Handling of Germline Findings in Clinical Comprehensive Cancer Genomic Profiling

Patients found to have presumed germline pathogenic variants (PGPVs) during comprehensive genomic profiling (CGP) require genetic counseling (GC) referrals. We retrospectively investigated the outcomes of patients with PGPVs. Among 159 patients who underwent CGP, we recommended GC for the 16 patients with PGPVs (3 with [FG group] and 13 without [G Group] a family/personal history of hereditary cancer) as well as for the 8 patients with no PGPVs, but a history (F group); 2 (67%), 5 (38%), and 3 (38%) patients received GC in the FG, G, and F groups, respectively. Germline testing results were positive in 1 and 2 patients of the FG and G groups, respectively. Among the patients recommended for GC, 58% did not receive GC due to lack of interest, poor performance status, or death. CGP contributes to the identification of germline variants in patients without a history of hereditary cancer. However, the proportion of patients who undergo GC should be improved

Genetic ancestry inference from cancer-derived molecular data across genomic and transcriptomic platforms

Genetic ancestry-oriented cancer research requires the ability to perform accurate and robust genetic ancestry inference from existing cancer-derived data, including whole exome sequencing, transcriptome sequencing, and targeted gene panels, very often in the absence of matching cancer-free genomic data. Here we examined the feasibility and accuracy of computational inference of genetic ancestry relying exclusively on cancer-derived data. A data synthesis framework was developed to optimize and assess the performance of the ancestry inference for any given input cancer-derived molecular profile. In its core procedure, the ancestral background of the profiled patient is replaced with one of any number of individuals with known ancestry. The data synthesis framework is applicable to multiple profiling platforms, making it possible to assess the performance of inference specifically for a given molecular profile and separately for each continental-level ancestry; this ability extends to all ancestries, including those without statistically sufficient representation in the existing cancer data. The inference procedure was demonstrated to be accurate and robust in a wide range of sequencing depths. Testing of the approach in four representative cancer types and across three molecular profiling modalities showed that continental-level ancestry of patients can be inferred with high accuracy, as quantified by its agreement with the gold standard of deriving ancestry from matching cancer-free molecular data. This study demonstrates that vast amounts of existing cancer-derived molecular data are potentially amenable to ancestry-oriented studies of the disease without requiring matching cancer-free genomes or patient self-reported ancestry

Germline BRCA testing in pancreatic cancer: improving awareness, timing, turnaround, and uptake

Prognosis is generally poor for patients with pancreatic ductal adenocarcinoma. However, patients with germline BRCA1 or BRCA2 mutations (gBRCAm) may benefit from first-line platinum-based chemotherapy and maintenance therapy with the poly(adenosine diphosphate-ribose) polymerase inhibitor olaparib following at least 16 weeks of first-line platinum-based chemotherapy without disease progression. Germline breast cancer gene (BRCA) testing is therefore important to ensure that patients receive the most effective treatment. In addition, testing for other DNA damage response gene mutations beyond gBRCAm may also guide treatment decisions. However, clinical pathways for genetic testing are often suboptimal, leading to delays in treatment initiation or missed opportunities for personalized therapy. Barriers to testing include low rates of referral and uptake, delays to referral and slow result turnaround times, cost, and biopsy and assay limitations if somatic testing is performed, leading to the requirement for subsequent dedicated germline testing. Low rates of referral may result from lack of awareness among physicians of the clinical value of testing, coupled with low confidence in interpreting test results and poor availability of genetic counseling services. Among patients, barriers to uptake may include similar lack of awareness of the clinical value of testing, anxiety regarding the implications of test results, lack of insurance coverage, fear of negative insurance implications, and socioeconomic factors. Potential solutions include innovative approaches to testing pathways, including ‘mainstreaming’ of testing in which BRCA tests are routinely arranged by the treating oncologist, with the involvement of genetic counselors if a patient is found to have a gBRCAm. More recently, the utility of multigene panel analyses has also been explored. Access to genetic counseling may also be improved through initiatives such as having a genetic counseling appointment for all new patient visits and telemedicine approaches, including the use of telephone consultations or DVD-assisted counseling. Educational programs will also be beneficial, and cost effectiveness is likely to improve as the number of targeted treatments increases and when the earlier detection of tumors in family members following cascade testing is considered

EM-mosaic detects mosaic point mutations that contribute to congenital heart disease.

BackgroundThe contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.MethodsWe developed a new computational method, EM-mosaic (Expectation-Maximization-based detection of mosaicism), to analyze mosaicism in exome sequences derived primarily from blood DNA of 2530 CHD proband-parent trios. To optimize this method, we measured mosaic detection power as a function of sequencing depth. In parallel, we analyzed our cohort using MosaicHunter, a Bayesian genotyping algorithm-based mosaic detection tool, and compared the two methods. The accuracy of these mosaic variant detection algorithms was assessed using an independent resequencing method. We then applied both methods to detect mosaicism in cardiac tissue-derived exome sequences of 66 participants for which matched blood and heart tissue was available.ResultsEM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The estimated true frequency of mosaic variants above 10% mosaicism was 0.14/person in blood and 0.21/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.ConclusionsWe estimate that ~ 1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants with relatively higher allele fraction. Although blood is a readily available DNA source, cardiac tissues analyzed contributed ~ 5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses

Talazoparib monotherapy in metastatic castration-resistant prostate cancer with DNA repair alterations (TALAPRO-1): an open-label, phase 2 trial

Poly(ADP-ribose) polymerase (PARP) inhibitors have antitumour activity against metastatic castration-resistant prostate cancers with DNA damage response (DDR) alterations in genes involved directly or indirectly in homologous recombination repair (HRR). In this study, we assessed the PARP inhibitor talazoparib in metastatic castration-resistant prostate cancers with DDR-HRR alterations. In this open-label, phase 2 trial (TALAPRO-1), participants were recruited from 43 hospitals, cancer centres, and medical centres in Australia, Austria, Belgium, Brazil, France, Germany, Hungary, Italy, the Netherlands, Poland, Spain, South Korea, the UK, and the USA. Patients were eligible if they were men aged 18 years or older with progressive, metastatic, castration-resistant prostate cancers of adenocarcinoma histology, measurable soft-tissue disease (per Response Evaluation Criteria in Solid Tumors version 1.1 [RECIST 1.1]), an Eastern Cooperative Oncology Group performance status of 0-2, DDR-HRR gene alterations reported to sensitise to PARP inhibitors (ie, ATM, ATR, BRCA1, BRCA2, CHEK2, FANCA, MLH1, MRE11A, NBN, PALB2, RAD51C), had received one or two taxane-based chemotherapy regimens for metastatic disease, and progressed on enzalutamide or abiraterone, or both, for metastatic castration-resistant prostate cancers. Eligible patients were given oral talazoparib (1 mg per day; or 0·75 mg per day in patients with moderate renal impairment) until disease progression, unacceptable toxicity, investigator decision, withdrawal of consent, or death. The primary endpoint was confirmed objective response rate, defined as best overall soft-tissue response of complete or partial response per RECIST 1.1, by blinded independent central review. The primary endpoint was assessed in patients who received study drug, had measurable soft-tissue disease, and had a gene alteration in one of the predefined DDR-HRR genes. Safety was assessed in all patients who received at least one dose of the study drug. This study is registered with ClinicalTrials.gov, NCT03148795, and is ongoing. Between Oct 18, 2017, and March 20, 2020, 128 patients were enrolled, of whom 127 received at least one dose of talazoparib (safety population) and 104 had measurable soft-tissue disease (antitumour activity population). Data cutoff for this analysis was Sept 4, 2020. After a median follow-up of 16·4 months (IQR 11·1-22·1), the objective response rate was 29·8% (31 of 104 patients; 95% CI 21·2-39·6). The most common grade 3-4 treatment-emergent adverse events were anaemia (39 [31%] of 127 patients), thrombocytopenia (11 [9%]), and neutropenia (ten [8%]). Serious treatment-emergent adverse events were reported in 43 (34%) patients. There were no treatment-related deaths. Talazoparib showed durable antitumour activity in men with advanced metastatic castration-resistant prostate cancers with DDR-HRR gene alterations who had been heavily pretreated. The favourable benefit-risk profile supports the study of talazoparib in larger, randomised clinical trials, including in patients with non-BRCA alterations. Pfizer/Medivation

A computational approach to distinguish somatic vs. germline origin of genomic alterations from deep sequencing of cancer specimens without a matched normal.

A key constraint in genomic testing in oncology is that matched normal specimens are not commonly obtained in clinical practice. Thus, while well-characterized genomic alterations do not require normal tissue for interpretation, a significant number of alterations will be unknown in whether they are germline or somatic, in the absence of a matched normal control. We introduce SGZ (somatic-germline-zygosity), a computational method for predicting somatic vs. germline origin and homozygous vs. heterozygous or sub-clonal state of variants identified from deep massively parallel sequencing (MPS) of cancer specimens. The method does not require a patient matched normal control, enabling broad application in clinical research. SGZ predicts the somatic vs. germline status of each alteration identified by modeling the alteration's allele frequency (AF), taking into account the tumor content, tumor ploidy, and the local copy number. Accuracy of the prediction depends on the depth of sequencing and copy number model fit, which are achieved in our clinical assay by sequencing to high depth (>500x) using MPS, covering 394 cancer-related genes and over 3,500 genome-wide single nucleotide polymorphisms (SNPs). Calls are made using a statistic based on read depth and local variability of SNP AF. To validate the method, we first evaluated performance on samples from 30 lung and colon cancer patients, where we sequenced tumors and matched normal tissue. We examined predictions for 17 somatic hotspot mutations and 20 common germline SNPs in 20,182 clinical cancer specimens. To assess the impact of stromal admixture, we examined three cell lines, which were titrated with their matched normal to six levels (10-75%). Overall, predictions were made in 85% of cases, with 95-99% of variants predicted correctly, a significantly superior performance compared to a basic approach based on AF alone. We then applied the SGZ method to the COSMIC database of known somatic variants in cancer and found >50 that are in fact more likely to be germline

ANO7 in aggressive prostate cancer

A significant proportion of men develop prostate cancer during their lifetime, which causes challenges for public health. PSA screening studies have shown that testing all men at specific ages causes overdiagnosis of prostate cancer and unnecessary treatments for individuals who have indolent disease. Additional methods are required alongside PSA testing to be able to prognosticate the disease outcome and to focus the treatments on aggressive prostate cancer cases. The chromosomal location 2q37.3, where the transmembrane protein coding gene Anoctamin 7 (ANO7) resides, is associated with prostate cancer susceptibility in linkage analyses and genome-wide association studies (GWAS). In addition, ANO7 has been considered to be expressed specifically in prostate tissue, and it has been suggested as a target for prostate cancer immunotherapy. This thesis work concentrates on investigating the role of ANO7 in prostate cancer. ANO7 expression was significantly higher in prostate tissue than in any other tissue type, and ANO7 expression was elevated in prostate cancer compared to benign tissue. Moreover, elevated expression was associated with poor survival among patients. Next-generation sequencing (NGS) analysis of the ANO7 gene revealed possible truncating rare germline variants, which were found only in prostate cancer patients and not in controls in the initial screening. These variants were genotyped from several different prostate cancer patient cohorts and sets of unaffected males. The stop-gain variant in exon 1 was associated with poor survival, and the variant in intron/exon 4 was associated with aggressive disease. Because the intron/exon 4 variant was related to aggressive cancer but not to poor survival, we investigated whether the variant carriers would have a good response to docetaxel treatment in castration-resistant prostate cancer. As was hypothesized, the variant carriers had a better response to docetaxel than the non-carriers. When investigating ANO7 protein interactions, we observed an enrichment specifically in proteins related to vesicle trafficking. This study indicates that ANO7 has a role in prostate cancer development and that truncating mutations in the gene predispose patients to aggressive prostate cancer. The variants reported in this study could facilitate precision medicine for prostate cancer patient care.ANO7 aggressiivisessa eturauhassyövässä Eturauhassyöpä on kansanterveydellinen haaste, koska suuri osa miehistä saa taudin vanhetessaan. PSA-seulontatutkimukset ovat osoittaneet, että kaikkien tietyn ikäluokan miesten testaaminen aiheuttaa ylidiagnosointia ja turhia hoitoja niille, joiden syöpä ei etene aggressiiviseksi. Sen lisäksi, että eturauhassyövän hoidosta aiheutuvat kustannukset terveydenhuoltojärjestelmälle ovat suuret, ylidiagnosointi aiheuttaa haittaa ja huolta myös potilaalle itselleen. PSA-testauksen rinnalle tarvitaankin muita menetelmiä, joiden avulla potilaiden taudin ennustetta voitaisiin paremmin määrittää. Kromosomaalinen alue 2q37.3, jossa solumembraaniproteiinia koodaava Anoktamiini 7 (ANO7) sijaitsee, on yhdistetty eturauhassyöpään genominlaajuisissa assosiaatioanalyyseissa (GWAS) ja kytkentäanalyyseissa. ANO7-geenin on ajateltu ilmentyvän spesifisesti eturauhasessa ja sitä on ehdotettu myös eturauhassyövän immunoterapian kohteeksi. Väitöskirjassa selvitettiin ANO7:n roolia eturauhassyövässä. Tutkimuksessa selvisi, että ANO7 ilmentyy enemmän eturauhasessa kuin muissa kudoksissa ja enemmän syövässä kuin normaalikudoksessa. Voimakas ilmentyminen oli lisäksi yhteydessä huonompaan eturauhassyövän ennusteeseen. ANO7-geenistä löytyi uuden sukupolven sekvensointimenetelmällä (NGS) proteiinille todennäköisesti haitallisia harvinaisia ituratavariantteja. Variantti ensimmäisessä eksonissa oli yhteydessä lyhentyneeseen elinaikaan ja intronialueella/eksonissa 4 oleva variantti assosioitui sairastumisriskiin ja aggressiiviseen tautiin. Koska intonin/eksonin 4 variantti oli yhteydessä aggressiiviseen tautiin, mutta ei lyhentyneeseen elinaikaan, tutkimme, hyötyvätkö variantin kantajat erityisen hyvin kastraatioresistentin syövän hoidossa käytettävästä docetaxel-lääkkeestä. Variantin kantajat saivat oletuksen mukaisesti paremman lääkevasteen. Kun tutkimme tarkemmin ANO7:n kanssa interaktoivia proteiineja, saimme selville, että solujen vesikkeliliikenteeseen osallistuvat proteiinit olivat yliedustettuina. Tämä tutkimus osoittaa, että ANO7 on osallisena eturauhassyövän kehityksessä ja proteiinille haitalliset variantit altistavat aggressiiviselle eturauhassyövälle. Tutkimuksessa löydettyjä variantteja voidaan mahdollisesti hyödyntää tulevaisuudessa eturauhassyövän yksilöllistetyssä hoidossa

Mosaicism and the genetic architecture of congenital heart disease

Congenital heart disease (CHD) is characterized by structural defects of the heart and great vessels. It is the most common birth defect, affecting an estimated 1% of live births, and is the leading cause of mortality among birth defects. Despite recent progress in genetic research, more than 50% of CHD cases remain unexplained. An estimated 23% are due to aneuploidies and copy number variants and up to 30% has been attributed to de novo variation, though that number ranges between 3-30% depending on CHD complexity. The contribution of somatic mosaicism, or de novo genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood due to limitations in sample size, detection method, and validation rate. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined. We developed a computational method, Expectation-Maximization-based detection of Mosaicism (EM-mosaic), to analyze mosaicism in exome sequences of 2530 CHD proband-parent trios. EM-mosaic accurately detected 309 mosaic mutations in blood, with 85 of 94 (90%) candidates tested independently confirmed. We found twenty-five likely damaging mosaics in plausible CHD-risk genes, affecting 1% of our cohort. Variants in these genes predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The frequency of protein-coding mosaic variants detectable in blood was 0.122 or roughly 1 in 8 individuals. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction. CHD patients often present with comorbid cardiac and extracardiac anomalies that further their impact quality of life. Neurodevelopmental disorders (NDDs) are especially prevalent in CHD cases compared to the general population, yet the underlying genetic causes remain poorly explained. Further, patients with single ventricle defects undergoing surgery often later develop arrhythmias and experience worsening ventricular function. We used a statistical approach to dissect the association between de novo variation and these clinical outcomes and found that pleiotropic mutations contribute a large fraction of the risk of acquiring NDD and abnormal ventricular function phenotypes in CHD patients. We developed a proof-of-concept rare variant risk score that combines information from de novo, rare transmitted, and copy- number variants and show that prediction of outcomes such as NDD can be improved, especially in complex CHD cases

Mainstreamed genetic testing in ovarian cancer: A case study of BRCA1/2 tumour testing

Background With the advent of targeted therapies in ovarian cancer (OC), there is an impetus to identify patients with a BRCA1/2 mutation. Germline testing has already been integrated into the oncology setting using a mainstreamed model (MGT). Tumour testing is now available to detect the presence of somatic BRCA1/2 mutations. Aim To explore the introduction of mainstreamed BRCA1/2 tumour testing (MTT) in OC, focusing on clinical outcomes and patient experience. Methods A case study approach, using different research methods, was taken to gain an in depth understanding of the case (MTT) within its context. Results A service evaluation of the current state of MGT at UCLH found that in the 122 patients who were tested over 12 months, germline BRCA1/2 mutation prevalence was 14.8%. Developing the MTT pathway was feasible but challenging; delays were related to retrieval and review of archived tumour tissue. First-line MTT was provided for fifty patients; one somatic and eight germline mutations were identified. More than half this sample (52.6%) required follow-up germline testing. A prospective study using validated measures found no change in distress or quality of life scores before, during and after MTT. Patients reported low decisional conflict scores and no decision regret over MTT. After results disclosure patients with a genetic alteration had significantly more testing-related distress. Qualitative interviews revealed MTT was a brief, transient experience in the context of facing OC. Genetic misconception was common, with patients incorrectly attributing a hereditary component to tumour testing. Primary motivations for testing were related to clarifying genetic risk information for family, rather than personal benefit for treatment options. Conclusion A more streamlined process of providing MTT is needed. While MTT appears to have little psychosocial impact, poor understanding of the distinction between germline and somatic mutations indicates the need for improved communication and information provision in OC