Germline Of Patients Research Articles

Evolution of predictive risk factor analysis for chemotherapy-related toxicity.

The causes of variation in toxicity to the same treatment regimen among seemingly similar patients remain largely unknown. There was tremendous optimism that the patient's germline genome would be strongly predictive of treatment-related toxicity and could be used to personalize treatment and improve therapeutic outcomes. However, there has been limited success in discovering robust pharmacogenetic predictors of treatment-related toxicity and even less progress in translating the few validated predictors into clinical practice. It is apparent that identification of toxicity predictors that can be used to predict and prevent treatment-related toxicity will require thinking beyond germline genomics. To that end, we propose an integrated biomarker discovery approach that recognizes that a patient's toxicity risk is determined by the cumulative effects of a broad range of "omic" and non-omic factors. This commentary describes the limited success in discovering and translating clinical and pharmacogenetic toxicity predictors into clinical practice. We illustrate the evolution of cancer toxicity biomarker discovery and translation through studies of taxane-induced peripheral neuropathy, which is one of the most common and debilitating side effects of cancer treatment. We then discuss the opportunities for discovering non-genomic (e.g., metabolomic, lipidomic, transcriptomic, proteomic, microbiomic, medical, behavioral, environmental) and integrated biomarkers that may be more strongly predictive of toxicity risk and the potential challenges with translating integrated biomarkers into clinical practice. This integrated biomarker discovery approach may circumvent some of the major limitations in toxicity biomarker science and move precision oncology treatment forward so that patients receive maximum treatment benefit with minimal toxicity.

Germline mutations in prostate cancer: a systematic review of the evidence for personalized medicine.

The goal of precision medicine in prostate cancer (PCa) is to individualize the treatment according to the patient's germline mutation status. PCa has a very high rate of genetic predisposition compared with other cancers in men, with an estimated rate of cancers ascribable to hereditary factors of 5-15%. A systematic search (PubMed, Web of Science, and ClinicalTrials.gov) of English literature from 2000 to 2022, using the keywords "prostate cancer", "germline mutations", "family history", and "inheritance" was conducted, according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. The search identified 980 publications. Of these, 200 papers were removed before screening (duplicates, non-English literature, and publication year before 2000) and 245 records were excluded after title/abstract screening. Finally, 50 articles were included in the final analysis. We analyze the latest evidence on the genetic basis of PCa predisposition and clinical implications for more personalized screening protocols and therapeutic management of this high-prevalent cancer. Emerging data show that germline mutations in homologous recombination genes (BRCA1/2, ATM, CHECK2), in mismatch repair genes (MLH1, MLH2, MSH6), and other additional genes are associated with the development and aggressiveness of PCa. Germline testing and genetic counseling have increasingly important implications in cancer screening and therapeutic decisions making for patients affected by PCa. Patients with localized PCa and some gene mutations are more likely to develop aggressive cancer, so active treatment may be preferable to active surveillance for these patients. Moreover, in patients with metastatic PCa, these gene alterations may be useful biomarkers for predicting response to specific therapy such as PARP inhibitors, recently approved for the treatment of metastatic castration-resistant PCa. The evidence supports recent guidelines and recommendations considering germline genetic testing for patients with a positive family history of PCa or men with high risk or metastatic disease.

A Novel Autosomal Dominant JAK2R715T Mutation Causes Polycythemia Vera in 3 Generations

Polycythemia vera (PV) has high hemoglobin (Hb) concentration caused by clonal proliferation of myeloid cells from a pluripotent stem cell (Prchal, N Engl J Med. 1974). Acquired somatic mutations of JAK2 gene, JAK2V617F and rarely exon 12, are present in virtually all PV patients (Ugo, Nature 2005, Scott N Engl J Med. 2007). The thromboses are the major causes of PV-related morbidity and mortality. A 38-year female suffered an ischemic stroke in 2017 at age 32 with negative thrombophilia work-up. She had elevated hemoglobin (Hb) 18 g/dL with intermittent elevation of leukocytes and normal platelets with borderline low erythropoietin (EPO) levels. JAK2V617F and exon 12 mutation analysis were negative. Her marrow was interpreted as compatible with PV and normal cytogenetics. Her hematologist treated her with phlebotomies. She was referred to us for an additional evaluation and management. Myeloid next generation sequencing (NGS) revealed a previously undescribed mutation JAK2R715T in the pseudokinase domain (JH2) with allelic burden ~50%. Some of her erythroid progenitors (BFU-Es) grew without extrinsic EPO, a hallmark of PV (Prchal, N Engl J Med. 1974). Phlebotomies were stopped; in two months, her Hb had risen to 17.4 g/dL and hematocrit had changed to 48.5% with normal leukocytes and platelets. We obtained JAK2 inhibitor fedratinib on compassionate basis from the manufacturer. On fedratinib, her Hb normalized to 15.2 g/dL with 42.8 % hematocrit. Her iron deficiency was corrected without increase of her Hb. The patient's germline mutation was confirmed by DNA sequencing of her nails. We encouraged her to have her only son and parents tested by NGS. The same JAK2R715T mutation was found in her 10-year-old son's blood sample. He also has elevated Hb at 16.2 g/dL (normal for age 11.5-15.5 g/dL), normal leukocytes and platelets were 400 K/µl. Her asymptomatic 63-year-old mother’ Hb was 17 g/dL, hematocrit 50.5%, and sequencing of her JAK2 also revealed JAK2R715T at ~50%. To assess how the R715T mutation may affect kinase activation, the JH1-JH2 domains of JAK2R715T and JAK2WT were modelled using AlphaFold 2.0 (Jumper, Nature 2021). The overall structure of JAK2R715T JH2 was more rigid than that of JAK2WT, in agreement with DynaMut in silico prediction (PMID: 29718330). -Helix C is shown in dark blue, Y570 and T555 in yellow, and ATP and T715 in red. Detailed comparison of JAK2R715T with JAK2WT JH2 (see Figure) indicated that the rigidification of the structure involved notably residues of the JH2-JH2 trans-dimerization domain centered around -Helix C. This has been suggested to mediate direct JH2-JH2 contacts (Wilmes, Science 2020) and a lengthening of the ß-sheet region surrounding the inhibitory phosphorylation site Tyr570 (Echarri, Mol Cell Biol. 2004). The R715 is in direct vicinity of the ATP binding pocket and promotes a closed lobe conformation through interaction with Thr555 of the nucleotide-binding loop (Bandaranayake, Nat Struct Mol Biol. 2012). We suggest that loss of this interaction may result in a more opened lobe conformation and disturbing of ATP binding to JH2, which regulates JAK2 kinase activity (Hammarén, Proc Natl Acad Sci USA 2015). To evaluate the effect of JAK2R715T in erythroid progenitor cells, the patient's erythroid progenitors derived from peripheral mononuclear cells were grown for 21 days with and without JAK inhibitor (ruxolitinib) following the previously published in vitro expansion protocol (Bruchova, Blood Cells Mol Dis, 2009). Compared to PVs and healthy controls, the patient cells showed increased proliferation. JAK inhibitor ruxolitinib treatment of patient cells induced decreased proliferation and differentiation compared to untreated cells. Assessment of STAT5 phosphorylation on patient's blood cells confirmed increased JAK2 activity to levels even higher than this of patients with confirmed JAK2V617F positive PV. X-chromosome based clonality studies of both females are ongoing. The EPO-independent colonies of her BFU-E is being evaluated for possible acquired conversion to homozygosity by uniparental disomy that is present in PV (Kralovics, Experimental Hematology, 2002). We conclude that this is the first example of dominantly inherited PV phenotype in 3 generations due to a novel germline JAK2R715T gain-of function mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A rare PALB2 germline variant causing G2/M cell cycle arrest is associated with isolated myelosarcoma in infancy

BackgroundIsolated myelosarcoma of infancy is a rare presentation of acute myelogenous leukaemia (AML). Because of its rarity and early onset in infancy underlying genetic predisposition is potentially relevant in disease initiation.Methods and ResultsWe report an oncologic emergency in an infant with thoracic and intraspinal aleukaemic myeloid sarcoma causing acute myelon compression and lower leg palsy. Whole‐exome sequencing of the patient's germline DNA identified a rare PALB2 (OMIM 610355) variant (p.A1079S), which is located in a domain critical for the gene's proper function within the homology‐directed repair pathway. In line with potential DNA damage repair defects mediated by the PALB2 deregulation, the patient's fibroblasts showed increased sensitivity towards radiation and DNA intercalating agents.ConclusionTherefore, we suggest PALB2 p.A1079S as a pathogenic variant potentially contributing to the here observed patient phenotype.

Abstract 261: Long-read sequencing characterization of a patient with bilateral Wilms tumor of unknown etiology

Abstract Wilms tumor is the most common childhood kidney cancer. 15% of patients with Wilms tumor have germline pathogenic variants in genes or regions such as WT1 or the 11p15 region. Variants in these regions can include structural or copy number alterations or alterations in methylation. In the majority of cases of Wilms tumor no known pathogenic variant could be found using the state-of-the-art technologies, including comprehensive approaches such as Illumina whole exome sequencing. One explanation for this is that such technologies have difficulties in detecting structural variants (SVs) in areas associated with repeat or low complexity sequence. In addition, Illumina technology does not immediately support direct methylation detection. Therefore, we hypothesized that analysis of bilateral Wilms tumor of unknown etiology using long-read sequencing could reveal molecular events of potential clinical interest. We performed in-depth genomic analysis on a whole blood DNA sample from a patient with a bilateral Wilms tumor. This patient had no significant family history of cancer, and previously tested negative for Beckwith-Wiedemann syndrome by methylation testing of the 11p15 region; clinical exome sequencing of the patient's germline detected no variants associated with Wilms tumors. We sequenced the genome at 40x depth using PromethION Nanopore sequencing. 29180 SVs (deletions or insertions larger than 30 base pairs) were detected using Sniffles and 26480 were detected with SVIM. Only SVs that were detected by both methods were considered for downstream analysis. Variants were annotated and filtered using a short-read catalog of SVs (gnomAD-SV), a long-read catalog, the Database of Genomic Variants catalog, and by comparison to 11 in-house genomes. We focused on SVs, copy number variants, and methylation events affecting genes previously associated with Wilms tumor. Our long-read sequencing approach detected compound heterozygotes using phased variant calls. A heterozygous missense mutation was identified in haplotype one, while a 300 base pair insertion in an ALU element was present in haplotype two. These two compound heterozygous variants overlap an exon of the OVCH2 gene, and the ALU element was not detected by the prior Illumina analysis. Additionally, we determined the frequency of methylation in CpG sites genomewide using nanopolish. Using a normal blood sample from an unrelated individual as a control, we searched for extreme differences across large and gene promoter regions. Hypermethylation in the promoter regions of genes in the 11p15.5 locus was observed in the patient as compared to the control. Hypomethylation in this region is associated with Beckwith-Wiedemann syndrome. In conclusion, nanopore technology is able to detect variants missed by Illumina sequencing, and has the potential to yield new findings of interest in a case of a child with suspected cancer predisposition syndrome. Citation Format: Allison R. Cheney, Jean Monlong, Holly C. Beale, Hugh Olsen, Ellen Towle Kephart, Katrina Learned, Shanna White, Julian A. Martinez-Agosto, Noah Federman, Mark Akeson, Miten Jain, Vivian Y. Chang, Olena M. Vaske. Long-read sequencing characterization of a patient with bilateral Wilms tumor of unknown etiology [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 261.

Interim Cell-Free Circulating Lymphoma DNA Analysis of the Phase 2 Accept Trial

Introduction Plasma cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) genotyping in Diffuse Large B-cell Lymphoma (DLBCL) holds great promise for early detection and response prediction. DLBCL is a highly heterogenous disease with diverse tumor behaviour and outcomes, yet the delineation of poor- risk groups remains challenging. Genomically driven molecular risk stratification, using ultradeep next generation sequencing (NGS) provides a comprehensive view of the mutational landscape of lymphoma and enables ultrasensitive disease detection. Here, we present an interim analysis of ctDNA kinetics from ACCEPT, a phase Ib/II study of acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for patients with previously untreated DLBCL. Methods Plasma samples were collected at four time points, at baseline and before the second, third and final cycles of chemotherapy. Extracted cell-free DNA (cfDNA) was quantified by fluorometry, prior to undergoing library preparation and next-generation sequencing using the CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) methodology. The targeted sequencing panel covered 799 regions over 50 genes (>200Kb) known to be involved in lymphomagenesis. Analysis was carried out with a custom bioinformatics pipeline using error-corrected deduplication and a database of known artefacts to reduce background noise and improve sensitivity for low-level somatic variant detection. Patients were closely monitored through the study period by clinical examination and PET-CT scanning to provide objective endpoints against which the ctDNA response could be correlated. Results Fifteen patients had samples analysed for all four time points at the time of the interim analysis and were included in the study. Ten (66%) had late stage disease, nine (60%) poor risk disease (IPI > 2). Early vs late stage of disease at presentation showed a strong correlation with baseline levels of both total cfDNA (3,946 HGE/ml vs 21,613 HGE/ml) and ctDNA (842HGE/ml vs 6,380 HGE/ml). Twelve patients (80%) had trackable variants detected at baseline. The majority of these cleared after the first cycle of chemotherapy (Figure A; representative patient). Two cases (Figures B and C) had variants persistently detectable at high variant allele frequency (VAF) (0.059 and 0.068). These patients both had primary refractory disease. One patient from the first acalabrutinib dose escalation cohort and the second patient was found to be ineligible and withdrawn from the primary analysis because of continuous co-administration of omeprazole during therapy which inhibits acalabrutinib absorption. All others achieved a metabolic complete metabolic remission (mCR) after the final cycle of therapy. A third pattern of response noted was (Figure D) showing disappearance of detectable variants at baseline but progressive emergence of an acquired TP53 variant throughout follow-up that was not detected in the patient's germline sample. This high-risk patient with IPI of 5 and triple-hit DLBCL achieved a mCR and remains in remission in follow-up. Conclusion The clearance of ctDNA from the plasma of the responding patients in our study is consistent with the prior literature, as is the poor prognostic significance of persisting variants after the first cycle of chemotherapy. The rapid kinetics of the changes in ctDNA is noted and is potentially of advantage over other modalities of monitoring, such as PET-CT scan. Data on the entire cohort (n=30) and longer follow-up will be available at the time of the meeting. Figure 1 Disclosures Davies: Roche: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Roche, Celgene, Kite Pharma, Acerta, Karyopharma, Regeneron, Incyte: Consultancy; Roche, Acerta Pharma, AstraZeneca, Celgene, Gilead, ADC Therapeutics, Gilead: Research Funding; Celegene, Roche, Kite Pharma, Celegene: Honoraria. Johnson:Oncimmune: Consultancy; Bristol-Myers: Honoraria; Celgene: Honoraria; Genmab: Honoraria; Incyte: Honoraria; Kite Pharma: Honoraria; Kymera: Honoraria; MorphoSys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Boehringer Ingelheim: Consultancy; Epizyme: Consultancy, Research Funding; Janssen: Consultancy; Oncimmune: Consultancy; Epizyme: Consultancy, Research Funding; Janssen: Consultancy. Schuh:Illumina: Other: Consulting fees; Roche: Other: Consulting fees; Gilead: Other: Consulting fees; Abbvie: Other: Consulting fees.

Targeted Therapy in Metastatic Breast Cancer-Which Molecular Tests Are Necessary?

In den letzten Jahren ist die zielgerichtete Therapie beim Mammakarzinom immer mehr in den Fokus gerückt. Neben den Hormonrezeptoren und dem humanen epidermalen Wachstumsfaktor 2 (HER2) sind derzeit für die Festlegung einer gezielten Therapie vor allem der immunhistochemische Nachweis des „programmed cell death ligand 1“ (PD-L1) bei fortgeschrittenen triple-negativen Mammakarzinomen sowie der Nachweis von Mutationen im Breast-cancer-1(BRCA1)- oder BRCA2-Gen in der Keimbahn der Patientinnen und von Mutationen im Phosphatidylinositol-3-Kinase(PI3K)-Weg relevant.

Igcaller: Reconstructing the Rearranged Immunoglobulin Gene in Lymphoid Neoplasms from Whole-Genome Sequencing Data

Introduction: Mature normal and tumor B cells express a unique rearranged immunoglobulin (IG) gene that can be used as a marker of the clonal expansion of the cell. Somatic hypermutation (SHM) in the V(D)J region of IG genes are acquired in the germinal center and are a surrogate imprint of the cell of origin of lymphoid neoplasms. In chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), the identification of SHM distinguishes subtypes of tumors with different clinical and biological behavior. Although still less studied, cases carrying highly similar IG sequences (i.e. stereotyped IG), specific light chain (LC) rearrangements, and the presence of class switch recombination (CSR) of the constant region of the heavy chains further sub-classify patients into potentially distinct clinico-biological subgroups. The analysis of the rearranged IG gene is currently performed by specific Sanger sequencing (SSeq) or next-generation sequencing protocols. Whole-genome sequencing (WGS) of B-cell neoplasms should store the information to reconstruct the entire rearranged IG gene [heavy (IGH) and kappa or lambda (IGK, IGL) LC]. However, the high genomic complexity and homology of these regions have prevented the analysis of the rearranged IG genes in WGS using standard bioinformatics pipelines. Aim: To assess the use of WGS data to fully characterize the rearranged IG gene in B-cell neoplasms. Methods: We developed IgCaller, a fast, easy-to-run program that uses already aligned WGS data to dissect the rearranged IGH V(D)J genes, IGK and IGL VJ genes, and the presence of constant heavy chain CSR. IgCaller also determines the homology of the rearranged sequences compared to the patient's germ line or reference genome. We demonstrated the accuracy of IgCaller using WGS data of 331 B-cell neoplasms [240 CLL (152 cohort 1 (C1)-CLL; 88 independent cohort 2 (C2)-CLL), 61 MCL, and 30 multiple myeloma (MM)] and compared with SSeq of the IGH V(D)J and/or LC and isotype expression. Results: IgCaller identified a complete IGH productive rearrangement [V(D)J] in 133 (88%) C1-CLL, 80 (91%) C2-CLL, 61 (100%) MCL, and 21 (70%) MM. A partial (VJ) rearrangement was detected in 8 (5%) C1-CLL and 1 (3%) MM. Available SSeq of the V(D)J or at least V gene for 131 C1-CLL, 10 C2-CLL, and 60 MCL successfully characterized by WGS highlighted only one discordant V(D)J rearrangement. Small discrepancies (only J or V disagreement) were found when the J (n=4) or V (n=1) gene identified by SSeq based on homology (IMGT/V-QUEST tool) did not correspond to the rearranged gene detected by WGS, which was the second scoring gene in IMGT/V-QUEST suggesting that our non-homology WGS-based approach might be more accurate. Of note, IgCaller identified the presence of two distinct IGH subclones in 1 case. Next, the comparison of the percentage of homology of the rearranged sequence to the germ line in 131 C1-CLL and 60 MCL with complete V gene both by SSeq and WGS showed a high correlation and concordance in both cohorts [R>0.95, p<1e-30; Passing Bablok regression: 0.05+1*SSeq (CLL), -0.19+1*SSeq (MCL)]. Only 1 CLL patient was differentially classified as mutated or unmutated between SSeq (complete rearrangement) and WGS (partial rearrangement) using the cut off of 98%. A productive IGK or IGL was found in 147 (97%) C1-CLL, 85 (97%) C2-CLL, 61 (100%) MCL, and 26 (87%) MM. These results fully agreed with the LC expression observed by flow cytometry (FC). Of clinical relevance, a total of 26/232 (11%) CLL carried IGLV3-21. The presence of IGLV3-21 was associated with a shorter time to first treatment (TTFT) independently of the IGHV status (p=0.045). Finally, IgCaller identified the CSR matching the isotype expressed by FC analysis in 26/30 (87%) MM. One of the 4 discordant cases with IgM by WGS expressed only LC by FC. Overall, IgCaller could not identify the isotype switch in 3 (10%) MM expressing IgG or IgA. In CLL, CSR was observed in 47/240 (20%) cases. Noteworthy, the presence of CSR identified 24% of M-CLL patients with a shorter TTFT than non-switched M-CLL (p=0.004), and similar to that of unmutated IGHV cases (p=0.15). Conclusions: IgCaller successfully characterized the entire IG gene of >90% B-cell neoplasms studied. The complete characterization of the rearranged IG gene based on WGS data, when available, could facilitate the analysis of LC rearrangements and CSR, and replace the traditional SSeq of the IG loci both in research and clinical settings. Disclosures No relevant conflicts of interest to declare.

Identification and characterization of a novel adenomatous polyposis coli mutation in adult pancreatoblastoma.

During next generation sequencing (NGS) analysis, many missense mutations were found in a well-known oncogene, many of which were variant of uncertain significance mutations. We recently treated an adult patient with pancreatoblastoma by chemotherapy. Using an NGS cancer panel, we found a previously unreported missense mutation in the 1835 codon of the adenomatous polyposis coli (APC) gene. We also found a heterogeneous mutation in the 1835 codon of the APC gene in the patient's germline by Sanger sequencing. Although this patient did not have a history of familial adenomatous polyposis, functional analysis suggested the R1835G mutant APC showed attenuated repression of Wnt/β-catenin signaling activity. This is the first report showing a novel APC missense mutation involved in the onset of adult pancreatoblastoma.

Precision oncology in the age of integrative genomics.

Precision oncology applies genomic and other molecular analyses of tumor biopsies to improve the diagnosis and treatment of cancers. In addition to identifying therapeutic options, precision oncology tracks the response of a tumor to an intervention at the molecular level and detects drug resistance and the mechanisms by which it occurs. Integrative genomics can include sequencing specific panels of genes, exomes, or the entire triad of the patient's germline, tumor exome, and tumor transcriptome. Although the capabilities of sequencing technologies continue to improve, widespread adoption of genomics-driven precision oncology in the clinic has been held back by logistical, regulatory, financial, and ethical considerations. Nevertheless, integrative clinical sequencing programs applied at the point of care have the potential to improve the clinical management of cancer patients.

Genomic analysis of the origins and evolution of multicentric diffuse lower-grade gliomas

Rare multicentric lower-grade gliomas (LGGs) represent a unique opportunity to study the heterogeneity among distinct tumor foci in a single patient and to infer their origins and parallel patterns of evolution. In this study, we integrate clinical features, histology, and immunohistochemistry for 4 patients with multicentric LGG, arising both synchronously and metachronously. For 3 patients we analyze the phylogeny of the lesions using exome sequencing, including one case with a total of 8 samples from the 2 lesions. One patient was diagnosed with multicentric isocitrate dehydrogenase 1 (IDH1) mutated diffuse astrocytomas harboring distinct IDH1 mutations, R132H and R132C; the latter mutation has been associated with Li-Fraumeni syndrome, which was subsequently confirmed in the patient's germline DNA and shown in additional cases with The Cancer Genome Atlas data. In another patient, phylogenetic analysis of synchronously arising grade II and grade III diffuse astrocytomas demonstrated a single shared mutation, IDH1 R132H, and revealed convergent evolution via non-overlapping mutations in ATRX and TP53. In 2 cases, there was divergent evolution of IDH1-mutated and 1p/19q-codeleted oligodendroglioma and IDH1-mutated and 1p/19q-intact diffuse astrocytoma, occurring synchronously in one case and metachronously in a second. Each tumor in multicentric LGG cases may arise independently or may diverge very early in their development, presenting as genetically and histologically distinct tumors. Comprehensive sampling of these lesions can therefore significantly alter diagnosis and management. Additionally, somatic IDH1 R132C mutation in either multicentric or solitary LGG identifies unsuspected germline TP53 mutation, validating the limited number of published cases.

A Novel Homozygous CTC1 Germline Mutation Associated with Bone Marrow Failure

Iron deficiency in young adult males is rare and necessitates prompt investigations. Here we describe a previously healthy 22-year-old male suffering from gastrointestinal bleeding leading to iron deficiency anemia and explained by a constitutional disease. His diagnosis was obtained after detecting a homozygous CTC1 mutation in his germline DNA. Conserved telomere maintenance component 1 (CTC1) is a part of the conserved telomere-associated complex (CST) participating in telomere replication. CST seems to have a role in resolving replication stress also in non-telomeric regions, but little is known about the exact mechanisms. Biallelic, typically compound heterozygous mutations in CTC1 are known to cause cerebroretinal microangiopathy with calcifications and cysts(CRMCC), which is considered as a telomere disease. It is a highly pleiotropic multi-organ disease characterized by abnormalities e.g. in the brain, retina, intestinal vasculature, as well as bone marrow failure (BMF). Some CRMCC patients have dyskeratosis congenita (DC) -like skin, nail, mucosal, and hair changes. Most patients are diagnosed in early childhood and die before teens. To date 37 patients with CRMCC and CTC1 mutations have been reported.The patient was referred to hematology due to iron deficiency anemia with coinciding thrombocytopenia. He also had a notable lymphopenia but no tendency for viral infections or other signs of immunodeficiency. Imaging revealed a patent umbilical vein, widened portal vein, and hepatosplenomegaly without parenchymal changes or lymphadenopathy. Gaucher's disease was excluded by glucocerebrosidase and genetic tests. Histology of a liver biopsy was normal. Based on these findings an idiopathic portal hypertension was diagnosed. Endoscopy revealed gastric antral vascular ectasia. The bone marrow was hypoplastic and pancytopenia persisted after supplementation with iron. He had no known family history of BMF. The patient's telomere length analyzed by a semi-quantitative PCR-test was within the normal range compared to healthy controls of the same age group. We then sequenced the patient's germline exome. Roche MedExome kit was used to target the coding regions of the genome and sequencing conducted using an Illumina HiSeq1500 sequencer. The quality of the data was assessed and the sequence data analyzed using an in-house developed analysis pipeline. The mutation analysis was targeted to genes previously reported to cause immune deficiencies, myelodysplastic syndrome and BMF. The average sequence coverage was 59x, translating into 95% of the exome being covered with a minimum of 20x.Common polymorphisms were excluded from the analysis. The patient was identified to carry a homozygous variation in CTC1 exon 12; c.1994T>G, p.V665G (rs199473676). The variant has previously been reported pathogenic, but all previously reported cases of p.V665G are compound heterozygous (in the majority of cases V665G and a frameshift mutation). The minor allele frequency for this variant in Finns is 0.0015 (10/6612 alleles) and 0.0001 (13/120608) in total (ExAC database) suggesting this mutation is a Finnish founder mutation. We validated the finding by capillary sequencing. The variant was detected heterozygous in both parents ruling out a larger deletion and verifying the mode of inheritance. A healthy sister did not carry the mutation. Identification of CTC1 p.V665G homozygosity raised a high suspicion of CRMCC and further investigations were indicated. Retinal examination as well as brain MRI/MRA revealed multiple lesions characteristic for CRMCC.This is the first report on homozygous CTC1 p.V665G and the second describing CTC1-homozygosity associated with CRMCC, strengthening the disease-causality of biallelic missense mutations in CTC1. The identification of a homozygous CTC1 p.V665G may also open new prospects for studying the effects of the mutation. Our case shows the importance and capacity of new genomic techniques in clinical management, as well: The patient's primary phenotype with iron deficiency anemia, thrombopenia and hepatosplenomegaly could have been explained by only an idiopathic portal hypertension. However, with exome sequencing we were able to reveal a more complex disease entity with major impact on patient's future care and genetic counseling, highlighting the value of germline sequencing in modern diagnostics. DisclosuresNo relevant conflicts of interest to declare.

1584P - Germline genetic background contribution to metastatic dissemination in breast cancer extreme phenotype patients

Previous studies suggest that breast cancer dissemination is not only driven by the tumor somatic alterations but also by the host's genetic background. The main objective of this study is to identify germinal genetic variations predictive of metastatic dissemination in breast cancer patients. Breast cancer patients with discordant extreme phenotypes were selected for a genome-wide association study (GWAS): low risk patients (<2cm and no lymph node involvement) that unexpectedly relapsed within 5 years after surgery and high risk patients (>10 positive lymph nodes) without relapse. Patients were distributed in a discovery set including controls (low risk without relapse and high risk with relapse) and a validation set. Around 4.3 million SNPs were genotyped with the HumanOmni5-Quad Beadchip (Illumina) from peripheral blood DNA. Data analysis was performed as an individual association analysis using Plink software, GenomeStudio (Illumina) and snpMatrix package from R and a pathways analysis with GSA-SNP (http://gsa.muldas.org). We successfully genotyped 145 patients, 95 in the discovery set (47 cases and 48 controls) and 50 cases in the validation set. In the individual association analysis, the strongest associations were located in SNPs in/close to genes related with metastatic dissemination. One of the top SNPs was rs28452734 (p = 3.43x10-5, OR = 9.86 in discovery set; p = 2.96x10-3, OR = 4.3 in validation set). This SNP is located near XYTL1, a gene involved in the biosynthesis of glycosaminoglycan chains which form the extracellular matrix. XYLT1 has been previously associated with adhesion, proliferation, angiogenesis and metastasis. In turn, the pathways analysis identified an enrichment of genes from KEGG pathways including ECM-receptor interaction and cell adhesion and in three pathways previously published in association with metastasis 1) PIK3CA multipotent genetic program 2) early-stage metastatic disease or low burden signature 3) exosomal integrins and organ-specific metastasis. These results suggest that there may be an association between patient's germline genetic background and breast cancer prognosis, independently or in conjunction with intrinsic tumor alterations.

The impact of mosaicism in preimplantation genetic diagnosis (PGD): approaches to PGD for dominant disorders in couples without family history

Mosaicism in certain dominant disorders may result in a 'non-Mendelian' transmission for the causative mutation. Preimplantation genetic diagnosis (PGD) is available for patients with inherited disorders to achieve an unaffected pregnancy. We present our experience for two female patients with different dominantly inherited autosomal disorders; neurofibromatosis type 1 (NF1) and tuberous sclerosis complex type 2 (TSC2). PGD protocol development was carried out using single cells from the patients. PGD was carried out on polar bodies and different embryonic cells. Protocol development for NF1 using lymphocytes from the patient suggested mosaicism for the mutation. This was supported further by quantitative fluorescent-PCR performed on genomic DNA. During PGD, polar bodies and blastomeres lacked the mutation that probably was absent or present at very low levels in the patient's germline. Single lymphocyte analysis during protocol development for TSC2 did not indicate mosaicism; however, analysis of single buccal cells and multiple embryo biopsies across two consecutive IVF/PGD cycles confirmed gonosomal mosaicism. The trend in PGD is for blastocyst biopsy followed by whole genome amplification, eliminating single cell analysis. In the case of certain dominantly inherited disorders, pre-PGD single cell analysis is beneficial to identify potential mosaicism that ensures robust protocols. © 2016 John Wiley & Sons, Ltd.

Abstract ES4-2: Tailoring chemotherapy

Abstract Numerous advances have been made in the management of advanced breast cancer over the last decade. Many of these have focused on the development of targeted agents, capitalising on our increased understanding of the molecular pathways involved in breast cancer biology. Nonetheless for many patients the backbone of treatment remains cytotoxic chemotherapy, either as a sole therapy (in those with cancer phenotypes where additional targeted therapy is not indicated) or combined with targeted therapy. Whilst most of the chemotherapy agents indicated in the treatment of advanced breast cancer have been available in the clinic for many years there remain controversies and variations in practice around the use of these standard agents. This presentation will discuss the indications and factors which should be taken into account in making the choice between combinations as compared sequential single agent chemotherapy. The clinical trial evidence will be overviewed with examples from clinical practice. The continuation of chemotherapy beyond maximal response and the advantages and disadvantages of maintenance chemotherapy will then be discussed. This will lead on to a discussion of whether there is a population of patients in whom metronomic low-dose chemotherapy might be indicated. In the final part of the presentation whether choice of chemotherapy should be influenced by tumour phenotype as defined by estrogen receptor status, HER2 status and the patient's germline BRCA mutation status will be discussed. At the conclusion of this educational session it is anticipated that the attendees will have a good understanding of the evidence underlying these areas, as well as practical knowledge which will help in clinical practice. It is hoped that better understanding of the optimal use of chemotherapy will maximise the benefit of these agents for our patients: gaining longest possible control of disease with best possible quality of life. Citation Format: Ring A. Tailoring chemotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr ES4-2.

MPTH-31CO-EXISTENCE OF TWO TERT PROMOTER REGION SOMATIC POINT MUTATIONS IN A RECURRENT GLIOBLASTOMA

BACKGROUND: Mutations in the promoter region of the telomerase reverse transcriptase gene (TERT) occur at a frequency of greater than 80% in primary GBM. The two most observed TERT promoter mutations in primary GBM, C250T and C228T, each create a de novo transcription factor consensus binding site, upregulating TERT mRNA expression, and are thought to occur in a mutually exclusive fashion. Here we describe the case of a recurrent GBM found to harbor both the C250T and C228T mutations. METHODS: A 57-year-old woman who presented with right hemiparesis and expressive dysphasia was found to have a left frontal contrast-enhancing tumor. She underwent subtotal resection, which revealed primary GBM, and went on to receive standard chemoradiation combined with intra-arterial and intravenous bevacizumab as part of an experimental protocol. She returned with disease progression ten months later and underwent second resection. Fresh tumor was preserved for histopathology, DNA extraction, and was cultured in both serum-free and adherent conditions. RESULTS: Sequencing of tumor DNA revealed that almost all cells harbored the C250T mutation, while a small fraction of cells harbored the C228T mutation. In adherent conditions at early passage, the C250T mutation continued to predominate while there was slight enrichment of C228T. Early and late passage spheres uniformly exhibited the C228T mutation, while C250T was undetectable. Late passage adherent cells exhibited only C228T and could form spheres when transferred into serum-free conditions. All cultures were genotyped and compared to the patient's germline DNA to exclude contamination. CONCLUSIONS: Our results suggest that ongoing convergent evolution gave rise to the C228T mutation in a clone of tumor cells that did not harbor the prevailing C250T mutation of the original tumor. The clone containing C228T exhibited greater proliferation and superior sphere-forming ability in vitro compared to clones containing C250T, and may have been selected through treatment.

A decision support framework for genomically informed investigational cancer therapy.

Rapidly improving understanding of molecular oncology, emerging novel therapeutics, and increasingly available and affordable next-generation sequencing have created an opportunity for delivering genomically informed personalized cancer therapy. However, to implement genomically informed therapy requires that a clinician interpret the patient's molecular profile, including molecular characterization of the tumor and the patient's germline DNA. In this Commentary, we review existing data and tools for precision oncology and present a framework for reviewing the available biomedical literature on therapeutic implications of genomic alterations. Genomic alterations, including mutations, insertions/deletions, fusions, and copy number changes, need to be curated in terms of the likelihood that they alter the function of a "cancer gene" at the level of a specific variant in order to discriminate so-called "drivers" from "passengers." Alterations that are targetable either directly or indirectly with approved or investigational therapies are potentially "actionable." At this time, evidence linking predictive biomarkers to therapies is strong for only a few genomic markers in the context of specific cancer types. For these genomic alterations in other diseases and for other genomic alterations, the clinical data are either absent or insufficient to support routine clinical implementation of biomarker-based therapy. However, there is great interest in optimally matching patients to early-phase clinical trials. Thus, we need accessible, comprehensive, and frequently updated knowledge bases that describe genomic changes and their clinical implications, as well as continued education of clinicians and patients.

High-resolution breakpoint analysis provides evidence for the sequence-directed nature of genome rearrangements in hereditary disorders.

Although most of the pertinent data on the sequence-directed processes leading to genome rearrangements (GRs) have come from studies on somatic tissues, little is known about GRs in the germ line of patients with hereditary disorders. This study aims at identifying DNA motifs and higher order structures of genome architecture, which can result in losses and gains of genetic material in the germ line. We first identified candidate motifs by studying 112 pathogenic germ-line GRs in hereditary colorectal cancer patients, and subsequently created an algorithm, termed recombination type ratio, which correctly predicts the propensity of rearrangements with respect to homologous versus nonhomologous recombination events.

Breast adenocarcinoma recurring as small cell carcinoma in a patient with a germline BRCA2 mutation: clonal evolution unchecked

BackgroundBecause up to 30% of breast cancer cases may relapse, understanding the biology of recurrent breast cancer is imperative in preventing these poor outcomes. Thus, we present this unusual case of a BRCA2 carrier who presented seven years after her initial diagnosis of breast adenocarcinoma with a new lump in the left axillary tail, which proved to be small cell carcinoma. The second cancer bore no morphologic or immunohistochemical resemblance to the first. However, we aimed to understand whether the two cancers could have been related.MethodsWe performed targeted Next Generation Sequencing on both cancer specimens in order to determine whether there was a genomic relationship between the two cancers.ResultsWe found that the initial breast adenocarcinoma was positive for a heterozygous mutation in PIK3CA (c. 1624 G > A, p.E42K) and a heterozygous 13-basepair deletion in TP53 (c.639-651del, p.H214fs). The small cell cancer was positive for the same mutation in PIK3CA, but negative for the mutation in TP53.ConclusionWe concluded that the small cell cancer may have arisen from a clone within the initial cancer, since they carried an identical genetic mutation. Furthermore, we postulate that the unusual morphology of the second cancer may be due, in part, to the patient’s germline BRCA mutation.

Abstract 4280: Integrative analyses of multi-omics sequencing data to guide treatment decisions in a patient with double malignancy

Abstract Multiple primary malignancies are becoming common. The number of second or higher-order cancers is burgeoning and accounted for about 16% of incident cancers. Double malignancy cases pose the problem of finding the best treatment for the patient. Next generation sequencing (NGS) was used in this study to characterize the genetic and genomic composition in a patient with multiple cancer syndromes. A lung adenocarcinoma brain metastasis sample (FFPE) naïve to any tumor therapy, and relapsed primary lung adenocarcinoma and ovarian serous carcinoma tumor biopsies (fresh frozen) were procured from a 51-year old Chinese female never-smoker. The primary lung and ovarian cancer samples were post to the treatments with both first line and EGFR-TKI targeted therapy. Multiple NGS approaches were conducted on the patient biopsies, including DNA whole genome/exome sequencing, RNA sequencing, and microRNA sequencing. Integrative analyses of the comprehensive NGS data indicated EGFR exon 19 deletions in-frame, which encode part of the kinase domain presented in both primary lung cancer and its brain metastases. EGFR T790M mutation which accounts for the resistance of lung tumor to the EGFR-TKI therapy was identified in the relapsed lung sample but not in the brain malignancy. These results were well aligned with the clinical response to EGFR-TKI treatment in the patient. Furthermore, we uncovered frequent occurrences of somatic copy number variations in both lung and ovarian tumors. A somatic non-sense mutation (COSMIC ID: 307331) in TP53 was uniquely identified in the ovarian tumor biopsy, suggesting a different mechanism driving tumorigenesis than that in the lung. Besides, we also identified a heterozygous insertion in DUSP16 and a stop code gained mutation in MAP3K15 in the patient's germline DNA, a potential predisposition of multiple cancer syndromes and early onset of tumor in this patient. Finally, structural variation and gene fusion, gene interaction, pathway analysis of the affected genes were integrated and evaluated. Overall, NGS approaches shed insights into the molecular phenotypes of tumors and thus have the potential to provide the physician key scientific rationale to design targeted therapeutic approaches in the treatment course. Citation Format: Wei Zhu, Jiaqi Huang, Brandon W. Higgs, Philip Brohawn, Shujun Dai, Laura Richman, Bahija Jallal, Liyan Jiang, Yihong Yao. Integrative analyses of multi-omics sequencing data to guide treatment decisions in a patient with double malignancy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4280. doi:10.1158/1538-7445.AM2014-4280