Normal Germline Research Articles

A maternal germline mutator phenotype in a family affected by heritable colorectal cancer.

Variation in DNA repair genes can increase cancer risk by elevating the rate of oncogenic mutation. Defects in one such gene, MUTYH, are known to elevate the incidence of colorectal cancer in a recessive Mendelian manner. Recent evidence has also linked MUTYH to a mutator phenotype affecting normal somatic cells as well as the female germline. Here, we use whole genome sequencing to measure germline de novo mutation rates in a large extended family containing both mothers and fathers who are affected by pathogenic MUTYH variation. By developing novel methodology that uses siblings as "surrogate parents" to identify de novo mutations, we were able to include mutation data from several children whose parents were unavailable for sequencing. In the children of mothers affected by the pathogenic MUTYH genotype p.Y179C/V234M, we identify an elevation of the C>A mutation rate that is weaker than mutator effects previously reported to be caused by other pathogenic MUTYH genotypes, suggesting that mutation rates in normal tissues may be useful for classifying cancer-associated variation along a continuum of severity. Surprisingly, we detect no significant elevation of the C>A mutation rate in children born to a father with the same MUTYH genotype, and we similarly find that the mutator effect of the mouse homolog Mutyh appears to be localized to embryonic development, not the spermatocytes. Our results suggest that maternal MUTYH variants can cause germline mutations by attenuating the repair of oxidative DNA damage in the early embryo.

Transgenerational Epigenetic DNA Methylation Editing and Human Disease.

During gestation, maternal (F0), embryonic (F1), and migrating primordial germ cell (F2) genomes can be simultaneously exposed to environmental influences. Accumulating evidence suggests that operating epi- or above the genetic DNA sequence, covalent DNA methylation (DNAme) can be recorded onto DNA in response to environmental insults, some sites which escape normal germline erasure. These appear to intrinsically regulate future disease propensity, even transgenerationally. Thus, an organism's genome can undergo epigenetic adjustment based on environmental influences experienced by prior generations. During the earliest stages of mammalian development, the three-dimensional presentation of the genome is dramatically changed, and DNAme is removed genome wide. Why, then, do some pathological DNAme patterns appear to be heritable? Are these correctable? In the following sections, I review concepts of transgenerational epigenetics and recent work towards programming transgenerational DNAme. A framework for editing heritable DNAme and challenges are discussed, and ethics in human research is introduced.

Evolution of the Mutation Spectrum Across a Mammalian Phylogeny.

Although evolutionary biologists have long theorized that variation in DNA repair efficacy might explain some of the diversity of lifespan and cancer incidence across species, we have little data on the variability of normal germline mutagenesis outside of humans. Here, we shed light on the spectrum and etiology of mutagenesis across mammals by quantifying mutational sequence context biases using polymorphism data from thirteen species of mice, apes, bears, wolves, and cetaceans. After normalizing the mutation spectrum for reference genome accessibility and k-mer content, we use the Mantel test to deduce that mutation spectrum divergence is highly correlated with genetic divergence between species, whereas life history traits like reproductive age are weaker predictors of mutation spectrum divergence. Potential bioinformatic confounders are only weakly related to a small set of mutation spectrum features. We find that clock-like mutational signatures previously inferred from human cancers cannot explain the phylogenetic signal exhibited by the mammalian mutation spectrum, despite the ability of these signatures to fit each species' 3-mer spectrum with high cosine similarity. In contrast, parental aging signatures inferred from human de novo mutation data appear to explain much of the 1-mer spectrum's phylogenetic signal in combination with a novel mutational signature. We posit that future models purporting to explain the etiology of mammalian mutagenesis need to capture the fact that more closely related species have more similar mutation spectra; a model that fits each marginal spectrum with high cosine similarity is not guaranteed to capture this hierarchy of mutation spectrum variation among species.

Somatic Sequencing and Microsatellite Instability Results From Mismatch Repair-deficient Endometrial Carcinoma Patients Without Lynch Syndrome ("Lynch-like" tumors): Implications for Heritable Cancer Screening, Molecular Prognostication, and Immunotherapeutic Vulnerability.

Immunostaining of endometrial carcinomas for mismatch repair (MMR) protein loss is standard-of-care for Lynch syndrome screening, but also identifies MMR-deficient tumors without germline pathogenic variants. While the majority show MLH1 hypermethylation (MLH1hm), somatic MMR pathogenic variants are increasingly recognized drivers of immunohistochemistry-germline discordance. Because MMR abnormalities with both germline and somatic origins have prognostic significance and impart susceptibility to immune checkpoint inhibitors, it is important to understand how frequently tumors with MMR immunohistochemical loss and normal germline testing ("Lynch-like" tumors) have underlying somatic MMR pathogenic variants. Somatic tumor sequencing±microsatellite instability (MSI) testing was performed on 18 endometrial cancers with MMR immunohistochemical loss but negative MMR germline results and negative MLH1hm where relevant. Tumor sequencing and MSI testing were successful in 94%. Where successful, 80% were MSI-high and 94% had a molecular correlate for the initial immunohistochemical interpretation. The single case without an identified somatic pathogenic variant was MSI-low and initially showed loss of MSH6 by immunohistochemistry but with extremely limited internal control staining. On review, MSH6 immunohistochemistry was reclassified as equivocal, and repeat staining revealed improved control expression with intact MSH6. Following reclassification of this case, 100% tumors with MMR deficiency by immunohistochemistry had at least 1 confirmed somatic MMR pathogenic variant, and 86% were MSI-high. These results demonstrate that when correctly interpreted immunohistochemistry is a strong surrogate for somatic MMR pathogenic variants and support its use as the frontline MMR biomarker in endometrial cancer for heritable screening, molecular prognostic classification, and immunotherapeutic biomarker testing purposes.

The compleX balancing act of controlling X-chromosome dosage and how it impacts mammalian germline development.

In female mammals, the two X chromosomes are subject to epigenetic gene regulation in order to balance X-linked gene dosage with autosomes and in relation to males, which have one X and one Y chromosome. This is achieved by an intricate interplay of several processes; X-chromosome inactivation and reactivation elicit global epigenetic regulation of expression from one X chromosome in a stage-specific manner, whilst the process of X-chromosome upregulation responds to this by fine-tuning transcription levels of the second X. The germline is unique in its function of transmitting both the genetic and epigenetic information from one generation to the next, and remodelling of the X chromosome is one of the key steps in setting the stage for successful development. Here, we provide an overview of the complex dynamics of X-chromosome dosage control during embryonic and germ cell development, and aim to decipher its potential role for normal germline competency.

Abstract 6689: Whole genome cell-free tumor DNA mutational signatures from blood for early detection of recurrence of low stage lung adenocarcinoma

Abstract Introduction: Lung cancer remains the leading cause of cancer-related deaths. Surgery is the best option for early lung cancer, and the role of adjuvant therapy remains controversial. Liquid biopsy offers a noninvasive approach to monitor cancer burden. Targeted sequencing of circulating cell free tumor DNA (ctDNA) in blood has shown success for diagnosis; however, low tumor burden and dynamic evolution of low stage disease is challenging for targeted panels. Thus, we hypothesized that a whole genome sequencing (WGS)-derived patient specific mutational signature from a matched tumor-normal WGS can provide sensitive and specific approach to detect mutations and copy numbers in ctDNA for monitoring of lung adenocarcinoma patients. Methods: We successfully profiled 50 Stage 1 or 2 lung adenocarcinomas. ctDNA was extracted from 1-2 mL of plasma, tumor DNA was extracted from pathology tissue and normal germline DNA from the white blood cells. WGS using was performed on matched tumor and normal DNA, and ctDNA extracted from plasma. WGS coverage was 40x for matched tumor-normal and 20x for ctDNA. We derived a personalized mutational pattern for each tumor and used an AI-based error suppression model for quantification and ultra-sensitive detection of ctDNA in plasma samples. A patient-specific personalized genome-wide compendium of somatic mutations and copy numbers was established and ctDNA tested at 3 to 18 available time points during the therapy or follow up. A personalized mutational signature for detection ctDNA from WGS was quantified and the ctDNA Tumor Fraction (TF) was compared to the clinical status and time to recurrence. Results: Tumor specific signatures were derived from matched tumor-normal samples with >5% tumor purity and <30% duplications rate. Out of all patients, 33 patients showed no recurrence and 12 recurred. Tumor-specific signatures detected the presence of the tumor signature in plasma with TF as low as 10−5. Based on positive minimal residual disease in plasma, the recurrence prediction sensitivity was 0.75 and specificity 0.82, with positive predictive value of 0.6 and negative predictive value 0.9. WGS ctDNA predicted recurrence with a median lead time of 508 days before clinical/imaging recurrence. In one case we were able to identify the second primary by deconvoluting known and novel ctDNA mutations. ctDNA mutational profiles enabled identification of smoking mutational signature matching clinical history, and APOBEC and ageing signatures as well as tumor mutational burden. Conclusions: Patient-specific WGS tumor signature from plasma derived ctDNA enables specific and ultrasensitive tracking of minimal residual disease in low stage lung adenocarcinoma patients. Molecularly positive status can be used to predict recurrence and identify patients with clinical low stage disease that may benefit from adjuvant therapy. Citation Format: Ivy Tran, Alejandro Vargas, Reid Wilkins, Isabella Pizzillo, Kenneth Tokoro, Danielle Afterman, Tomer Lauterman, Maja Kuzman, Santiago Gonzalez, Dunja Glavas, James Smadbeck, Dillon Maloney, Jurica Levatic, Samuel Phillips, Sunil Deochand, Michael Yahalom, Ryan Ptashkin, Iman Tavassoly, Zohar Donenhirsh, Eric White, Ravi Kandasamy, Ury Alon, Paz Polak, Boris Oklander, Asaf Zviran, Matija Snuderl, Harvey I. Pass. Whole genome cell-free tumor DNA mutational signatures from blood for early detection of recurrence of low stage lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6689.

Unmatched Whole-Genome Sequencing As a Clinical Tool for Hematological Neoplasms with Significant Utility in Cases with Tumor-in-Normal Contamination

Introduction For patients with acute leukemias, mapping of clinical biomarkers is critical for disease classification, risk stratification and treatment decisions. At diagnosis, molecular profiling is primarily based on cytogenetic analysis, SNP-arrays and increasingly complemented with targeted assays to include Fluorescent In Situ Hybridization (FISH), and DNA/RNA panel assays. Recently, Duncavage et al. showed that for acute myeloid leukemia, whole genome sequencing (WGS) can replace cytogenetics and detect the hotspot mutations in the ELN2017 guidelines (Duncavage et al., 2021). However, the 2022 revision of disease classification and risk stratification led to the incorporation of additional events characterized by a diverse spectrum of non-hotspot mutations (Khoury et al. 2022, Bataller et al. 2022). To this end, the diagnostic utility of WGS for comprehensive characterization of relevant biomarkers across hematological neoplasms is not established. A further limitation for matched WGS (mWGS) is the requirement of a normal sample at diagnosis, as sources of control tissue can be contaminated with tumor cells. Methods To resolve this unmet clinical need, we developed a computational framework for the analysis of WGS data without the use of a normal germline control (uWGS). We applied this uWGS in 2 independent cohorts of patients (Table 1). In cohort 1 of 49 patients, representative of a broad range of hematological neoplasms, uWGS findings were compared against standard-of-care molecular assays to include cytogenetics, FISH, SNP-arrays, targeted-gene panels and RNA sequencing for findings of clinical relevance (disease classification, risk stratification, therapy informing) as annotated by OncoKB (Chakravarty et al. 2017). To further evaluate whether an uWGS can detect the same spectrum of acquired mutations that a matched WGS workflow (mWGS), we compared findings for 52 B-ALL patients in cohort 2 with mWGS data, and we estimated the tumor-in-normal contamination (TiN) with the algorithm deTiN (Taylor-Weiner et al., 2018). Results In Cohort 1, uWGS detected 100% (24/24) of highly confident findings (cancer cell fraction >20) of clinical relevance described by standard-of-care assays (0.49 average, 0-3 range) (Fig.1). Moreover, uWGS identified 9 mutations in 8 patients that were not detected by standard-of-care. These include 2 disease-defining P2RY8::CRLF2 in B-ALL, 2 canonical t(7;9)(q34;q34.3) NOTCH1-intergenic in T-ALL, a TCF3 D561V point mutation and a IGH::MYC event in Burkitt lymphoma, 2 focal deletions in CDKN2A and IKZF1 in B-ALL, and a focal NF1 deletion in JMML. Importantly, 5/8 of these cases did not have a clinically relevant alteration identified by standard-of-care. In Cohort 2, we evaluated what proportion of relevant events identified by mWGS can be detected by uWGS. Putative biomarkers had been identified by mWGS in 88% of patients (46/52). These included delineations of aberrant karyotypes where chromosome banding failed (5/52), newly described fusion genes (i.e. UBTF::ATXN7L3, EP300::ZNF384, in 27/52) and recurrent gene mutations (i.e. PAX5 P80R, ZEB2 H1038R, in 14/52). As a result, uWGS workflow captured 100% of biomarkers identified in the mWGS (5/5 ploidy, 27/27 fusion, 14/14 coding). Remarkably, evaluation of TiN levels reveals that 4 driver events had been missed in the mWGS workflow, affecting genetic alterations across variant classes. For example, a focal BTG1 loss detected by uWGS was not called by mWGS. Overall, 25% of B-ALL cases had evidence of TiN >2% (14/55, ranges 2-57%), resulting in lower sensitivity of detection of copy number variants (CNVs) in mWGS. When looking into the whole spectrum of CNVs detected only by uWGS, we found lower levels of precision in mWGS that were associated with evidence of TiN (median 0.45 vs 0.64, p-value 0.03). Additionally, the other 3 drivers missed by mWGS were indels in CHEK2, JAK1 and TCF3. Conclusions We have developed and validated an uWGS workflow for the detection of clinically relevant alterations in leukemia across variant classes to include CNVs, translocations and point mutations. We demonstrated that for hematological neoplasms, uWGS rescues events that can be missed by mWGS workflows owing to TiN, which shows how uWGS enables the detection of clinically relevant biomarkers, and the opportunity to discover new clinical findings using a single test and a single biopsy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 3401: Whole genome cell-free tumor DNA mutational signatures for noninvasive monitoring of pediatric brain cancers

Abstract Introduction: Liquid biopsy offers a noninvasive approach to monitor cancer burden during therapy and surveillance period. However, in pediatric brain cancers, liquid biopsy methods from the blood have been unsuccessful due to a low tumor burden and low number of mutations in coding regions. We hypothesized that a whole genome sequencing (WGS)-derived patient specific mutational signature from a matched tumor-normal WGS can provide a sensitive and specific approach to detect mutations in circulating cell free tumor DNA (ctDNA) and provide blood-based monitoring in pediatric patients with brain tumor. Methods: All tumors were analyzed and molecularly subclassified using whole genome DNA methylation profiling and machine learning classifier. Tumor DNA was extracted from pathology tissue and normal germline DNA from the white blood cells, while ctDNA was extracted from 1-2 mL of post-surgery or follow-up plasma samples, WGS was applied to sequence DNA from matched tumor-normal and plasma samples. WGS coverage was 40x for matched tumor-normal DNA and 20x for ctDNA. Using the C2i assay, we derived a personalized mutational pattern for each tumor and used an AI-based error suppression model for quantification and ultra-sensitive detection of ctDNA in plasma samples. A patient-specific personalized genome-wide compendium of somatic mutations was established and ctDNA tested at 1 to 3 available time points during the therapy or surveillance period. An AI-based error suppression model was implemented to filter out the noise in the cell free DNA (cfDNA) while the personalized mutational signature was used to detect the ctDNA in the cfDNA and to amplify the somatic signal contained in it. The ctDNA Tumor Fraction (TF) was compared to the clinical status and MR-based imaging. Results: We profiled 7 pediatric brain tumors, including 2 medulloblastomas (one Group 3, one Group 4), 3 pediatric glioblastomas IDH wild-type, 1 ependymoma PFA subtype and one low grade ganglioglioma. Tumor specific signatures were identified and detected in the plasma of 5 patients with clinical disease with a TF range 0.02-0.0005 but not in 2 patients with no tumor at the time of blood collection. In two children with a medulloblastoma and glioblastoma, the decrease of tumor fraction in ctDNA over 2 (TF: 0.002 to 0.0009) and 3 time points (TF: 0.0005 to undetectable), respectively, correlated with response to therapy based on imaging. Conclusions: Patient-specific WGS tumor signature in ctDNA from blood can be used for sensitive monitoring of children with brain tumors. Citation Format: Ivy Tran, Kristyn Galbraith, Guisheng Zhao, Robyn Borsuk, Joyce Varkey, Sharon Gardner, Jeffrey Allen, David Harter, Jeffrey Wisoff, Eveline T. Hidalgo, Sunil Deochand, Dillon Maloney, Danielle Afterman, Tomer Lauterman, Noah Friedman, Imane Bourzgui, Nidhi Ramaraj, Zohar Donenhirsh, Ronel Veksler, Jonathan Rosenfeld, Ravi Kandasamy, Iman Tavassoly, Boris Oklander, G. Praveen Raju, Theodore Nicolaides, Asaf Zviran, Matija Snuderl. Whole genome cell-free tumor DNA mutational signatures for noninvasive monitoring of pediatric brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3401.

Abstract CT182: Phase I trials of personalized cancer vaccine TG4050 in surgically treated high-risk head and neck squamous cell carcinoma (HNSCC) and relapsing ovarian cancer (OvC) patients

Abstract Background: Cancer mutations acquired during carcinogenesis are not subject to central tolerance and thus, are cornerstone in cancer immunosurveillance. The use of mutation-derived neoepitopes to design cancer vaccines is an unprecedented opportunity to target efficient antigens, but the stochastic occurrence of mutations implies that a tailored vaccine needs to be designed and manufactured for each patient. We used next generation sequencing, machine learning based genomic data analysis and advances in genome editing to systematically identify private neoepitopes in high-risk HPV negative HNSCC and relapsing OvC patients. Methods: Mutations were identified by DNA and RNA sequencing and putative epitopes were selected for vaccine design on their likelihood to elicit a class I or II response based on HLA binding, allelic frequency, prediction of processing and expression at RNA level. The vaccine was a recombinant virus of the modified Vaccinia Ankara (MVA) strain encoding for up to 30 sequences of 29 amino acids centred on the predicted epitope and flanked with the normal germline sequence in case of missense mutations, and of the mutated protein sequence in case of frameshift mutations. The viral vaccine was amplified under GMP conditions and administered to patients after curative intent treatment in two phase I trials. Treatment was started for OvC patients at relapse defined as elevation of CA-125 or onset of suspicious radiological findings and, after first line therapy for HNSCC patients with locally advanced disease with either extracapsular extension or positive resection margins following upfront surgery. The vaccine was administered for 6 weeks and a booster dose every three weeks over a year, in absence of other cancer therapy. Results: Six patients (4 OvC and 2 HNSCC) were treated and experienced only mild related adverse events. After 6 doses of vaccine, we measured immunoreactive T cells in PBMCs against each of the targeted epitopes using an ex vivo IFNγ ELISPOT showed robust responses in all evaluable patients. Positive responses to vaccination were observed for a total of 46 epitopes over 115 tested with a median of 11 epitopes (6 to 18) per patient. Responses were directed against both class I and II epitopes and were either de novo responses (64%) or amplification of pre-existing responses (36%). In parallel, phenotyping of patient PBMCs showed, in all treated patients, a decline in naïve CD4 and CD8 cells and the onset of effector CD27- CD4 and CD27-/CD57+ CD8; similarly, CD56dim NK cells increased suggesting a stimulation of antitumor innate immunity. In one ovarian cancer patient, these immune changes correlated with a normalization of CA-125 that lasted 7 months. Conclusions: These data show that the personalized approach with TG4050 is feasible, safe and immunogenic. The two phase I trials are ongoing. Citation Format: Matthew S. Block, Jean-Pierre Delord, Christian H. Ottensmeier, Christophe Le Tourneau, Ana Lalanne, Olivier Lantz, Keith L. Knutson, Gisèle Lacoste, Annette Tavernaro, Maud Brandely, Nathalie Silvestre, Benoit Grellier, Yoshiko Yamashita, Onoue Kousuke, Naoko Yamagata, Eric Quemeneur, Kaidre Bendjama. Phase I trials of personalized cancer vaccine TG4050 in surgically treated high-risk head and neck squamous cell carcinoma (HNSCC) and relapsing ovarian cancer (OvC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT182.

TMC-SNPdb 2.0: an ethnic-specific database of Indian germline variants.

Cancer is a somatic disease. The lack of Indian-specific reference germline variation resources limits the ability to identify true cancer-associated somatic variants among Indian cancer patients. We integrate two recent studies, the GenomeAsia 100K and the Genomics for Public Health in India (IndiGen) program, describing genome sequence variations across 598 and 1029 healthy individuals of Indian origin, respectively, along with the unique variants generated from our in-house 173 normal germline samples derived from cancer patients to generate the Tata Memorial Centre-SNP database (TMC-SNPdb) 2.0. To show its utility, GATK/Mutect2-based somatic variant calling was performed on 224 in-house tumor samples to demonstrate a reduction in false-positive somatic variants. In addition to the ethnic-specific variants from GenomeAsia 100K and IndiGenomes databases, 305 132 unique variants generated from 173 in-house normal germline samples derived from cancer patients of Indian origin constitute the Indian specific, TMC-SNPdb 2.0. Of 305 132 unique variants, 11.13% were found in the coding region with missense variants (31.3%) as the most predominant category. Among the non-coding variations, intronic variants (49%) were the highest contributors. The non-synonymous to synonymous SNP ratio was observed to be 1.9, consistent with the previous version of TMC-SNPdb and literature. Using TMC SNPdb 2.0, we analyzed a whole-exome sequence from 224 in-house tumor samples (180 paired and 44 orphans). We show an average depletion of 3.44% variants per paired tumor and significantly higher depletion (P-value < 0.001) for orphan tumors (4.21%), demonstrating the utility of the rare, unique variants found in the ethnic-specific variant datasets in reducing the false-positive somatic mutations. TMC-SNPdb 2.0 is the most exhaustive open-source reference database of germline variants occurring across 1800 Indian individuals to analyze cancer genomes and other genetic disorders. The database and toolkit package is available for download at the following: Database URL http://www.actrec.gov.in/pi-webpages/AmitDutt/TMCSNPdb2/TMCSNPdb2.html

Developmental potential of cryopreserved gonadal germ cells from 7-day-old chick embryos recovered using the PBS(-) method

ABSTRACT 1. A series of experiments were conducted to examine the developmental potential of cryopreserved gonadal germ cells (GGCs) recovered from both males and females on embryo day 7 (7 d-GGCs) using the PBS(-) method. Germline chimeras were produced by transferring 200 frozen/unfrozen 7 d-GGCs recovered from female/male Rhode Island Red (RIR) embryos into the dorsal aorta of 2-day-old female and male white leghorn (WL) embryos. 2. Germ-cell recipient embryos were hatched and raised to sexual maturity and progeny testing was conducted by mating with RIR of the opposite sex. Brown-feathered progeny chicks were hatched in all eight possible progeny testing combinations, except for male GGC recipients produced by transferring female GGCs. Furthermore, brown-feathered progeny chicks were hatched when frozen-thawed sperm from male germline chimeras, produced by transferring unfrozen 7d-GGCs, were inseminated in normal female RIR and female WL germline chimeras. 3. The results indicated that cryopreserved female/male GGCs from 7-day-old chick embryos, recovered using the PBS(-) method, were fully capable of developing into normal spermatozoa and ova in the gonad of recipient embryos under appropriate GGC donor/recipient combinations.

The RNA-binding protein Igf2bp3 is critical for embryonic and germline development in zebrafish.

The ability to reproduce is essential in all branches of life. In metazoans, this process is initiated by formation of the germline, a group of cells that are destined to form the future gonads, the tissue that will produce the gametes. The molecular mechanisms underlying germline formation differs between species. In zebrafish, development of the germline is dependent on the specification, migration and proliferation of progenitors called the primordial germ cells (PGCs). PGC specification is dependent on a maternally provided cytoplasmic complex of ribonucleoproteins (RNPs), the germplasm. Here, we show that the conserved RNA-binding protein (RBP), Igf2bp3, has an essential role during early embryonic development and germline development. Loss of Igf2bp3 leads to an expanded yolk syncytial layer (YSL) in early embryos, reduced germline RNA expression, and mis-regulated germline development. We show that loss of maternal Igf2bp3 function results in translational de-regulation of a Nodal reporter during the mid-blastula transition. Furthermore, maternal igf2bp3 mutants exhibit reduced expression of germplasm transcripts, defects in chemokine guidance, abnormal PGC behavior and germ cell death. Consistently, adult igf2bp3 mutants show a strong male bias. Our findings suggest that Igf2bp3 is essential for normal embryonic and germline development, and acts as a key regulator of sexual development.

Abstract 3017: Advancing PDX research through model, data, and bioinformatics with the PDXNet Portal

Abstract We created the PDX Network (PDXNet) Portal to provide an intuitive way for researchers to explore and understand the models, sequencing data, and bioinformatics workflows generated by NCI's PDXNet consortium for research access (https://portal.pdxnetwork.org/). The portal also provides metrics for PDXNet's activities, data processing protocols, and training materials for processing PDX data. The PDXNet Portal highlights model and data resources that include 216 new models across 29 cancer types. The most prevalent cancers represented in the PDX model dataset include invasive breast carcinoma (30.6%), melanoma (18.1%), and adenocarcinoma (14.4%). PDXNet teams have provided 2263 sequencing files from 356 samples across 204 patients, comprising whole exome (82.9%) and RNA seq files (17.1%). The most prevalent cancers represented in the PDXNet sequencing data set include Breast Pleural Effusion (27.2%), Breast Poorly Differentiated (12.5%), and Lung Adenocarcinoma (9.6%). The portal also provides access to 9492 sequencing files across 78 disease types that include 2594 samples across 463 patients uploaded from the NCI Patient-Derived Model Repository. The dataset includes both whole exomes (52.8%) and RNA seq (47.2%) data. The PDMR samples include PDX (82.7%), primary tumor (5.7%), normal germline (5.5), organoid culture (3.2), and Mixed Tumor Culture (2.9). The PDMR dataset also has multiple passages: P0 (21.8%), P1(39.5%), P2 (25.6%), and P3 (8.5%). These models and data resources support ten PDXNet Pilot activities, multiple publications, and international collaborations. PDXNet has also developed a set of 13 robust, validated, and standardized workflows for processing PDXNet whole-exome and RNA seq data. Collectively, these workflows allow for the standardized processing of PDX and complementary human tissues (normal and tumor). Our plan is to continuously update the model and data lists on the PDX portal as resources are generated. We expect that the PDXNet generated models, scheduled to grow to 1000 new models by 2022, will support multi-agent treatment studies, determination of mechanisms of sensitivity and resistance, and pre-clinical trials for example through the COMBO-MATCH program. The robust standard workflows currently processing all PDX sequencing data may also facilitate harmonizing data across studies. Lastly, we expect that the generated sequencing data will support computational approaches for studying cancer evolution and the mechanisms underlying cancer treatments. Citation Format: Soner Koc, Mike Lloyd, Steven Neuhauser, Javad Noodbakhsh, Anuj Srivastava, Xing Yi Woo, Ryan Jeon, Jeffrey Grover, Sara Seepo, Christian Frech, Jack DiGiovanna, PDXNet Consortium, Yvonne A. Evard, Tiffany Wallace, Jeffrey Moscow, James H. Doroshow, Nicholas Mitsuade, Salma Kaochar, Chong-xian Pan, Moon S. Chen, Luis Carvarjal-Carmona, Alana Welm, Bryan Welm, Michael T. Lewis, Govindan Ramaswamy, Li Ding, Shunquang Li, Meenherd Herlyn, Mike Davies, Jack Roth, Funda Meric-Bernstam, Peter Robinson, Carol J. Bult, Brandi Davis-Dusenbery, Dennis A. Dean, Jeffrey H. Chuang. Advancing PDX research through model, data, and bioinformatics with the PDXNet Portal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3017.

Plasma Cell Disease Genotyping with the Liquid Biopsy

IntroductionEasily accessible, real-time genotyping is desirable for patients suffering from plasma cell (PC) disorders for diagnostic, prognostic and therapeutic purpose. However, PC disorders usually lack a leukemic phase, and up to now genotyping of tumor cells required purified material from bone marrow (BM) or tissue biopsies. Circulating cell-free DNA (cfDNA) might be an accessible source of tumor material in patients with PC diseases to identify cancer-gene somatic mutations. Accessing the peripheral blood (PB) has clear advantages with respect to the sampling procedure itself, and has the potential to better reflect tumor heterogeneity. Here we aimed at tracking the genetic profile of different PC dyscrasias using plasma cfDNA.MethodsTwenty-eight consecutive patients (median age= 64; male:female ratio=15:13) with PC disorders [two with monoclonal gammopathy of undetermined significance (MGUS), five with smoldering MM (SMM), and 21 with symptomatic MM] were included between September 2016 and May 2017. The following material was collected: (1) cfDNA isolated from plasma, (2) tumor genomic DNA (gDNA) from CD138+ purified BM PCs for comparative purposes, and (3) normal germ line gDNA extracted from PB granulocytes after Ficoll separation, to filter out polymorphisms. The sampling was done at diagnosis in 25 patients, and during the course of the disease in three MM cases. Median BM PC infiltration was 40% (range: 7-90%). A targeted resequencing gene panel, including coding exons and splice sites of 14 genes (target region: 31 kb) was specifically designed and optimized to allow a priori the recovery of at least one clonal mutation in 68% (95% confidence interval: 58-76) of MM patients. Ultra-deep next-generation sequencing (NGS) of the gene panel was performed on MiSeq (Illumina) using the CAPP-seq library preparation strategy (NimbleGen). The somatic function of VarScan2 was used to call non-synonymous somatic mutations, and a stringent bioinformatic pipeline was developed and applied to filter out sequencing errors (detection limit 3x10-3). The sensitivity and specificity of plasma cfDNA genotyping were calculated in comparison with tumor gDNA genotyping as the gold standard.ResultsIn this prospectively collected cohort of 28 unselected PC dyscrasia patients, circulating tumor cfDNA was detectable in plasma samples with a median of 12011.60 haploid genome-equivalents per mL. The application of our targeted ultra-deep NGS approach for plasma cfDNA genotyping resulted in ≥90% of the target region covered ˃1000X in all plasma samples, and ≥90% of the target region covered ˃2000X in 23/28. Overall, within the interrogated genes, 18/28 (64%) patients harbored somatic mutations (total number: 28; range: 1-4 mutations per patient) that were detectable in plasma cfDNA. Quite consistent with the typical spectrum of mutated genes in MM, plasma cfDNA genotyping revealed somatic variants of NRAS in 25%; KRAS in 14%; TP53, TRAF3 and FAM46C in 11%, respectively; CYLD and DIS3 in 7%, respectively; and BRAF and IRF4 in 4% of cases, respectively (Figure 1A). By comparing the genotype of circulating tumor cfDNA with that of gDNA from purified malignant BM PCs of the diagnostic biopsy (gold standard) (Figure 1B), we found that cfDNA genotyping correctly identified 72% of the mutations confirmed in the tumor biopsy (Figure 1C). Notably, the remaining mutations not discovered in cfDNA had a low representation in the purified BM PCs (median allelic abundance=2.5%; range 1.1-4.96%). ROC analysis showed that circulating tumor cfDNA genotyping had the highest sensitivity (92.9%) if mutations were represented in >5% of the alleles of the purified BM PCs (Figures 1D-E). In none of the cases, cfDNA genotyping identified additional somatic mutations not detected in the purified BM PCs.ConclusionsOverall, our results provide the proof of principle that circulating tumor cfDNA genotyping is a feasible, non-invasive real-time approach that reliably detects clonal and subclonal somatic mutations represented in at least 5% of alleles in tumor PCs. [Display omitted] DisclosuresBertoni:Acerta Pharma: Research Funding; Bayer: Research Funding; Cellestia: Research Funding; Menarini: Research Funding; Piqur: Research Funding; Immunogen: Research Funding. Zucca:Celgene: Honoraria, Research Funding; Jannsen: Consultancy, Honoraria, Other: Advisory role; Mundipharma: Research Funding; Celgene: Honoraria, Research Funding; Roche: Advisory role, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Mundipharma: Research Funding; Gilead Science: Consultancy, Other: Advisory role; Sandoz: Consultancy, Other: Advisory role; Jannsen: Consultancy, Honoraria, Other: Advisory role; Gilead Science: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Bayer: Consultancy, Other: Advisory Role; Sandoz: Consultancy, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Takeda: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role.

Genotyping of Classical Hodgkin Lymphoma on the Liquid Biopsy

INTRODUCTION. In classical Hodgkin lymphoma (cHL), the low representation (~5%) of Hodgkin-Reed-Sternberg cells (HRS) challenged tumor genotyping on the tissue biopsy. Cell free DNA (cfDNA) is shed into the blood by tumor cells and can be used as source of tumor DNA for the identification of somatic mutations, track clonal evolution of tumors and detect minimal residual disease during therapy. AIMS. The study aimed at: i) showing that cfDNA mirrors the genetics of HRS cells in cHL patients; ii) characterizing the mutational profile of a large cohort of newly diagnosed and chemorefractory cHL; iii) identifying molecular prognostic subtypes; iv) early detecting residual disease during therapy; and v) longitudinally tracking tumor clonal evolution under different treatment modalities. METHODS. The study included 80 newly diagnosed cHL and 32 chemorefractory cHL. The following biological material was analyzed: i) cfDNA from plasma collected at diagnosis, during ABVD courses, at refractory progression, before and during therapy with brentuximab or nivolumab; and ii) normal germline genomic DNA (gDNA) from granulocytes. For comparative purposes, paired tumor gDNA from microdissected HRS cells of 13 cases was also analyzed. A targeted resequencing panel optimized to include the coding exons and splice sites of 77 genes recurrently mutated in B-cell lymphomas was used for genotyping. Ultra-deep next-generation sequencing (NGS) of the gene panel was performed on NexSeq 500 (Illumina) using the CAPP-seq library preparation strategy (NimbleGen). RESULTS. In cHL patients, cfDNA surrogated gDNA from HRS cells, since it harbored 87.5% of the tumor confirmed mutations. Genes recurrently affected by non-synonymous somatic mutations in >20% of cHL included STAT6 (37.5%), TNFAIP3 (35%), and ITPKB (27.5%) (Fig. 1A). Mutations clustered in major pathways, including NF-κB, PI3K-AKT, cytokine and NOTCH signaling, and immune evasion. ITPKB mutations: i) were quite specific for cHL, being rare or absent in other lymphomas; ii) caused the subcellular delocalization of the protein in primary HRS cells of mutated patients; iii) correlated with clues of PI3K-AKT signaling activation both at gene expression and protein levels; and iv) consistent with the positioning of ITPKB downstream PI3K in the pathway, associate with resistance to PI3K inhibitors. Mutations of CD58, encoding a co-stimulatory molecule for T-cells, associated with short PFS independent of interim PET/CT results, pointing to immune escape genetic lesions as biomarkers of aggressive disease (Fig. 1B). Newly diagnosed and chemorefractory cHL shared a largely overlapping mutational landscape. TP53 mutations were not enriched in refractory cHL as instead commonly found in other types of refractory B-cell tumors. Conversely, more TET2 mutations were documented in refractory cHL, including newly acquired mutation, thus signaling towards aberrant DNA methylation programming as a mechanism of resistance in cHL with potential therapeutic implications. By longitudinal analysis, in patients relapsing under/after chemotherapy or brentuximab vedotin, pre-treatment/relapse tumor pairs branched through the acquisition of phase specific mutations from an ancestral clone, that always persisted (Fig. 1C). Conversely, in patients maintained under nivolumab, clones were cyclically suppressed and replaced by completely novel clones. We utilized the change in circulating tumor cfDNA load from baseline to interim timepoint to predict the best response to ABVD and to complement interim PET/CT in anticipating cure. A drop of 100-fold or 2-log drop in tumor cfDNA after 2 ABVD courses associated with an eventual complete response and cure. All cured patients that were inconsistently judged as interim PET/CT positive turned out to have a >2 log drop in tumor cfDNA. A drop of less than 2-log in tumor cfDNA after 2 ABVD courses associated with an eventual progression. All relapsed patients that were inconsistently judged as interim PET/CT negative turned out to have a <2 log drop in tumor cfDNA (Fig. 1D). CONCLUSIONS. Circulating tumor cfDNA allows to noninvasively detect tumor-specific mutations: i) identify prognostic subtypes; ii) early detect residual disease during therapy; and iii) longitudinally track tumor clonal evolution under different treatment modalities. [Display omitted] DisclosuresStathis:Celgene: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Roche: Consultancy, Other: Advisory board; Amgen: Honoraria. Bertoni:Acerta Pharma: Research Funding; Bayer: Research Funding; Cellestia: Research Funding; Menarini: Research Funding; Piqur: Research Funding; Immunogen: Research Funding. Santoro:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zucca:Bayer: Consultancy, Other: Advisory Role; Jannsen: Consultancy, Honoraria, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celgene: Honoraria, Research Funding; Sandoz: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role; Gilead Science: Consultancy, Other: Advisory role; Roche: Honoraria, Research Funding; Jannsen: Consultancy, Honoraria, Other: Advisory role; Roche: Advisory role, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Research Funding; Mundipharma: Research Funding; Gilead Science: Consultancy, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Sandoz: Consultancy, Other: Advisory role. Gaidano:Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Carlo-Stella:ADC Therapeutics: Research Funding.

Analytic validation and clinical utilization of the comprehensive genomic profiling test, GEM ExTra®.

We developed and analytically validated a comprehensive genomic profiling (CGP) assay, GEM ExTra, for patients with advanced solid tumors that uses Next Generation Sequencing (NGS) to characterize whole exomes employing a paired tumor-normal subtraction methodology. The assay detects single nucleotide variants (SNV), indels, focal copy number alterations (CNA), TERT promoter region, as well as tumor mutation burden (TMB) and microsatellite instability (MSI) status. Additionally, the assay incorporates whole transcriptome sequencing of the tumor sample that allows for the detection of gene fusions and select special transcripts, including AR-V7, EGFR vIII, EGFRvIV, and MET exon 14 skipping events. The assay has a mean target coverage of 180X for the normal (germline) and 400X for tumor DNA including enhanced probe design to facilitate the sequencing of difficult regions. Proprietary bioinformatics, paired with comprehensive clinical curation results in reporting that defines clinically actionable, FDA-approved, and clinical trial drug options for the management of the patient’s cancer. GEM ExTra demonstrated analytic specificity (PPV) of > 99.9% and analytic sensitivity of 98.8%. Application of GEM ExTra to 1,435 patient samples revealed clinically actionable alterations in 83.9% of reports, including 31 (2.5%) where therapeutic recommendations were based on RNA fusion findings only.

The mitochondrial localized CISD-3.1/CISD-3.2 proteins are required to maintain normal germline structure and function in Caenorhabditis elegans.

Reproductive organs and developing tissues have high energy demands that require metabolic functions primarily supported by mitochondria function. The highly conserved CISD/NEET iron-sulfur (Fe-S) protein family regulates iron and reactive oxygen homeostasis, both of which are important for mitochondrial function. Disruption of iron and reactive oxygen homeostasis typically leads to detrimental effects. In humans, CISD dysfunction is associated with human health issues including Wolfram syndrome 2. Using C. elegans, we previously determined that the cisd-1, cisd-3.1 and cisd-3.2 have an overlapping role in the regulation of physiological germline apoptosis through the canonical programmed cell death pathway. Here, we isolated the cisd-3.2(pnIs68) mutant that resulted in physiological and fitness defects including germline abnormalities that are associated with abnormal stem cell niche and disrupted formation of bivalent chromosomes. The cisd-3.2(pnIs68) mutation led to complete disruption of the cisd-3.2 gene expression and a decrease in expression of genetically intact cisd-1 and cisd-3.1 genes suggesting an indirect impact of the cisd-3.2(pnIs68) allele. The CISD-3.2 and CISD-3.1 proteins localize to the mitochondria in many tissues throughout development. The cisd-3.2(pnIs68) mutant displays phenotypes associated with mitochondrial dysfunction, including disruption of the mitochondrial network within the germline. These results further support the idea that the CISD protein family is required for mitochondrial function that supports important functions in animals including overall fitness and germline viability.

Expression of Mst89B and CG31287 is Needed for Effective Sperm Storage and Egg Fertilization in Drosophila

In Drosophila, male reproductive fitness can be affected by any number of processes, ranging from development of gametes, transfer to and storage of mature sperm within the female sperm storage organs, and utilization of sperm for fertilization. We have previously identified the 89B cytogenetic map position of D. melanogaster as a hub for genes that effect male paternity success when disturbed. Here, we used RNA interference to test 11 genes that are highly expressed in the testes and located within the 89B region for their role in sperm competition and male fecundity when their expression is perturbed. Testes-specific knockdown (KD) of bor and CSN5 resulted in complete sterility, whereas KD of CG31287, Manf and Mst89B, showed a breakdown in sperm competitive success when second to mate (P2 < 0.5) and reduced fecundity in single matings. The low fecundity of Manf KD is explained by a significant reduction in the amount of mature sperm produced. KD of Mst89B and CG31287 does not affect sperm production, sperm transfer into the female bursa or storage within 30 min after mating. Instead, a significant reduction of sperm in female storage is observed 24 h after mating. Egg hatchability 24 h after mating is also drastically reduced for females mated to Mst89B or CG31287 KD males, and this reduction parallels the decrease in fecundity. We show that normal germ-line expression of Mst89B and CG31287 is needed for effective sperm usage and egg fertilization.

SeqURE \u2013 a new copy-capture based method for sequencing of unknown Retroposition events

BackgroundRetroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated.ResultsHere we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions.ConclusionsThis study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.

Extensive germline genome engineering in pigs.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.