Germline Sequence Research Articles

Generation and Next-Generation Sequencing-Based Characterization of a Large Human Combinatorial Antibody Library.

Antibody phage display is a key technology for the discovery and development of target-specific monoclonal antibodies (mAbs) for use in research, diagnostics, and therapy. The construction of a high-quality antibody library, with larger and more diverse antibody repertoires, is essential for the successful development of phage display-derived mAbs. In this study, a large human combinatorial single-chain variable fragment library (1.5 × 1011 colonies) was constructed from Epstein-Barr virus-infected human peripheral blood mononuclear cells stimulated with a combination of two of the activators of human B cells, the Toll-like receptor 7/8 agonist R848 and interleukin-2. Next-generation sequencing analysis with approximately 1.9 × 106 and 2.7 × 106 full-length sequences of heavy chain variable (VH) and κ light chain variable (Vκ) domains, respectively, revealed that the library consists of unique VH (approximately 94%) and Vκ (approximately 91%) sequences with greater diversity than germline sequences. Lastly, multiple unique mAbs with high affinity and broad cross-species reactivity could be isolated from the library against two therapeutically relevant target antigens, validating the library quality. These findings suggest that the novel antibody library we have developed may be useful for the rapid development of target-specific phage display-derived recombinant human mAbs for use in therapeutic and diagnostic applications.

Abstract P6-02-15: Don’t get lost in translation: Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) recommendations for reporting germline cancer susceptibility gene variants in 19 languages – breast cancer as a model

Abstract Genetic testing for cancer susceptibility is a cornerstone of precision cancer prevention and care. Major communication hurdles remain for the differently specialized professionals involved in the identification, counselling, and clinical management of at-risk individuals. This may be ascribed to gaps in the genetic/genomic literacy of health care providers and to an ambiguous lexicon used for variant description. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) international consortium endorses controlled terminology and a framework for interpretation and reporting of germline variants in cancer susceptibility genes (PMID: 30962250). However, for most ENIGMA affiliates a language other than English is used for written and verbal communication of genetic test results, potentially confounding local application of the published framework. The ENIGMA Clinical Working Group thus launched a Vocabulary Translation Project (VTP) to translate the ENIGMA recommendations into the various languages spoken by the membership. The VTP involved 65 ENIGMA members from 22 countries organized into 19 language-specific teams, covering Catalan, Chinese, Czech, Danish, Dutch, Finnish, French, Galician, German, Greek, Italian, Japanese, Malay, Norwegian, Polish, Portuguese, Spanish (Castilian), Swedish, and Tagalog. Excerpts from the original publication were selected for translation based on a majority consensus and included a glossary of terms and recommendations for interpreting and reporting germline sequence variants in (breast) cancer susceptibility genes. Using a two-step process, each team conducted the relevant translation followed by independent back-translation to English. The VTP proved useful to reappraise the reference text. It disclosed transnational issues, which prompted revision of the original source to emphasize that risk estimates and actionability were based on breast cancer as an exemplar. It also highlighted country-specific differences with regards to breast cancer risk assessment (e.g. different absolute/relative breast cancer risk cut points) and management. As a secondary outcome, via electronic survey of the participating teams we documented the perceived high value of the translation effort and its expected positive impact on more consistent clinical management of carrier individuals. The identified target audience encompasses medical geneticists, physicians of other specialties participating in multidisciplinary teams, genetic counselors, primary care physicians, as well as non-health care professionals, e.g. journalists and science communicators. The outreach program includes dissemination of the translations via local, regional, and especially national networks and their use for education and training purposes. Because French, Portuguese, and Spanish are widely used as official, co-official, or secondary languages, the reach of the VTP potentially extends to a greater number of countries and territories, mostly in Central and South America, Caribbean, and Africa. By moving a step forward towards terminological coherence across disciplines and borders, we will facilitate more precise delivery and clinical application of genetic test results for breast cancer predisposition. Our translated recommendations will improve interdisciplinary cross-talk and carriers’ awareness of the risks and implications associated with their status, contributing to more informed decision-making. We used breast cancer as a blueprint. Application of the model to other cancer types will require calibration on the cancer-specific absolute and relative risks. Citation Format: Arcangela De Nicolo, Diana M. Eccles, Sarah Louise Ariansen, Michela Biancolella, Miguel de la Hoya, Orland Diez, Hans Ehrencrona, Florentia Fostira, Tiara Hassan, Issei Imoto, Artur Kowalik, Fabienne Lesueur, Arjen R. Mensenkamp, Heli Nevanlinna, Joanne Ngeow, Edenir I. Palmero, Inge Søkilde Pedersen, Frances Que, Jana Soukupová, Yen Tan, Ana Vega, Amanda B. Spurdle, Paolo Radice. Don’t get lost in translation: Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) recommendations for reporting germline cancer susceptibility gene variants in 19 languages – breast cancer as a model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-02-15.

The Conjusome—A Transient Organelle Linking Genome Rearrangements in the Parental and Developing Macronuclei

The conjusome plays an important role in the conjugation events that occur in Tetrahymena thermophila. The conjusome appears in the anterior of conjugant pairs during the early stages of new macronuclei (anlagen) development. It lacks a membrane, and is composed of a network of fibrous, electron dense material, containing background cytoplasm and ribosomes. Several proteins localize to this organelle, including Pdd1p, a chromodomain protein that participates in the formation of chromatin-containing structures in developing macronuclear anlagen, and is associated with the elimination of specific germ-line sequences from developing macronuclei. Conjugants lacking the PDD1 allele in the parental macronucleus do not show Pdd1p antibody staining in conjusomes. Investigations were performed using mutant cell lines, uniparental cytogamy and drug treatment, and show that the conjusome appears to be dependent on parental macronuclei condensation, and is a transitory organelle that traffics nuclear determinants from the parental macronucleus to the developing anlagen. These data, taken together with Pdd1p knockout experiments, suggest the conjusome is involved in the epigenetic phenomena that occur during conjugation and sexual reorganization. This is likely a conserved organelle. Conjusome-like structures were also observed in another Ciliate, Stylonichia. In general, conjusomes have features that resemble germ line P-granules.

From Samples to Germline and Somatic Sequence Variation: A Focus on Next-Generation Sequencing in Melanoma Research.

Next-generation sequencing (NGS) applications have flourished in the last decade, permitting the identification of cancer driver genes and profoundly expanding the possibilities of genomic studies of cancer, including melanoma. Here we aimed to present a technical review across many of the methodological approaches brought by the use of NGS applications with a focus on assessing germline and somatic sequence variation. We provide cautionary notes and discuss key technical details involved in library preparation, the most common problems with the samples, and guidance to circumvent them. We also provide an overview of the sequence-based methods for cancer genomics, exposing the pros and cons of targeted sequencing vs. exome or whole-genome sequencing (WGS), the fundamentals of the most common commercial platforms, and a comparison of throughputs and key applications. Details of the steps and the main software involved in the bioinformatics processing of the sequencing results, from preprocessing to variant prioritization and filtering, are also provided in the context of the full spectrum of genetic variation (SNVs, indels, CNVs, structural variation, and gene fusions). Finally, we put the emphasis on selected bioinformatic pipelines behind (a) short-read WGS identification of small germline and somatic variants, (b) detection of gene fusions from transcriptomes, and (c) de novo assembly of genomes from long-read WGS data. Overall, we provide comprehensive guidance across the main methodological procedures involved in obtaining sequencing results for the most common short- and long-read NGS platforms, highlighting key applications in melanoma research.

Inherited Cancer Susceptibility Gene Sequence Variations Among Patients With Appendix Cancer

Germline sequence variations in APC, BMPR1A, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, PMS2, PTEN, SMAD4, STK11, and TP53 genes are associated with susceptibility to gastrointestinal cancers. As a rare cancer, the evaluation of appendiceal cancer (AC) predisposition has been limited. To assess the prevalence and spectrum of inherited cancer susceptibility gene sequence variations in patients with AC and the utility of germline genetic testing for this population. This cohort study included patients with AC who underwent germline genetic testing of 14 cancer susceptibility genes performed by a clinical testing laboratory between March 1, 2012, and December 31, 2016. Data were analyzed from March to August 2022. Clinical, individual, and family histories were obtained from clinician-completed test requisition forms. Multigene panel testing was performed by targeted custom capture and sequencing and chromosome rearrangement analysis. The main outcomes were germline sequence variation prevalence and spectrum in patients with AC. Among the 131 patients with AC in the cohort (90 female [68.7%]), a total of 16 deleterious sequence variations were identified in 15 patients (11.5%). Similarly, when limited to the 74 patients with AC as the first and only primary tumor, a total of 8 patients (10.8%) had at least 1 deleterious sequence variation in a cancer susceptibility gene. Overall, 6 patients (4.6%) had a deleterious sequence variation observed in MUTYH (5 with monoallelic MUTYH and 1 with biallelic MUTYH). All 4 patients with Lynch syndrome (3.1%) had a sequence variation in the MLH1 gene, of whom 3 were aged 50 years or older at AC diagnosis. Five patients (3.8%) had deleterious sequence variations in other cancer predisposition genes (1 with APC [c.3920T>A, p.I1307K], 2 with CHEK2 [c.470T>C, p.I157T], 1 with SMAD4 [c.263 287dup, p.L98IFS*14], and 1 with TP53 [c.524G>A, p.R175H]). In this cohort study, 1 in every 10 patients with AC who underwent testing for hereditary cancer predisposition carried an inherited gene sequence variation associated with cancer susceptibility. Given the high frequency and broad spectrum of germline gene sequence variations, these data suggest that genetic evaluation might be warranted for all patients diagnosed with this rare malignant tumor. A systemic sequencing effort for all patients with AC may also identify cancer vulnerabilities to exploit for therapeutic development in a cancer type for which clinical trials are limited.

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.

Blockade effect of avian-derived anti-VISTA antibodies on immunosuppressive responses

B7 family members and their receptors have been confirmed to participate in T cell response signaling through the induction of immunosuppressive effects. The V-domain Ig suppressor of T-cell activation (VISTA) is expressed in myeloid cells as well as T lymphocytes and plays a crucial role in immune responses. In this study, the phage display technique was employed to isolate specific antibodies targeting VISTA in chickens. In in vitro experiments, VISTA could trigger the inhibition of T-cell activation through interaction during binding. However, isolated single-chain variable fragments (scFvs) effectively inhibited immune cells from producing exhaustion responses, freeing the T cells in peripheral blood mononuclear cells from inhibition and allowing them to remain activated. In addition, the isolated scFvs can block the interaction between VISTA and the VSIG3 ligand. Further analysis on one scFv VS10 revealed that a significant insertion mutation occurred at the CDR3 of the VH domain compared to the chicken germline sequence. Finally, the complex structures of scFv VS10 and VISTA were simulated using molecular docking, and the interaction produced was analyzed. The experimental results support the applicability of VISTA as a target for immune checkpoints and contribute to explaining geometric structures and properties in chicken antibodies.

Results of an immunohistochemistry-based universal screening strategy for Lynch syndrome in patients with colorectal cancer treated in a public hospital from Latin-America

BackgroundThere is limited evidence regarding the implementation of an immunohistochemistry-based universal screening for Lynch Syndrome (LS) in Latin America. The aim of this study was to evaluate the results of a universal screening strategy in colorectal cancer in a public hospital from Latin-America. MethodsSingle center prospective study including patients who underwent colorectal resection for CRC between March 1, 2016 and March 1, 2017. Mismatch Repair status (MMR) based on immunohistochemistry (IHC) was obtained. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for revised Bethesda and modified Amsterdam II criteria. Kaplan-Meier analysis was performed for overall and relapse free survival. Results65 patients were enrolled (35 women). Median age 58 years (22-88). Main tumor location was left colon (48%). Nineteen patients (29%) had positive Bethesda Criteria. Eight patients (12%) with MMR deficiency were detected by IHC: 5 MLH1/PMS2, 2 MSH2/MSH6 and 1 MSH6. The sensibility and specificity for Bethesda Criteria to assess MMR loss were 50% and 73.8%, respectively. Among the patients with MLH1 loss, one patient had a mutated BRAF and two MLH1 promoter hypermethylation. Of the five eligible patients for genetic study, one did not present mutations, three corresponded to a variable of uncertain significance and one had a pathogenic variant of MSH2. The 5-year overall survival was 63.1% without any difference according MMR status. ConclusionsDespite the barriers in public health from Latin America, higher detection rates of patients who should undergo germline sequence study for LS supports the implementation of a universal LS screening strategy.

Prevalence of Germline Sequence Variations Among Patients With Pancreatic Cancer in China

A higher incidence of pancreatic cancer has been reported in the Chinese population compared with the White population, but genetic differences are unknown to date. Large-sample germline testing for both familial and sporadic pancreatic cancers has been conducted predominantly in White populations, whereas similar studies in Chinese populations are limited. To assess the prevalence of germline sequence variations in patients with pancreatic diseases in China. This genetic association study was a case series that included genetic data from patients with pancreatic ductal adenocarcinoma (PDAC) or non-PDAC pancreatic diseases seen at The First Affiliated Hospital of Nanjing Medical University in Nanjing, China, between January 2006 and December 2017 (Nanjing cohort). Comparator group data were obtained for a US cohort from Johns Hopkins Hospital (JHH), a population from East Asia from the Exome Aggregation Consortium (ExAC) database, and the larger population from China from the ChinaMAP database. Data were updated and analyzed in July 2021. Next-generation sequencing technology was used to examine the prevalence of deleterious variations in 59 genes of the included Chinese patients with DNA extracted from peripheral blood samples. The Fisher exact test was used to assess differences among the frequencies of germline variations in the study patients vs the comparator groups. A total of 1009 patients with PDAC (627 [62.1%] male; mean [SD] age, 62.8 [10.2] years) and 885 with non-PDAC diseases (477 [53.9%] male; mean [SD] age, 52.0 [15.9] years) from the Nanjing cohort were included for genetic analysis; all were Han Chinese individuals. Pathogenic variations were detected in 63 patients with PDAC (6.2%; 95% CI, 4.7%-7.7%). Variations in BRCA2 (odds ratio [OR], 3.2; 95% CI, 1.4-7.7; P = .008) and PALB2 (OR, 5.2; 95% CI, 1.6-17.0; P = .007) were significantly associated with pancreatic risk in the Nanjing cohort. Pathogenic variants of genes associated with homologous recombination DNA damage repair, including ATM, BRCA1/2, PALB2, BRIP1, FANCA, FANCC, RAD51D, and XRCC2, were found in 34 patients with PDAC (3.4%). No Ashkenazi Jewish-specific BRCA2 variation (p.Ser1982fs) was detected. The odds ratio of a SPINK1 variation in patients with PDAC was 3.2 (95% CI, 1.8-5.7; P < .001) in the Nanjing cohort compared with the ExAC cohort. Variations in the pancreatic secretory enzyme genes CPA1 and CPB1 were not detected in the Nanjing cohort. In this genetic association study, sporadic pancreatic cancer was associated with pathogenic germline variations in a cohort from China. These findings provide insights into the genetic background of pancreatic cancer in the Han Chinese population with PDAC.

Germline sequence variants contributing to cancer susceptibility in South African breast cancer patients of African ancestry

Since the discovery of the breast cancer susceptibility genes, BRCA1 and BRCA2, various other genes conferring an increased risk for breast cancer have been identified. Studies to evaluate sequence variants in cancer predisposition genes among women of African ancestry are limited and mostly focused on BRCA1 and BRCA2. To characterize germline sequence variants in cancer susceptibility genes, we analysed a cohort of 165 South African women of self-identified African ancestry diagnosed with breast cancer, who were unselected for family history of cancer. With the exception of four cases, all others were previously investigated for BRCA1 and BRCA2 deleterious variants, and were negative for pathogenic variants. We utilized the Illumina TruSight cancer panel for targeted sequencing of 94 cancer susceptibility genes. A total of 3.6% of patients carried a pathogenic/likely pathogenic variant in a known breast cancer susceptibility gene: 1.2% in BRCA1, 0.6% in each of BRCA2, ATM, CHEK2 and PALB, none of whom had any family history of breast cancer. The mean age of patients who carried deleterious variant in BRCA1/BRCA2 was 39 years and 8 months compared to 47 years and 3 months among women who carried a deleterious variant in other breast cancer susceptibility genes.

An optimized protocol for evaluating pathogenicity of VHL germline variants in patients suspected with von Hippel-Lindau syndrome: Using somatic genome to inform the role of germline variants

The interpretation of hereditary genetic sequencing variants is often limited due to the absence of functional data and other key evidence to assess the role of variants in disease. Cancer genetics is unique, as two sets of genomic information are often available from a cancer patient: somatic and germline. Despite the progress made in the integrated analysis of somatic and germline findings, the assessment of pathogenicity of germline variants in high penetrance genes remains grossly underutilized. Indeed, standard ACMG/AMP guidelines for interpreting germline sequence variants do not address the evidence derived from tumor data in cancer. Previously, we have demonstrated the utility of somatic tumor data as supporting evidence to elucidate the role of germline variants in patients suspected with VHL syndrome and other cancers. We have leveraged the key elements of cancer genetics in these cases: genes with expected high disease penetrance and those with a known biallelic mechanism of tumorigenicity. Here we provide our optimized protocol for evaluating the pathogenicity of germline VHL variants using informative somatic profiling data. This protocol provides details of case selection, assessment of personal and family evidence, somatic tumor profiles, and loss of heterozygosity (LOH) as supporting evidence for the re-evaluation of germline variants.

1008. Presence of Antibody Dependent Cell Cytotoxicity (ADCC) Functional Antibodies that Target a Complex Gp41 Epitope Correlates with Long-term Non-progression and ADCC is Maintained with Mutants Using Germline Heavy Chain Variable Gene Sequence of VH1-02 Gene

BackgroundRecent data supports that improved qualitative antibody responses correlate with elite controllers (EC) of HIV. As ADCC has been associated with protection in vaccine studies, thorough exploration of antibodies that facilitate ADCC is warranted. In studies on monoclonal antibodies from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated antibodies against a complex conformational epitope with contributions from both gp41 heptad repeat regions. Despite using the VH1-02 gene segment, known to contribute to some of the broadest neutralizing antibodies against HIV, members of these antibodies, termed group 76C antibodies, did not exhibit broad neutralization. MethodsOur goal was to characterize the non-neutralizing functions of antibodies of group 76C, to assess targeting of the epitope in various clinical presentations, and to assess the development of these antibodies by comparison to their predicted common ancestor. Serum samples were obtained from HIV+ clinical groups: EC, LTNP, stable CD4 counts on therapy, and those off therapy.ResultsIn antibody/serum competition assays, comparison to VRC01 which also uses VH1-02, showed that antibodies targeting the 76C group epitope were enriched in LTNPs. We then show recombinant antibodies of 76C members 6F5 and 6F11 both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc also shows comparable ADCC to 6F5 and 6F11 on both clade B and C constructs.Common ancestor antibodies maintain function and these types of antibodies correlate to a non-progressive clinical state. (A) Serum from long-term non-progressors (LTNPs) compared to serum from a group of HIV infected with lower CD4 levels as a control for viral load were used to compete against biotinylated CD4 binding site (VRC01) and 76C Gp41 conformational epitope (6F11) targeting antibodies. Serum dilutions were chosen to align means near 50%. Means with 95% confidence intervals are shown. (B) Monoclonal antibody 76Canc was created using the germline sequence of the heavy chain variable region with the CDR3 and light chain of 76C member. Antibody dependent cell cytotoxicity flow cytometric based assays were performed using gp41 proteins from clade B (MN) and clade C (ZA1197).ConclusionCertain antibodies present early on in infection may contribute to overall clinical course. Variable gene germline sequences that support functional activity against HIV could be targeted in vaccine regimens.Disclosures All Authors: No reported disclosures

Sequence and functional characterization of a public HIV-specific antibody clonotype

SummaryPublic antibody clonotypes shared among multiple individuals have been identified for several pathogens. However, little is known about the determinants of antibody “publicness”. Here, we characterize the sequence and functional properties of antibodies from a public clonotype targeting the CD4 binding site on HIV-1 Env. Our results showed that HIV-1 specificity for the public antibodies studied here, comprising sequences from three individuals, was modulated by the VH, but not VL, germline gene. Non-native pairing of public heavy and light chains from different individuals suggested functional complementation of sequences within this public antibody clonotype. The strength of antigen recognition appeared to be dependent on the specific antibody light chain used, but not on other sequence features such as native-antibody or germline sequence identity. Understanding the determinants of antibody clonotype “publicness” can provide insights into the fundamental rules of host-pathogen interactions at the population level, with implications for clonotype-specific vaccine development.

A Small Step, a Giant Leap: Somatic Hypermutation of a Single Amino Acid Leads to Anti-La Autoreactivity.

The anti-La mab 312B, which was established by hybridoma technology from human-La transgenic mice after adoptive transfer of anti-human La T cells, immunoprecipitates both native eukaryotic human and murine La protein. Therefore, it represents a true anti-La autoantibody. During maturation, the anti-La mab 312B acquired somatic hypermutations (SHMs) which resulted in the replacement of four aa in the complementarity determining regions (CDR) and seven aa in the framework regions. The recombinant derivative of the anti-La mab 312B in which all the SHMs were corrected to the germline sequence failed to recognize the La antigen. We therefore wanted to learn which SHM(s) is (are) responsible for anti-La autoreactivity. Humanization of the 312B ab by grafting its CDR regions to a human Ig backbone confirms that the CDR sequences are mainly responsible for anti-La autoreactivity. Finally, we identified that a single amino acid replacement (D > Y) in the germline sequence of the CDR3 region of the heavy chain of the anti-La mab 312B is sufficient for anti-La autoreactivity.

Aberrant Dntt Expression, TdT-Aided Gene Length Mutations, and Prognosis Relevance in ATG/Alg Based Regimen Allo-HSCT in Acute Myeloid Leukemias

DNTT encodes the most template-independent DNA polymerases TdT. The canonical function of TdT is to boost the diversity of immunoglobulin and T cell receptors by incorporating non-templated nucleotides (NTN) to their variable regions via RAG1/2 mediated DNA breaks and non-homologous end joining (NHEJ) rearrangement process. This study aimed to investigate the relationship between aberrant DNTT expression and illegitimately TdT-aided microhomology-mediated replication-dependent recombination (MMRDR) with the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and their prognosis relevance.A cohort of 578 AML cases was enrolled. Fifty healthy donors for allogeneic hematopoietic stem cell transplantation (allo-HSCT), 393 B cell acute lymphoblastic leukemia (B-ALL) cases, 78 T-ALL cases, and 25 mixed-phenotype acute leukemia (MPAL) cases were used as control. Next-generation sequencing was performed for mutation analysis of 86 leukemia driver genes. RNA-seq was used to analyze the expression of DNTT and other non-homologous end joining (NHEJ) associated genes. Prognostic was investigated in a subset of 239 AML cases who underwent anti-thymocyte globulin (ATG) or anti-lymphocyte globulin (ALG) based regimen allo-HSCT.Based on sequence anatomy that considers the MMRDR mechanism and nucleotides characteristics of TdT mediated NTN incorporation, we formulate a classification algorithm for LMs and divide them into four subtypes (type I-IV). Type-I indicates pure duplicated/triplicated germline sequences with identifiable ≥2bp canonical triple microhomology (MH) sequences; type-II indicates pure duplicated/triplicated germline sequences without ≥2bp canonical MH sequences; type-III indicates any LMs with NTN insertions; type-IV indicates any other LMs, mainly deletions.FLT3-LMs has the highest overall incidence and occur across multiple lineage leukemias. We observed a significant FLT3-LMs subtypes distribution bias among acute leukemia subtypes (Figure 1A). Type-I FLT3 LMs are only observed in AML; there are mainly type-III FLT3 LMs in T-ALL and MPAL; type-II, III, IV FLT3 LMs are predominant in B-ALL. The overall DNTT expression was significantly lower in AML than in other leukemia subtypes and control groups (P < 0.001). This supports that FLT3-LMs subtypes distribution bias might be attributed to the difference in the overall DNTT expression among leukemia lineages.A total of 458 LMs events were observed in 295 cases (51.0%) within 25 genes (FLT3, NPM1, CEBPA, RUNX1, KIT, etc.) in our AML cohort. The incidence of type-II and type-III LMs, both of which the mutagenesis relies on TdT-aided MMRDR in theoretically speculate, were 31.2 % and 47.8 %, respectively. Type-I and type-II, which manifested as pure germline sequence duplications, account for 43.6% of the FLT3 LMs; type-III LMs, which with additional inserted NTN sequences, account for as high as 50.4% of the FLT3 LMs.We analyzed the G/C nucleotide contents adjacent to LMs junctions. A significantly high G/C bias was observed at +1 nucleotide position in type-II and type-III subsets (Figure 1B), suggesting that the TdT-aided MMRDR mechanism plays a role in the mutagenesis in these cases. We also observed a strong positive correlation between fragment length and G/C content of the inserted NTN sequences (P < 0.001) within the type-III subset of the 25 LMs genes (Figure 1C), suggesting a higher TdT activity mediates longer inserted sequences.DNTT expressions level of type-III LMs cases were significantly higher than that of type-I, II, IV LMs cases and cases without LMs in the total 25 LMs genes (Figure 1D) and the FLT3 LMs subset. Similar expression signatures of other NHEJ associated genes RAG2, XRCC4, and XRCC6 were also observed. For the survival analysis in the ATG/ALG based regimen allo-HSCT AML subset, we observed a significantly better overall survival (P = 0.024) in cases positive for type-III FLT3-LMs than that of type-I, II (Figure 1E).In this study, we proposed a subclassification algorithm for LMs (type I-IV) in AML. Both DNTT gene expression and sequence character suggesting that TdT-aided MMRDR plays a role in the mutagenesis of type-III and type-II LMs. We also observed AML cases with type-III FLT3 LMs benefit more from ATG/ALG based regimen allo-HSCT than cases with other FLT3 LMs types, which may be attributed to the aberrant lymphoid lineage antigen expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pathogenic Germline Variants in Cancer Susceptibility Genes in Children and Young Adults With Rhabdomyosarcoma.

Genome sequencing of the discovery cohort (n = 273) and exome sequencing of the secondary cohort (n = 121) were conducted on germline DNA. Analyses were performed on 130 cancer susceptibility genes (CSG). Pathogenic or likely pathogenic (P/LP) variants were predicted using the American College of Medical Genetics and Genomics (ACMG) criteria. Structural variation and survival analyses were performed on the discovery cohort. We found that 6.6%-7.7% of patients with RMS harbored P/LP variants in dominant-acting CSG. An additional approximately 1% have structural variants (ATM, CDKN1C) in CSGs. CSG variants did not influence survival, although there was a significant correlation with an earlier age of tumor onset. There was a nonsignificant excess of P/LP variants in dominant inheritance genes in the patients with FOXO1 fusion-negative RMS patients versus the patients with FOXO1 fusion-positive RMS. We identified pathogenic germline variants in CSGs previously (TP53, NF1, DICER1, mismatch repair genes), rarely (BRCA2, CBL, CHEK2, SMARCA4), or never (FGFR4) reported in RMS. Numerous genes (TP53, BRCA2, mismatch repair) were on the ACMG Secondary Findings 2.0 list. In two cohorts of patients with RMS, we identified pathogenic germline variants for which gene-specific therapies and surveillance guidelines may be beneficial. In families with a proband with an RMS-risk P/LP variant, genetic counseling and cascade testing should be considered, especially for ACMG Secondary Findings genes and/or with gene-specific surveillance guidelines.

Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies.

Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs (CR9114, 16 heavy-chain mutations; CR6261, 11 heavy-chain mutations), reconstructing all possible evolutionary intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR6261 display moderate affinity to diverse antigens, those of CR9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR6261 and different for CR9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR9114, while the acquisition of breadth to more similar antigens for CR6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.

Exome Sequencing Of Paired Chronic-Blast Phase Samples In Myeloproliferative Neoplasms Reveals High Frequency Of Rare Germline Sequence Variants In TYK2 and Novel Blast Phase-Restricted Mutations

The molecular underpinnings of leukemic transformation in myeloproliferative neoplasms (MPN) are not well characterized. We used Whole Exome Sequencing (WES) as a screening tool to gain further insight into the genomic origin of MPN progression to blast phase disease. WES analysis was performed in 6 JAK2V617F(+) MPN patients for which paired samples from chronic and blast phase disease were available. Respective clinical data were incorporated into the evaluation, and variations of interest were validated by conventional Sanger sequencing. Single-nucleotide-polymorphism database (dbSNP) was used to exclude/include established germline aberrations. Using WES filtering for non-synonymous variations as predicted deleterious, we detected a significant number of genetic abnormalities in both chronic and blast phase of MPN; these could be functionally linked to “tumorigenesis” (157 genes), “cancers and tumors” (159 genes) and “hematological neoplasia” (48 genes). In both phases of MPN, we could find mutations in genes already known to be linked with the development and/or transformation of MPN (ie, MPL, TET2, EZH2, IDH1, NRAS, LNK, TP53, WT1). Notably, eight genes were identified exclusively at onset of leukemic transformation and could be classified as potential driver mutations with damaging translational impact; these novel mutations could be grouped into “transcriptional regulators” (CUX2, ORC2, KDM6B, CNOT3, TBL1XR1), “NOTCH-signaling pathway” (NOTCH2NL, NOTCH1) and “tyrosine kinases” (STAP2). Moreover, we also identified several single-nucleotide polymorphisms encoding amino acid substitutions (ie, non-synonymous changes) in receptor as well as cytoplasmatic tyrosine kinase genes. Interestingly, the JAK-family member TYK2 showed the largest number of rare non-synonymous polymorphisms (A165T, V362F, G363S, I684S) leading to missense mutations in 67% (4/6) of the MPN patients; in contrast, none of 30 control samples with other types of cancer showed sequence variances in the TYK2 gene. Deficiency of TYK2 has shown to impair tumor surveillance and increase the susceptibility for neoplasia including AML. The present study demonstrates recurrent germline sequence variants in the non-receptor tyrosine kinase TYK2, and novel functionally-relevant somatic mutations with potential pathogenetic contribution to leukemic transformation in MPN. Disclosures:No relevant conflicts of interest to declare.

Leveraging three-dimensional chromatin architecture for effective reconstruction of enhancer-target gene regulatory interactions.

A growing amount of evidence in literature suggests that germline sequence variants and somatic mutations in non-coding distal regulatory elements may be crucial for defining disease risk and prognostic stratification of patients, in genetic disorders as well as in cancer. Their functional interpretation is challenging because genome-wide enhancer–target gene (ETG) pairing is an open problem in genomics. The solutions proposed so far do not account for the hierarchy of structural domains which define chromatin three-dimensional (3D) architecture. Here we introduce a change of perspective based on the definition of multi-scale structural chromatin domains, integrated in a statistical framework to define ETG pairs. In this work (i) we develop a computational and statistical framework to reconstruct a comprehensive map of ETG pairs leveraging functional genomics data; (ii) we demonstrate that the incorporation of chromatin 3D architecture information improves ETG pairing accuracy and (iii) we use multiple experimental datasets to extensively benchmark our method against previous solutions for the genome-wide reconstruction of ETG pairs. This solution will facilitate the annotation and interpretation of sequence variants in distal non-coding regulatory elements. We expect this to be especially helpful in clinically oriented applications of whole genome sequencing in cancer and undiagnosed genetic diseases research.

Absolute Percent Deviation of IGHV Mutation As a Continuum Is Prognostically Relevant in Patients with CLL

Abstract Introduction: The percent deviation of immunoglobulin heavy chain gene variable region from the germline sequence (IGHV% or mutation status of IGHV) is commonly used as a prognostic factor in CLL. A 98% cutoff in IGHV %is used to classify CLL patients into mutated (≤98% identity or ≥2% deviation) with a favorable treatment outcome, and unmutated (>98% identity or Methods: We analyzed the data of 203 patients with CLL enrolled on the original frontline FCR clinical trial. Karyotype analysis, exome sequencing and stereotypy status was not available for these patients as most patients were enrolled in the protocol 17 years ago. Testing of IGHV mutation status was performed by comparing the resulting IGHV sequence with the consensus germline sequence in the IMGT/V-QUEST database and the percent change of base pairs was calculated. Cox proportional hazard regressionmodels were used to identify any association between covariates and survival (OS and PFS). Using the IGHV% as a continuous variable, we also analyzed the outcome of patients treated with frontline FCR off protocol (n=332) and of patients treated with ibrutinib (n=166). Statistical analysis was performed using the Stata/SE version 14.2 statistical software (Stata Corp. LP, College Station, TX). Results: The clinical characteristics of 203 patients treated with FCR on protocol and of 332 patients treated with FCR off protocol were similar. The median follow-up of patients enrolled on the original FCR trial was 10.7 years (range 0.1-15.5) and of patients treated with FCR off protocol was 5.6 (0.2-12.1) years. The mean number of treatment cycles was 6 (range 1-6) in both cohorts. Cox proportional hazard models showed the association between IGHV% , as a continuous variable, and survival (PFS and OS) in original FCR trial cohort (Figure 1A and B). The IGHV% was significantly associated with PFS (hazard ratio (HR) 95%; confidence interval (CI): 0.81 (0.76-0.87); P Conclusions: A continuous increment of IGHV% deviation predicts the survival of CLL patients treated with FCR. The higher the percent deviation, the better is treatment outcome, suggesting that the prognostic relevance of IGHV% is a continuous, rather than a dichotomized, variable. In sharp contrast, the IGHV% deviation does not appear to predict the survival of patients treated with ibrutinib. Download : Download high-res image (145KB) Download : Download full-size image Disclosures Burger: Gilead: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; TG Therapeutics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses. Tam: Abbvie: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding. Wierda: The University of Texas MD Anderson Cancer Center: Employment; AbbVie: Consultancy, Honoraria, Research Funding; GSK/Novartis: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Juno: Research Funding; Emergent: Consultancy, Honoraria, Research Funding; Kite: Research Funding; Genentech/Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Karyopharm: Research Funding; Acerta: Research Funding; Celgene: Consultancy, Honoraria; Merck: Consultancy, Honoraria. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jain: Genentech: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; BMS: Research Funding; Verastem: Research Funding; Celgene: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Bristol-Meyers Squibb: Research Funding; Amgen: Research Funding. Cortes: ImmunoGen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding; Teva: Research Funding; BMS: Consultancy, Research Funding. O'Brien: Astellas: Consultancy; Vaniam Group LLC: Consultancy; Janssen: Consultancy; AbbVie: Consultancy; Gilead Sciences, Inc.: Consultancy, Other: Research Support: Honorarium, Research Funding; Pharmacyclics: Consultancy, Other: Research Support: Honorarium, Research Funding; Acerta: Other: Research Support: Honorarium, Research Funding; Sunesis: Consultancy; CLL Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Aptose Biosciences, Inc.: Consultancy; GSK: Consultancy; Celgene: Consultancy; Regeneron: Other: Research Support: Honorarium, Research Funding; TG Therapeutics: Consultancy, Other: Research Support: Honorarium, Research Funding; ProNAI: Other: Research Support: Honorarium, Research Funding; Alexion: Consultancy; Pfizer: Consultancy, Research Funding.