. elegans germline fecundity, which begins even in early adulthood, is mainly due to an age-induced disruption in spatial regulation of Notch-dependent transcription in the germline combined with a moderate reduction in Notch transcription at both tissue and cellular levels. Consequently, the Notch-responsive GSC pool shifts from the distal end of the gonad to a more proximal region, disrupting the distal-to-proximal germline polarity. We find that this GSC pool shift is due to a dislocation of the DTC/niche nucleus, which is associated with age-induced changes in the structure and morphology of the DTC/niche. Our findings reveal a critical link between physiological changes in the aging niche, their consequences in stem cell regulation, and germline tissue functions.

Characterization of the distribution and dynamics of chromatin states in the C. elegans germline reveals substantial H3K4me3 remodeling during oogenesis.

Chromatin organization in the C. elegans germline is tightly regulated and critical for germ cell differentiation. Although certain germline epigenetic regulatory mechanisms have been identified, how they influence chromatin structure and ultimately gene expression remains unclear, in part because most genomic studies have focused on data collected from intact worms comprising both somatic and germline tissues. We therefore analyzed histone modification and chromatin accessibility data from isolated germ nuclei representing undifferentiated proliferating and meiosis I populations to define chromatin states. We correlated these states with overall transcript abundance, spatiotemporal expression patterns, and the function of small RNA pathways. Because the essential role of the germline is to transmit genetic information and establish gene expression in the early embryo, we compared epigenetic and transcriptomic profiles from undifferentiated germ cells to those of embryos to define the epigenetic changes during this developmental transition. The active histone modification H3K4me3 shows particularly dynamic remodeling as germ cells differentiate into oocytes, which suggests a mechanism for establishing early transcription of essential genes during zygotic genome activation. This analysis highlights the dynamism of the chromatin landscape across developmental transitions and provides a resource for future investigation into epigenetic regulatory mechanisms in germ cells.

Germ Line/Multipotency Genes Show Differential Expression during Embryonic Development of the Annelid Enchytraeus coronatus.

Germ line development and the origin of the primordial germ cells (PGCs) are very variable and may occur across a range of developmental stages and in several developmental contexts. In establishing and maintaining germ line, a conserved set of genes is involved. On the other hand, these genes are expressed in multipotent/pluripotent cells that may give rise to both somatic and germline cells. To begin elucidating mechanisms by which the germ line is specified in Enchytraeus coronatus embryos, we identified twenty germline/multipotency genes, homologs of Vasa, PL10, Piwi, Nanos, Myc, Pumilio, Tudor, Boule, and Bruno, using transcriptome analysis and gene cloning, and characterized their expression by whole-mount in situ hybridization. To answer the question of the possible origin of PGCs in this annelid, we carried out an additional description of the early embryogenesis. Our results suggest that PGCs derive from small cells originating at the first two divisions of the mesoteloblasts. PGCs form two cell clusters, undergo limited proliferation, and migrate to the developing gonadal segments. In embryos and juvenile E. coronatus, homologs of the germline/multipotency genes are differentially expressed in both germline and somatic tissue including the presumptive germ cell precursors, posterior growth zone, developing foregut, and nervous system.

Localization and tissue tropism of the symbiont Microsporidia MB in the germ line and somatic tissues of Anopheles arabiensis.

Microsporidia MB is a symbiont with a strong malaria transmission-blocking phenotype in Anopheles arabiensis. It spreads in mosquito populations through mother-to-offspring and sexual transmission. The ability of Microsporidia MB to block Plasmodium transmission, together with its ability to spread within Anopheles populations and its avirulence to the host, makes it a very attractive candidate for developing a key strategy to stop malaria transmissions. Here, we report tissue tropism and localization patterns of Microsporidia MB, which are relevant to its transmission. We find that Microsporidia MB accumulates in Anopheles arabiensis tissues, linked to its sexual and vertical transmission. Its prevalence and intensity in the tissues over the mosquito life cycle suggest adaptation to maximize transmission and avirulence in Anopheles arabiensis. These findings provide the foundation for understanding the factors that may affect Microsporidia MB transmission efficiency. This will contribute to the development of strategies to maximize Microsporidia MB transmission to establish and sustain a high prevalence of the symbiont in Anopheles mosquito populations for malaria transmission blocking.

Effects of paternal and chronological age on BEGAIN methylation and its possible role in autism.

Children from old fathers carry an increased risk for autism spectrum (ASD) and other neurodevelopmental disorders, which may at least partially be mediated by paternal age effects on the sperm epigenome. The brain enriched guanylate kinase associated (BEGAIN) protein is involved in protein-protein interactions at and transmission across synapses. Since several epigenome-wide methylation screens reported a paternal age effect on sperm BEGAIN methylation, here we confirmed a significant negative correlation between BEGAIN promoter methylation and paternal age, using more sensitive bisulfite pyrosequencing and a larger number of sperm samples. Paternal age-associated BEGAIN hypomethylation was also observed in fetal cord blood (FCB) of male but not of female offspring. There was no comparable maternal age effect on FCB methylation. In addition, we found a significant negative correlation between BEGAIN methylation and chronological age (ranging from 1 to 70 years) in peripheral blood samples of male but not of female donors. BEGAIN hypomethylation was more pronounced in male children, adolescents and adults suffering from ASD compared to controls. Both genetic variation (CC genotype of SNP rs7141087) and epigenetic factors may contribute to BEGAIN promoter hypomethylation. The age- and sex-specific BEGAIN methylation trajectories in the male germ line and somatic tissues, in particular the brain, support a role of this gene in ASD development.

Pediatric Burkitt Leukemia with Underlying MSH2, MUTYH and POT1 Germline Variants: The Complexities and Relevance of Understanding Cancer Predisposition

Monoallelic germline variants in POT1 can lead to POT1-tumor predisposition (POT1-TPD), an autosomal dominant disorder leading to a spectrum of neoplasia, predominantly, melanoma, glioma, angiosarcoma and chronic lymphocytic leukemia (CLL). Recently, germline POT1 variants have been described to be associated with familial clonal hematopoiesis syndrome.The POT1 protein forms a part of shelterin complex, which has been implicated in DNA damage protection, telomere length regulation and chromosomal stability. The proposed mechanism of oncogenesis by POT1 variants is presumed to be secondary to increased telomere length causing genomic instability and cellular immortality. While POT1-TPD can cause CLL in adulthood, childhood leukemias have not been commonly observed. Here, we present a case of a previously healthy 12-year-old male diagnosed with Burkitt leukemia. He presented with leukocytosis and tumor lysis syndrome. Bone marrow evaluation revealed hypercellular marrow involved by Burkitt lymphoma, comprising over 90% marrow cellularity. Flow cytometry showed an abnormal lambda monotypic B-lymphoid population positive for CD10, CD19, CD20, CD22 and CD45. FISH studies demonstrated low-level MYC rearrangement (9%); negative for BCL2 and BCL6 rearrangement. EBV staining was negative. Acute leukemia NGS panel (63 genes) revealed a Tier 1 somatic variant in PHF6. RNA sequencing did not detect additional fusions. His chemotherapy course was complicated by significant toxicities including prolonged count recovery, severe peripheral neuropathy, neurotoxicity, kidney injury and several infectious complications. Given extensive family history of cancers on the maternal side including colorectal, duodenal, brain and skin cancers, a germline cancer predisposition NGS panel was sent from cultured skin fibroblast and identified two heterozygous pathogenic variants in MSH2 (c.942+3A&amp;gt;T) and MUTYH (c.1187G&amp;gt;A) and a heterozygous likely pathogenic variant in POT1 (c.233T&amp;gt;C). To delineate potential roles of these germline variants in oncogenesis, additional testing was performed. Immunohistochemical staining of bone marrow revealed intact nuclear expression of MLH1, PMS2, MSH2,and MSH6,suggesting microsatellite stability. Extended tumor profiling through whole exome sequencing did not identify additional somatic variants or copy loss of heterozygosity in MSH2, MUTYH and POT1 but additional somatic loss of function variants were seen in CCND3, CASP8, ARID1, and PTPRD.To assess potential functional consequences of the POT1 germline variant on DNA double-strand break (DSB) damage repair, the patient lymphocytes were exposed to 2 Gy ionizing radiation and mobilization and kinetics of DNA DSB repair was assessed at 1h and 24h post-irradiation (IR) using phosphorylation (p) of ATM, SMC1 and H2AX (gamma), and cell death and apoptosis was also assessed at 24h post-irradiation by flow cytometry. There was normal mobilization and repair of DNA DSBs in T, B and NK cells although there was a subset of CD19dim B cells that showed constitutive DNA DSBs at1h with partial repair at 24h. It is not evident that this defect can be attributed to the germline defects as they are also observed in several other clinical contexts, including post-chemotherapy. Telomere length assay from peripheral blood demonstrated telomere length at 50 th percentile in both lymphocytes and granulocytes. Children in families with adult-onset cancer predisposition syndromes may present with cancers but the potential contribution of these germline variants to these childhood cancers is unknown. Our molecular and functional studies on both germline and tumor tissues did not suggest direct contribution of these germline variants on lymphomagenesis, however there might be other mechanism of inactivation not identified from this study. This case highlights the complexities of understanding genetic susceptibility in childhood cancers. We propose a multifactorial oligogenic inheritance of cancer susceptibility in this patient through several mechanisms of genomic instability.

Genomic Characterization of Locally Recurrent Prostate Cancer after Radiation Therapy.

Radiation therapy (RT) is a backbone of treatment for patients with prostate cancer (PCa). However, locally recurrent disease after definitive RT (i.e., radiorecurrent PCa) is not uncommon and is associated with a higher risk of distant metastases and death from PCa. While the genomic landscape of primary PCa is well-characterized, little is known regarding the genomic landscape of radiorecurrent PCa or how this compares to that of primary PCa. We hypothesized that the genomic landscape of radiorecurrent PCa differs significantly from primary PCa and that these differences have clinical relevance. We examined this hypothesis by performing whole exome sequencing (WES) of radiorecurrent PCa. We identified 25 patients with radiorecurrent PCa with available post-RT tissue obtained from biopsy or radical prostatectomy, as well as germline tissue. The tumor and germline tissue for 19 patients successfully underwent WES. We identified genomic variants including single nucleotide variants (SNVs), insertions/deletions, and copy number alterations. Furthermore, we estimated the tumor mutational burden (TMB; number of nonsynonymous mutations per megabase [Mb]) and contribution of individual mutational signatures. We compared our samples to a publicly available large cohort of primary PCa (n = 680) to define genomic alterations unique to radiorecurrent PCa. In the overall cohort of 25 patients, the RT modality included external beam RT (56%), brachytherapy (36%), and combination of both (8%). 40% of patients received upfront androgen deprivation therapy with RT. The median time to local recurrence was 6.5 years. For the 19 radiorecurrent patients with WES data, the median TMB was 2.7 mutations/Mb, which was significantly higher than the median TMB of 0.7 mutation/Mb for primary PCa (P = 0.002 after multivariable adjustment). Radiorecurrent PCa demonstrated an enrichment of short deletions, with a significantly higher deletion/SNV ratio compared to primary PCa (P = 0.006). TP53 was the most frequently mutated gene in radiorecurrent PCa (n = 6), and the TP53 mutation prevalence was significantly higher compared to primary PCa (32% vs 10%, P = 0.016 by Fisher's exact test). TP53 was also determined to be recurrently mutated using MutSigCV (Q = 0.0003). Additionally, 3 samples demonstrated evidence of whole genome doubling. Radiorecurrent PCa has a distinct genomic profile compared to primary PCa, characterized by a higher TMB with an enrichment of short deletions as part of the mutational composition, which may be a scar of nonhomologous end joining subsequent to RT-induced DNA double-stranded breaks. In addition, TP53 mutations may be of functional consequence in radiorecurrent PCa. Further efforts are underway to examine other genomic features apparent in WES data, as well as perform whole transcriptome sequencing to provide complementary insights into radiorecurrent PCa.

Vasa, Piwi, and Pl10 Expression during Sexual Maturation and Asexual Reproduction in the Annelid Pristina longiseta.

Naidids are tiny, transparent freshwater oligochaetes, which are well known for their ability to propagate asexually. Despite the fact that sexually mature individuals and cocoons with embryos are sometimes found in nature, in long-period laboratory cultures, worms reproduce agametically only. In this paper, we showed, for the first time, the expression of Vasa, Piwi, and Pl10 homologs in mature Pristina longiseta worms with well-developed reproductive system structures and germ cells. Although the animals have been propagated asexually by paratomic fission for over 20 years in our lab, some individuals become sexualized under standard conditions for our laboratory culture and demonstrate various stages of maturation. The fully matured animals developed a complete set of sexual apparatus including spermatheca, atrium, seminal vesicles, and ovisac. They also had a clitellum and were able to form cocoons. The cues for the initiation of sexual maturation are still unknown for P. longiseta; nevertheless, our data suggest that the laboratory strain of P. longiseta maintains the ability to become fully sexually mature and to establish germline products even after a long period of agametic reproduction. On the other hand, many of the sexualized worms formed a fission zone and continued to reproduce asexually. Thus, in this species, the processes of asexual reproduction and sexual maturation do not preclude each other, and Vasa, Piwi, and Pl10 homologs are expressed in both somatic and germline tissue including the posterior growth zone, fission zone, nervous system, germline cells, and gametes.

Germline progenitors and oocyte production in the Honeybee Queen Ovary.

Understanding the reproduction of honeybee queens is crucial to support populations of this economically important insect. Here we examine the structure of the honeybee ovary to determine the nature of the germline progenitors in the ovary. Using a panel of marker genes that mark somatic or germline tissue in other insects we determine which cells in the honeybee ovary are somatic and which germline. We examine patterns of cell division, and demonstrate that, unlike Drosophila, there is no evidence of single germline stem cells that provide the germline in honeybees. Germline progenitors are clustered in groups of 8 cells, joined by a polyfusome, and collections of these, in each ovariole, appear to maintain the germline during reproduction. We also show that these 8-cell clusters can divide, and that their division occurs such that the numbers of germline progenitors are relatively constant over the reproductive life of queen honeybees. This information helps us to understand the diversity of structures in insect reproduction, and provide information to better support honeybee reproduction.

Maternal effect senescence via reduced DNA repair ability in the three-spined stickleback.

Maternal effect senescence, a decline in offspring viability with maternal age, has been documented across diverse animals, but its mechanisms remain largely unknown. Here, we test maternal effect senescence and explore its possible molecular mechanisms in a fish. We compared the levels of maternal mRNA transcripts of DNA repair genes and mtDNA copies in eggs and the levels of DNA damage in somatic and germline tissues between young and old female sticklebacks. We also tested, in an in vitro fertilization experiment, whether maternal age and sperm DNA damage level interactively influence the expression of DNA repair genes in early embryos. Old females transferred less mRNA transcripts of DNA repair genes into their eggs than did young females, but maternal age did not influence egg mtDNA density. Despite a higher level of oxidative DNA damage in the skeletal muscle, old females had a similar level of damage in the gonad to young females, suggesting the prioritization for germline maintenance during ageing. The embryos of both old and young mothers increased the expression of DNA repair genes in response to an increased level of oxidative DNA damage in sperm used for their fertilization. The offspring of old mothers showed higher rates of hatching, morphological deformity and post-hatching mortality and had smaller body size at maturity. These results suggest that maternal effect senescence may be mediated by reduced capacity of eggs to detect and repair DNA damages, especially prior to the embryonic genomic activation.

Genomic characterization of locally recurrent prostate cancer after radiation therapy.

e17002 Background: Radiation therapy (RT) is a backbone of treatment for patients with prostate cancer (PCa). However, locally recurrent disease after definitive RT (i.e. radiorecurrent PCa) is not uncommon and is associated with a higher risk of distant metastases and death from PCa. While the genomic landscape of primary PCa is well-characterized, little is known regarding the genomic landscape of radiorecurrent PCa or how this compares to that of primary PCa. We hypothesized that the genomic landscape of radiorecurrent PCa differs significantly from primary PCa and that these differences have clinical relevance. We examined this hypothesis by performing whole exome sequencing (WES) of radiorecurrent PCa. Methods: We identified 25 patients with radiorecurrent PCa with available post-RT tissue obtained from biopsy or radical prostatectomy, as well as germline tissue. The tumor and germline tissue for 19 patients successfully underwent WES. We identified genomic variants including single nucleotide variants (SNVs), insertions/deletions, and copy number alterations. Furthermore, we estimated the tumor mutational burden (TMB; number of nonsynonymous mutations per megabase [Mb]) and contribution of individual mutational signatures. We compared our samples to a publicly available large cohort of primary PCa (n = 680) to define genomic alterations unique to radiorecurrent PCa. Results: In the overall cohort of 25 patients, the RT modality included external beam RT (56%), brachytherapy (36%), and combination of both (8%). 40% of patients received upfront androgen deprivation therapy with RT. The median time to local recurrence was 6.5 years. For the 19 radiorecurrent patients with WES data, the median TMB was 2.7 mutations/Mb, which was significantly higher than the median TMB of 0.7 mutation/Mb for primary PCa ( P= 0.002 after multivariable adjustment). Radiorecurrent PCa demonstrated an enrichment of short deletions, with a significantly higher deletion/SNV ratio compared to primary PCa ( P= 0.006). TP53 was the most frequently mutated gene in radiorecurrent PCa (n = 6), and the TP53 mutation prevalence was significantly higher compared to primary PCa (32% vs 10%, P= 0.016 by Fisher’s exact test). TP53 was also determined to be recurrently mutated using MutSigCV (Q = 0.0003). Additionally, 3 samples demonstrated evidence of whole genome doubling. Conclusions: Radiorecurrent PCa has a distinct genomic profile compared to primary PCa, characterized by a higher TMB with an enrichment of short deletions as part of the mutational composition, which may be a scar of nonhomologous end joining subsequent to RT-induced DNA double-stranded breaks. In addition, TP53 mutations may be of functional consequence in radiorecurrent PCa. Further efforts are underway to examine other genomic features apparent in WES data, as well as perform whole transcriptome sequencing to provide complementary insights into radiorecurrent PCa.

Phenotypic differences related to homologous recombination repair (HRR) gene-mutated metastatic castration-resistant prostate cancer (mCRPC) treated in routine care settings in the United States (US).

e17047 Background: Recent evidence suggests mutations in HRR genes predict clinical benefit from treatment with PARP inhibitors in patients (pts) with mCRPC. Little is known about the prevalence, clinical and prognostic phenotypes associated with HRR-mutated (HRRm) mCRPC pts receiving routine oncology care in the US. We assessed HRRm positivity rate and described difference in phenotypic factors between HRRm+ and HRRm- mCRPC patients receiving routine care in community oncology settings. Methods: In this retrospective cohort study, we identified biologically male pts with mCRPC, ≥18 years, who received HRRm testing and on systemic anti-cancer treatment from 5/1, 2020 – 3/31, 2022, from the Integra Connect database, an electronic medical record (EMR) and claims database of US cancer pts in community oncology. We defined 2 cohorts based on their HRRm status (defined by the presence of an alteration in at least 1 of the following genes: BRCA1/2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, RAD54L). We described age and race (at diagnosis), cancer stage and Gleason score (at staging), and line of therapy (LOT), Eastern Cooperative Oncology Group (ECOG) performance status, prostate-specific antigen (PSA) values and microsatellite instability (MSI) status using values closest to mCRPC diagnosis. HRR tests were stratified by germline, ctDNA, and solid tumor. Results: The study cohort comprised 376 HRRm-tested pts: by ctDNA only – 29%, germline only – 16.2%, solid tissue only – 18.4% and &gt; 2 testing types – 33.5%. 96 had HRRm+ mCRPC (25.5%). Median (IQR) age at diagnosis for both cohorts was 67 (43-89) years and there were no statistically significant differences in age, race, stage, Gleason score, PSA value, LOT or ECOG by HRRm status. HRRm+ rates by ctDNA, germline, and solid tissue testing were 26.6%, 13.1% and 21.7%, respectively. Of the 126 patients with &gt; 2 testing types, HRRm+ rate was 30.9%. A higher, but not statistically significant, proportion of HRRm+ pts had Gleason scores 9-10 compared to HRRm- pts (54.8% vs 48.6%). Of HRRm- pts, 10.2% had MSI-H status compared to 6.5% in HRRm+ pts. Conclusions: In this real-world cohort of HRR gene-tested pts with mCRPC, we observed a 25.5% HRRm+ rate, which suggests the prevalence for HRR mutation is significant enough to warrant testing for all pts with mCRPC. Further research is needed to examine the impact of these findings on overall survival and other clinical outcomes.