NGS Testing Research Articles

Abstract 2183: Interrogating the molecular profile of colorectal cancer: detection of clinically actionable alterations in Hispanics

Abstract Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths among men and women and the third cause of cancer deaths in the US. Tumor profiling techniques have led to the identification of actionable alterations to guide treatment and improve survival in cancer patients. However, comprehensive genetic profiling of colorectal cancer tumors from Hispanics living in Puerto Rico (PRH) has not been performed. Our study aimed at understanding the common CRC carcinogenic molecular pathways that affect our Hispanic population by evaluating colorectal tumors from molecularly characterized ethnically diverse cohorts. Methods: We retrospectively evaluated the mutational profile of CRC tumors from 218 PRH that underwent NGS testing from 2015 to 2020 (provided by CARIS Life Sciences and the Precision Oncology Alliance). We estimated the prevalence of somatic mutations of PRH CRC tumors and compared them with those reported in The Cancer Genome Atlas (TCGA) Pan-Cancer Atlas Clinical Data and the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE), both available through the cBioPortal for Cancer Genomics. Descriptive statistics were performed to characterize the database. Results: Among the top mutated genes for CRC tumors in PRH were APC (81.9%, n=182), TP53 (75.3%, n=178), KRAS (56.2%, n=185), PIK3CA (18.0%, n=178) and SAMD4 (12.6%, n=175). For PRH, the most frequent gene amplifications were: CDX2 (20.1%, n=184), CDKN1B (6.6%, n=183), and HNRNPA2B1 (5.4%, n=184). Significant differences in the mutational frequencies of CRC driver genes, such as APC, TP53, PIK3CA, FBXW7, CDX2, CDKN1B, and HNRNPA2B1, and actionable genes, such as KRAS/NRAS, BRAF and for ERBB2 amplifications, among PRH were observed when compared to the TCGA and GENIE datasets. The prevalence of MSI tumors for PRH was 2.0%. Conclusion: This is the first study to report the mutational profile of CRC tumors from PRH and to describe differences in mutational frequencies when compared other non-Hispanic and U.S. mainland Hispanic populations using data from TCGA and GENIE. Differences in prevalence of immunotherapy and targeted associated biomarkers may contribute to the observed disparities in survival and response to therapies. Understanding the most common carcinogenic molecular pathways that affect Hispanics with CRC is crucial to guide research efforts in the development of new therapeutic modalities incorporating genomically diverse populations. Citation Format: Ingrid M. Montes-Rodriguez, Hilmaris Centeno-Girona, Camila Rivera-Lynch, Noridza Rivera, Marcia Cruz-Correa. Interrogating the molecular profile of colorectal cancer: detection of clinically actionable alterations in Hispanics [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 2183.

JAK2 Unmutated Polycythaemia-Real-World Data of 10 Years from a Tertiary Reference Hospital.

(1) Background: Polycythaemia is defined by an increase in haemoglobin (Hb) concentration, haematocrit (Hct) or red blood cell (RBC) count above the reference range adjusted to age, sex and living altitude. JAK2 unmutated polycythaemia is frequent but under-investigated in original publications. In this retrospective cohort study, we investigated the clinical and laboratory data, underlying causes, management and outcomes of JAK2 unmutated polycythaemia patients. (2) Methods: The hospital database was searched to identify JAK2 unmutated patients fulfilling WHO 2016 Hb/Hct criteria for PV (Hb >16.5 g/dL in men and >16 g/dL in women, or Hct > 49% in men and >48% in women, or RBC mass > 25% above mean normal predicted value) between 2008 and 2019. Clinical and laboratory data were collected and analysed. (3) Results: From 727,731 screened patients, 294 (0.04%) were included, the median follow-up time was 47 months. Epo and P50 showed no clear pattern in differentiating causes of polycythaemia. In 30%, the cause remained idiopathic, despite extensive work-up. Sleep apnoea was the primary cause, also in patients under 30. Around 20% had received treatment at any time, half of whom had ongoing treatment at the end of follow-up. During follow-up, 17.2% developed a thromboembolic event, of which 8.5% were venous and 8.8% arterial. The mortality was around 3%. (4) Conclusions: Testing for Epo and P50 did not significantly facilitate identification of underlying causes. The frequency of sleep apnoea stresses the need to investigate this condition. Idiopathic forms are common. A diagnostic flowchart based on our data is proposed here. NGS testing should be considered in young patients with persisting polycythaemia, irrespective of Epo and P50 levels.

A Scoring Model and Protocol to Adapt Universal Screening for Lynch Syndrome to Identify Germline Pathogenic Variants by Next Generation Sequencing from Colorectal Cancer Patients and Cascade Screening.

Simple SummaryLynch syndrome (LS) is an autosomal-dominantly inherited form of cancer predisposition dominated by colorectal cancer (CRC). LS is caused by germline pathogenic variants (PV) occurring in known mismatch repair genes. For effective cascade screening, it is critical to identify PV for LS predisposition. When limited resources are available, next generation sequencing (NGS) of an entire cohort of colorectal cancer (CRC) patients, even those under 50 or 60 years of age, places a huge burden on the system. Here, we present an innovative LS ascertainment and follow-up program that includes LS molecular analysis, PV screening with NGS technology, and cascade screening. The goal is to improve LS ascertainment in light of the growing burden of early-onset CRC, particularly in low- and middle-income countries.Identification of germline pathogenic variants (PV) predisposing to Lynch syndrome (LS) is an important step for effective use of cascade screening of extended at-risk lineages, leading to reduced morbidity and mortality due to colorectal cancer (CRC). As a general rule, however, next generation sequencing (NGS, either of gene panels or whole exomes) is relatively expensive and unaffordable for general clinical use. In resource-poor settings, performing NGS testing on an entire cohort of CRC patients, even if limited to those under 50 or 60 years of age, still places an enormous burden on limited resources. Although family history can be a good indicator for LS testing, identifying at-risk family members and offering cascade screening may not benefit many patients/probands without an obvious family history. This article presents a novel program called Modified Ascertainment and follow-up Program (MAP) with a scoring model for LS ascertainment and molecular screening by NGS with diagnosis confirmation of PV and cascade screening. The goal is to improve LS ascertainment in light of the growing burden of early-onset CRC, particularly in low- and middle-income countries. Through MAP, judiciously applied molecular genetics will improve identification of PV predisposing to LS and cascade screening.

Addressing cancer health disparities (CHD) in an independent community cancer clinic: All solutions are local.

e18522 Background: The COVID-19–associated global pandemic has brought many public and population health issues to light. A glaring issue is the disparities among and differences in susceptibility of diverse ethnicities. Viral infection is not the only area of healthcare facing this issue: disparities in prevalence, biology, prognosis, and outcomes of cancer exist, often based on socioeconomics and social determinants of health (SDoH). CHD have been debated and discussed at several levels, from local counties to the congress to the Centers for Medicare and Medicaid Services. Amid the debates and recommendations, it has been difficult to carve out a clear path forward. The complex factors involved include access to care because of financial challenges, biological and genetic factors, impact of SDoH, and access to screening, NGS testing, and clinical trials. CHD may contribute to almost 34% of deaths among adult cancer patients and additional spending of $230 billion. Addressing CHD could result in an indirect savings of as much as $1 trillion over 3 years (AACR, CDR, 2020). A comprehensive approach is needed to generate a groundswell of resources. This should include support for public policies aimed at improving understanding of the issue by all parts of the ecosystem (pharma, researchers, government agencies, physicians), adequate funding for federal and local initiatives, considerations of health in community planning and development, and collection of real-world data and evidence. At Carolina Blood and Cancer Care (CBCCA), the team decided to study this issue and initiate solutions one step at a time. Methods: CBCCA prioritized solutions in 5 categories. The top priority was to address issues impacting patients: access to care, NGS testing, cancer screening, SDoH, and clinical trial access. To address access to care issue (financial constraints), 2.5 FTEs (full-time equivalents) were allocated to carry out needs assessments and identify resources, including Medicaid eligibility, dual eligibility, other foundations, and sources for free drugs. A not-for-profit entity, No One Left Alone, was started to address CHD. Results: During 2021 CBCCA physicians saw 1787 unique cancer patients (both established and new). Of these, 319 needed IV anticancer treatment and an additional 104 needed oral oncolytics. Fifty-three could not pay for their cancer treatment. Another 101 patients needed assistance for out-of-pocket costs. Financial counselors procured free drugs worth $1,633,588 and an additional $135,931 in cash assistance for high out-of-pocket costs. The pharmacy team raised $253,218 for 64 patients (374 transactions) for out-of-pocket costs. As a result, not a single patient was left without treatment. Conclusions: Financial toxicity was addressed by allocating 0.5 FTE per oncologist to ensure access to care. In the next phase, CBCCA will address access to NGS testing.

Evolution of biomarker testing in advanced non-small cell lung cancer (aNSCLC) and metastatic breast cancer (mBC) in U.S. community practices.

e18778 Background: Biomarker testing has advanced from single-gene to NGS. This study examined aNSCLC and mBC biomarker testing ≤ 90d of advanced (adv) or metastatic (met) diagnosis (Dx) and treatment (tx) patterns at US practices. Methods: A retrospective observational study used Flatiron Health electronic health-record-derived de-identified database at OneOncology (OO) community and non-OO Flatiron Health nationwide (NAT) sites (̃90% community, ̃10% academic). Patients (Pts) ≥ 18y, with Dx of aNSCLC or mBC from 1/1/18 - 4/30/21, with ≥ 1 visit ≤ 90d after adv/met Dx and ≥ 90d follow-up were evaluated. Descriptive analyses and logistic regression were used. Results: A total of 16,882 pts with aNSCLC (2366 OO; 14,516 NAT), and 6500 pts with mBC (1026 OO; 5474 NAT) were included. Overall testing was high and stable (OO: 85% aNSCLC, 98% mBC; NAT: 84%, 97%) with higher NGS testing at OO (58% aNSCLC; 28% mBC) vs NAT (49%; 16%) (Table), which reflected more pts with aNSCLC tested for all 6 mutations (ALK, BRAF, KRAS, ROS-1, EGFR, PD-L1; 54% OO vs 50% NAT, p<0.001) and more pts with mBC tested for PIK3CA (27% OO vs 16% NAT, p<0.001). In aNSCLC, NGS testing increased similarly for OO and NAT over time (p>0.05); mBC NGS testing increased faster at NAT vs OO (p<0.05). Of pts tested and treated, 16% aNSCLC (1945 OO; 11,376 NAT) and < 3% mBC (14 OO; 108 NAT) received tx before test results were available. For pts with aNSCLC with ≥ 1 actionable mutation (ALK, BRAF, ROS-1, EGFR), 18% OO and 22% NAT had tx before test results. Cancer immunotherapy plus chemotherapy was the most common tx (36 % OO vs 40 % NAT); after test results, 33% vs 56% OO and 45% vs 44% NAT pts stayed on tx vs switched to targeted tx. For pts with aNSCLC with ≥ 1 aforementioned actionable mutations who waited until test results were available, 65% received targeted tx at OO and NAT. Conclusions: Biomarker testing has become standard of care in aNSCLC and mBC in US community settings. NGS rates increased over time and were higher at OO vs NAT. Differences in pts treated before test results reflects the need to wait for NGS results to inform initial tx in aNSCLC vs non-NGS results for mBC. This study shows NGS testing in US community practices has increased since 2018, particularly in mBC, but opportunities remain to optimize NGS results into tx decisions.[Table: see text]

BRCAAWAY: A randomized phase 2 trial of abiraterone, olaparib, or abiraterone + olaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) with DNA repair defects.

5018 Background: The PARP-inhibitor olaparib is approved for mCRPC patients (pts) with deleterious germline or somatic homologous recombination repair gene mutations (HRRm). PARP1 interacts with androgen signaling, and castration-resistant tumor cells exhibit increased PARP1 activity. Preclinically PARP1-inhibition synergizes with androgen receptor (AR) targeted therapy. BRCAAway is a biomarker selected, randomized, open-label, multicenter phase 2 trial evaluating efficacy of targeting AR vs PARP vs combination in first line mCRPC patients with germline and/or somatic HRRm in BRCA1, BRCA2, or ATM. Methods: Eligible mCRPC pts underwent tumor next generation sequencing and germline testing. Pts with inactivating BRCA1, BRCA2 and/or ATM alterations were randomized 1:1:1 to Arm 1 abiraterone (1000 mg daily) + prednisone (5mg bid) (Abi/pred), Arm 2 olaparib (300 mg bid) or Arm 3 olaparib + Abi/pred. The primary end point is progression-free survival (PFS) analyzed using Kaplan-Meier estimates and Cox regression. Secondary endpoints include measurable disease response rate (RR) by RECIST, PSA-RR, undetectable PSA (≤ 0.2 ng/ml) and toxicity. Arms 1 and 2 pts were allowed to cross over at progression. Pts with other HRRm were treated with olaparib; Arm 4 (ongoing). Results: 161 pts were registered and had NGS testing; 60 pts were randomized to Arms 1-3; to date 59 are evaluable for toxicity and 53 are evaluable for PFS. Baseline median age 67 (range 42-85) years; 54 pts were White, 6 were Black; sites of disease: bone only (n=31), soft tissue only (n=18), bone and soft tissue (n=10); median PSA 14.61 ng/ml (range 0.15-4036.8). Mutational status: BRCA1 only n = 2, BRCA2 only n = 39, ATM only n = 8, and > 1 HRRm n = 11. 34 pts had germline and 26 had somatic mutations. Median (range) follow-up time: 8.3 (0.8, 33.3), 12.2 (2.7, 21.8) and 16.8 (2.9, 41.7) months in Arms 1, 2 and 3. 43 pts had treatment-related adverse events; most common were fatigue (23 pts; 1 Grade (G) 3, 22 G1/2), nausea (17 pts, G1/2), and anemia (9 pts, 2 G3, 7 G1/2). ≥50% PSA decline was 79%, 67%, and 85% of pts in Arms 1, 2, and 3, respectively. Median PSA nadir (ng/mL) (95% CI) Arms 1-3: 2.17 (0.44, 49.27), 3.10 (0.83, 12.01), and 0.50 (0.10, 2.13), respectively. Undetectable PSA, median PFS, and 12-month PFS by Arm are listed in the table. Conclusions: In mCRPC pts with inactivating BRCA1, BRCA2 and/or ATM alterations Abi/pred + olaparib was well tolerated and resulted in longer PFS and better PSA response vs either agent alone. Clinical trial information: NCT03012321. [Table: see text]

AI-enabled identification prediction of homologous recombination deficiency (HRD) from histopathology images.

3019 Background: Homologous recombination deficient (HRD) tumors are highly responsive to platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitor (PARPi) therapy. Pathogenic BRCA-1 and BRCA-2 (BRCA1/2) alterations are key members of the HR DNA repair pathway but genomic instability status, including loss of heterozygosity, telomeric allelic imbalance and large scale state transitions across the genome are also predictive of HRD. HRD testing is currently performed by next generation sequencing which can take 2-4 weeks for results, has a high failure rate, requires significant tissue and is costly. We developed and tested the ability of an AI enabled platform to predict HRD status from the analysis of whole slide imaging of the diagnostic H&E slide. This platform, iPREDICT-HRD is rapid, precise, and cost effective. Methods: The AI engine was trained on 120 H&E slides that were used to identify tumor prior to manual microdisseection for HRD assessment by NGS. Histopathological features were extracted, followed by feature mapping to predict HRD status based on the results of NGS testing. ResNet AI algorithm was trained to segment, annotate and predict HRD status. 10 lac tiles of 256x256 size at 40x magnification were generated per pathological class. 70% of the data set was used for training and 30% for validation of the AI model. Results: Using single blinded clinical samples, iPREDICT-HRD tool detected HRD + ve samples with 99.3% accuracy with 100% sensitivity and 99% specificity in the test set. Patch-level predictions of HRD status demonstrated intra-tumor heterogeneity within the H&E slides. Visual inspection of the heatmap suggested the presence of patches with high predictive ability of HRD status and this outperformed an average HRD score for slides with heterogeneity. Conclusions: AI-enabled prediction of HRD status can be accurately performed on diagnostic H&E slides potentially yielding results quickly and afforadably, even when limited tissue is available for testing.

Dissection of the expressed actionable fusions’ repertoire in solid tumors tested serially in the clinical setting across 14 cancer types.

e18804 Background: More frequently, select gene fusions have been tested in clinical practice as more approvals of very effective therapies targeting oncogenic chimeric fusion proteins. Large studies based on DNA-based NGS testing alongside clinical trials have established the prevalence of clinically actionable gene fusions such as NTRK1/2/3. However, much less is known about the broader repertoire and prevalence of bonafide expressed gene fusions. Methods: 6009 tumors across 14 cancer types were tested in our clinical laboratory between January and October 2021. Enrichment was performed using a custom hybridization-based capture after RNA to cDNA conversion with custom designed probes targeting > 1104 different genes and enriched for 2230 specific RNA fusions to maximize sensitivity and breadth before sequencing ̃50 million 150x150 reads/sample on a NovaSeq 6000. Data was deidentified and aggregated according an approved IRB. Statistical significance was calculated by Fisher’s exact test. Results: We found that 37% of tumors tested carry > 1 expressed chimeric transcript and that 7.2% of tumors (n = 431) express a gene fusion involving one of 19 actionable gene families/pathways targeted by an approved therapy. The most frequent involved FGFR1/2/3/4 (15%), ALK (14%), NTRK1/2/3 (12%), BRAF, ERBB2, RET and MET (6% each), RAS/RAF/MAPK and CDK4/6 pathway (5%). These fusions imply intra- and inter-chromosomal translocations on all chromosomes, especially on chromosomes 1, 3, 6, 7, 11, 12 and 17. NTRK1/2/3 fusions prevalence (0.9%) is significantly higher (p = 1.1e-10) than the 0.3% reported on a recent study from a large dataset (296K samples) based on DNA-sequencing testing (1). Moreover, we found that 32% of the NTRK partners were unique vs. 10% (p = 6.5e-08) from the Westphalen et al. study (1). 14 of 25 unique NTRK partners have not been described on large datasets (1) and only 3 have been reported on the literature as NTRK partners. Among them, we identified the hypoxia-induced neovascularization gene HIF1A, as a novel in frame 3’ fusion partner of NTRK3 [t(15;14)(15q25.3; 14q23.1). Finally, NTRK fusions were detected across 14 cancer types with higher frequency on brain tumors (17%), followed by lung cancers, colorectal and head and neck cancer (13% each) and sarcomas (12%). Conclusions: Gene fusions involving common actionable genes showed a high prevalence in the clinical setting. This study confirms that the number and breadth of gene fusions can present with common actionable information in > 7% of clinical cases. We report here 14 new unique NTRK partners. Despite a 50 times smaller dataset, 3-fold more actionable expressed NTRK fusions involving novel partners were detected with a targeted RNA based hybridization approach in lieu of DNA based testing supporting the benefit of this strategy. Overexpression and translation can be confirmed only by this approach.

FGFR2 fusion detection in plasma: A new era in the clinical monitoring of iCCA.

4085 Background: Actionable genetic alterations can be identified in over 50% of intrahepatic cholangiocarcinoma (iCCA). Since EMA approved Pemigatinib, a selective Fibroblast growth factor receptor 1-3 (FGFR1-3) inhibitor for the treatment of CCA with FGFR2 fusions or rearrangements, the screening of patients who may benefit from such targeted therapies is especially relevant. In addition, novel FGFR inhibitors that are effective for treatment of resistant mutant clones are under development. In patients with no available tissue for genomic profiling or at different timepoints after targeted therapy, NGS testing of circulating cell-free DNA (ccfDNA) would be the most convenient option. Hence, two important issues must be addressed: i) technical set-up and validation of detection of FGFR2 rearrangements in plasma, and ii) patient shedding in iCCA, in order to consider liquid biopsy for routine clinical care. Methods: We conducted a retrospective study in a cohort of 18 iCCA patients with known FGFR2 fusion or rearrangement events previously identified in tumor FFPE by NGS (FoundationOne CDx test). A custom-designed capture-based NGS panel for use in plasma or tissue (VHIO-iCCA test) was developed to detect FGFR2 rearrangements and other common altered genes in iCCA. After validating our VHIO-iCCA panel with fusion positive FFPE samples, a concordance study was conducted to evaluate the detection of FGFR2 fusion and rearrangements in matched-to-tissue timepoint plasmas. Finally, additional serial plasma samples taken during FGFR inhibitor treatment were also analyzed. Results: From the 18 rearrangement events previously detected with FoundationOne CDx, we were able to identify all 18 in FFPE samples and 15 in the paired plasma using our VHIO-iCCA panel, representing a 100% and 83% concordance, respectively. In the 3 discordant cases, no additional alterations were detected in plasma, indicating a lack of circulating tumor DNA (ctDNA) shedding. Serial sampling of these patients indicated persistent non-shedding. The analysis of fusion allele fraction (FAF) in serial plasma samples revealed that detection of fusion FGFR2 changed during treatment and correlated with best response. In general, patients with a stable FAF showed a SD, while patients which reduced FAF presented a PR. Conclusions: This extremely valuable set of cases has allowed us to validate our VHIO-iCCA panel to be used in tissue and plasma, and to determine that the sensitivity in plasma is >80%, making this a feasible option to avoid tissue biopsies, whenever patients cannot undergo the procedure and even to aid in cancer monitoring. Patient shedding is high in iCCA, yet a fraction of patients may not find a useful resource in liquid biopsy. For those who shed ctDNA, monitoring through the FAF may guide clinical management of iCCA.

Associations between genomic instability score (GIS), molecular characterization, and clinical outcomes in high grade serous ovarian cancers (HGSOC).

e17596 Background: To describe the molecular profiles of tumors with a positive GIS status (GI+) in patients with newly diagnosed BRCA wild-type HGSOC and to examine possible associations between genomic mutations and survival outcomes in these patients. Methods: Genetic testing results of patients who underwent both somatic BRCA1/BRCA2 and germline multigene panel testing between 1/1/2015 and 12/1/2020 were prospectively collected. Patients had multigene panel germline testing in tandem with Myriad somatic BRCA1/2 testing and GIS assay. Expanded somatic NGS testing (108 gene panel) was then performed on GIS+ (GIS ≥42) tumor samples from women without somatic or germline BRCA mutations. The primary outcomes were the type and frequency of genomic mutations, which were grouped according to the following pathways: DNA-repair related (separated into homologous recombination (HR), Fanconi anemia (FA), other DNA repair related pathway) or non-DNA repair related. GIS and molecular profiles of tumors from patients who recurred versus those who did not recur were compared. Results: 143 women with HGSOC underwent both germline and somatic testing. 18/143 (12.5%) tumors with negative germline multigene panel and negative somatic BRCA1/2 testing were GIS+. 17/18 (94.4%) GIS+ tumors had pathogenic TP53 mutations. While all samples had at least one DNA repair related variant, only 22.2% had an HR-associated variant, and 16.7% had an FA variant. At time of analysis, 10 patients had recurred and 8 patients had not recurred. Median PFS of those who recurred was 15.8 months (range 4.8-30.1 months). Patients who recurred had lower GIS (median 47.5 vs. 69.5, p = 0.04), but they had no appreciable differences in gene or pathway alterations when compared to those who did not recur. Conclusions: Known pathogenic alterations in DNA repair pathways commonly associated with HR were rare in this cohort of BRCA negative HGSOC patients with GIS+ tumors. However, there was a high frequency of other DNA repair pathway alterations. Further investigation of the molecular signatures associated with recurrence are needed to identify potential novel targets. Lower GIS in this cohort were associated with higher risk of recurrence, although this finding must be validated in the setting of larger studies. [Table: see text]

Molecular characterization and subsequent treatments after encorafenib-cetuximab +/- binimetinib in BRAF V600E-mutated colorectal cancer.

3562 Background: The BRAF-V600E mutation confers poor prognosis in patients with mCRC. E-C+/-B improve clinical outcomes compared with standard of care. Data about subsequent treatment lines and the impact of NGS in the treatment choice after the BRAF targeted treatment are still limited. We have analyzed the global overall survival (OS) and the impact of NGS to guide subsequent treatments (approved targeted therapies (TT) or clinical trials (CT)) after E-C+/-B. Methods: Patients who had received E-C+/-B were included. Clinical data was collected prospectively. Tissue NGS was performed prospectively and retrospectively using an in-house NGS panel (VHIO-300). Subsequent treatments’ clinical outcomes were described. Percentage of patients who received TT or CT based on NGS results is reported. Clinical outcomes were calculated using survival Kaplan-Meier curves. Results: From 2017 to 2021, 59 patients with refractory mCRC received E-C+/-B at our institution. 50 patients have already progressed to the BRAF inhibitor combination and 9 patients remain on BRAF inhibitor treatment. Tumor tissue for NGS testing before BRAF inhibitor treatment was available in 70% of patients (41/59). BRAF-V600E mutation was confirmed in tissue using ddPCR in all patients. The most frequent alterations were: BRAF-V600E 93%, TP53 64%, RNF43 30%, APC 20%, PIK3CA 14%, NOTCH1/2 9%, RANBP2 8%, PTEN 8%, MET amplification 5%. MSI incidence was 10%, 2 out of 6 (33%) received subsequent immunotherapy. After the BRAF inhibitor combination, 23 patients (38%) did not receive subsequent treatment (clinical deterioration or death). The 27 remaining patients (62%) received 41 subsequent treatments (37% of them were TT or CT). Median subsequent lines after the BRAF inhibitor combination were 1 (0-3). NGS led to targeted therapy in 20% of the patients: BRAF inhibitors combos (20%), microtubule inhibitors (13%), antiPD1 (13%), NOTCH inhibitors (6%), and MET inhibitor (6%). ORR among patients who received matched TT or CT was 6% and DCR (CR+PR+SD) was 47%. The median OS after the BRAF inhibitor was 3.8 months; 10.1 months vs 3.0 months (HR 0.43 (95%CI 0.2-0.95), p = 0.04) for those patients who received TT or CT vs patients who did not receive TT or CT. When MSI patients were excluded mOS was 5.2 vs 3.0 months (HR 0.61 (95%CI 0.27-1.37), p = 0.23) respectively. There were no differences in terms of OS regarding the number of previous lines (0-1 vs 2-3, HR 0.75 (95%CI 0.4-1.42), p = 0.37) or with the BRAF inhibitor regimen used (E-C vs E-C-B) (HR 0.84, (95%CI 0.44-1.6), p = 0.61) Conclusions: This study suggests that patients with BRAF-V600E mutant mCRC could have benefit when treated with TT and/or in CT after a BRAF inhibitor-based therapy, including immunotherapy. Genomic analysis might help to guide subsequent treatments. Because of molecular heterogeneity, these patients should be discussed in Molecular Tumor Boards.

Reducing pre-analytical sample QC failure rates for cancer molecular genetic assays with SLIMamp technology.

e15034 Background: Identification of somatic variants in cancer by high-throughput sequencing has become common clinical practice largely because many of these variants may be predictive biomarkers for targeted therapies. However, there can be high sample QC failure rates for some assays (sometimes up to 40%), preventing the return of results that may affect patient treatment decisions. Pillar Biosciences has incorporated their patented SLIMamp technology into commercially available cancer NGS testing kits with the claim that these kits can successfully interrogate challenging formalin-fixed paraffin-embedded tissue (FFPET) samples with low tumor purity, poor DNA quality, and/or low input DNA, resulting in a high sample QC pass rate. The aim of this study was to substantiate that claim using Pillar’s amplicon-based oncoReveal Solid Tumor Panel. Methods: We acquired 48 tumor samples that had failed one or more pre-analytical QC sample parameters for whole exome sequencing (WES) from ATGC’s ISO15189 accredited diagnostic genomics laboratory. XING Genomic Services performed an exploratory data analysis using our pre-analytical QC assays to characterise the samples and then sequenced the samples in our ISO15189 accredited laboratory using the validated oncoReveal Solid Tumor Panel. Results: We were able to achieve high sequencing coverage (>3000X) for all 48 samples and explored the determinants of sample “success”. We were able to generate clinical reports for 45 samples (94%), of which 38 (79%) contained clinically actionable or significant variants that would not have otherwise been identified. Ten samples had a higher number of total variant calls with over-representation of C>T transitions representing stochastically-amplified, formalin-induced artefacts in samples with very low input template DNA. Of these, 7 cases had reportable variants and 3 were deemed unreportable. We demonstrated that DNA integrity is the major determinant of success even in samples with low input DNA or low tumor purity and were able to further refine pre-analytical and post-analytical QC metrics to better identify samples with poor quality DNA that can be sequenced reliably. Conclusions: In this study, we showed that the Pillar Biosciences oncoReveal Solid Tumor Panel, which uses SLIMam technology, was able to generate reliable, interpretable results for 94% of samples that failed pre-analytical QC for WES, substantiating Pillar’s claim.

Landscape of tumor genetic testing and targeted therapies over an eight-year span in a rural population.

e15036 Background: Advances in tumor molecular profiling and bioinformatics are adding new insights to support real-world research. In this study, paired tumor NGS testing data was curated and analyzed from electronic medical records (EMR) to understand mutational and targeted therapy landscape. Methods: A database initiated in 2013 to collect data, including outcomes, on all patients referred to the molecular tumor board at the Avera Cancer Institute was curated and analyzed. A data pipeline that combined results from curated NGS data with curated EMR data from Avera was used to correlate targetable mutations to a corresponding therapy and assessed treatment patterns. Results: Data were from 1804 patients with 44 cancer types including breast (462; 26%), lung (338;19%), colorectal (198;11%), ovarian (168;9%), and uterine (100;5%). Overall, 83% of patients had a positive NGS result and 67% had a targetable mutation per NCCN guidelines. 32% of patients with a targetable mutation received targeted therapy per guideline. Overall, 381 patients with 28 tumor types received targeted therapies based on NGS results and targetable biomarkers. Of these, 288/381 (76%) were stage III-IV and 93/381 (24%) early stage/stage unknown. In non-small cell lung cancer, 76% of stage III-IV patients with mutations in EGFR (exon 19 deletion, L858, L861, S768, G719), MET (exon 14 skipping, amplification), BRAF V600 and ALK/ ROS1/ RET fusions received a targeted therapy per NCCN guidelines. In advanced breast cancer, 97% with ERBB2 amplification received targeted therapy (trastuzumab, pertuzumab, neratinib, lapatinib, tucatinib). 8% of advanced breast cancer patients with PIK3CA mutation received alpelisib, with all patients receiving the drug since 2019. 38% advanced breast cancer patients with BRCA1/2 mutation received Olaparib. In ovarian cancer, 48% of advanced stage patients received targeted therapy for BRCA 1/2, ATM, CDK12, CHEK2, ERBB2, FANCL, KRAS, ALK, BRAF alterations. Olaparib was used in 70% of advanced prostate cancer patients with BRCA1/2 mutations. Dabrafenib combined with Trametinib were most preferred for BRAF V600 mutation in melanoma and colorectal tumors respectively. Off label use of targeted therapy was observed in about 6% of advanced patients; examples include trametinib for KRAS/NRAS mutation [non-small cell lung cancer, colorectal, pancreatic, ovarian], PARP inhibitors for BRCA1/2, ATM mutations [urothelial, peritoneal], anti-ERBB2 inhibitors for ERBB2 in-frame, missense, exon 20 insertion mutations [lung and breast]. Conclusions: These study results demonstrate that automated data mining can provide insights into the genomic and therapeutic landscape of a real-world patient population. Across all tumor types, most patients are being given appropriate targeted therapies as per guidelines. Off-label use of targeted therapy was also observed.

Molecular profiling of dedicated lung cancer biopsy tissue sample collected at time of diagnostic bronchoscopy.

e20587 Background: The combined use of molecular biomarker testing and targeted precision therapeutics has led to improved survival in lung cancer. Broadening access to national guideline recommended comprehensive molecular testing requires overcoming the challenges of inadequate tissue biopsies, which can lead to the need for additional procedures and ultimately, delays in initiation of care. We show Percepta Genomic Atlas identifies key molecular alterations in transbronchial needle aspirate (TBNA) and transbronchial biopsy (TBB) samples of lung or lymph node collected during the initial diagnostic bronchoscopy. Methods: Percepta Genomic Atlas combines the whole exome TruSeq RNA Exome and targeted AmpliSeq Focus DNA assays (Illumina) for a comprehensive gene panel including ALK, RET, ROS1, NTRK1/3, MET, EGFR, BRAF, KRAS and HER2. TruSight Oncology 500 DNA and AmpliSeq Focus RNA assays (Illumina) were used as reference assays. DNA and RNA were extracted from samples with the AllPrep Micro kit (Qiagen) and analyzed by Percepta Genomic Atlas and reference assays. 94 biopsy samples (73 TBNA and 21 TBB from 71 patients undergoing a diagnostic bronchoscopy for suspected lung cancer were collected into RNAprotect (Qiagen) under an IRB approved protocol. Local molecular testing results from FFPE biopsy samples taken during the same bronchoscopy procedure were collected. Results: RNA and DNA in sufficient amounts to run the Percepta Genomic Atlas and reference assays was obtained from 85 of 94 lung biopsies from 63 of 71 patients. Percepta Genomic Atlas identified pathogenic variants in 29 bronchoscopy biopsy samples from 23 patients including single nucleotide variants in EGFR, KRAS, BRAF and PIK3CA, an EGFR exon 19 deletion/insertion and copy number amplications in AR, EGFR, CDK4, CCND1, MYC, MYCN and PIK3CA. No fusions were identified. This results in a 100% sensitivity for detecting pathogenic alterations when compared to reference assay results. When comparing Percepta Genomic Atlas results to local molecular testing of bronchoscopy samples performed as part of routine clinical care, we found 100% concordance with 7 of 7 alterations detected in 7 patients, including mutations in EGFR, KRAS and BRAF. In a further 4 patients, Percepta Genomic Atlas and local multi-gene NGS testing results agreed, with neither assay identifying guideline recommended alterations. Conclusions: Using a combination of whole exome RNA and targeted DNA sequencing, Percepta Genomic Atlas detects the clinically actionable mutations in patients with non-small cell lung cancer using fresh tissue specimens collected during bronchoscopic tissue sampling, with high concordance to standard of care testing. By initiating broad molecular testing at the time of the bronchoscopy, Percepta Genomic Atlas may provide timelier results for patients with lung cancer.

MP02-14 RE-IMAGINING URINARY TRACT INFECTION IN WOMEN: CLUES FROM THE UROBIOME OF ASYMPTOMATIC HEALTHY CONTROLS VS VACCINATED WOMEN WITH RECURRENT URINARY TRACT INFECTION

You have accessJournal of UrologyCME1 May 2022MP02-14 RE-IMAGINING URINARY TRACT INFECTION IN WOMEN: CLUES FROM THE UROBIOME OF ASYMPTOMATIC HEALTHY CONTROLS VS VACCINATED WOMEN WITH RECURRENT URINARY TRACT INFECTION J. Curtis Nickel, R. Christopher Doiron, Kerri-Lynn Kelly, Nicolle Diaz, Caleb D. Phillips, and Craig D. Tipton J. Curtis NickelJ. Curtis Nickel More articles by this author , R. Christopher DoironR. Christopher Doiron More articles by this author , Kerri-Lynn KellyKerri-Lynn Kelly More articles by this author , Nicolle DiazNicolle Diaz More articles by this author , Caleb D. PhillipsCaleb D. Phillips More articles by this author , and Craig D. TiptonCraig D. Tipton More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002514.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: A comparison of the urobiome of healthy controls with no UTI to women with recurrent UTI (rUTI) before and after MV140 vaccination would suggest that our traditional concept of rUTI in women may be more complicated than envisioned. METHODS: Female subjects with rUTI (≥3 UTIs/year) enrolled in the MV140 North American early clinical experience study and age–matched asymptomatic females (about 1:2 ratio) with no UTI or antibiotics in the previous 12 and 3 months respectively provided midstream urine specimens for culture, NGS and quantitative PCR testing (MicroGenDx) at baseline and for the rUTI group, following the 3-month vaccination period. RESULTS: The urobiome of 13 rUTI patients with sterile urine did not differ significantly in number of species (Anova; p = 0.96, Fig 1a) although species composition differed significantly (nested ADONIS; p<0.001, R2=0.068, Figs 1b,c) from 23 age-matched healthy controls. This composition difference was driven by some species more abundant in the rUTI patients (e.g., Finegoldia magna), though other potentially pathogenic species were similarly abundant or more abundant in controls (eg. E. coli and Enterococcus faecalis, respectively). There was no significant change in NGS detected number of species (Anova; p = 0.91, R2 = 0.001, Fig 1a) or species detected (Fig 1c) 3 months after vaccination in the rUTI group. Similarly, there was no difference in qPCR estimated bacterial load following 3-month vaccination period in the rUTI group (Anova; p = 0.83, R2 = 0.001). CONCLUSIONS: The urobiome comparison between healthy and rUTI subjects and lack of impact of a successful vaccine on the urobiome of rUTi patients would suggest that we need to re-look at interaction of bacterial virulence and the local bladder immune mechanisms and even origin of the organisms that cause UTI. This small study suggests that UTI may arise from the patients’ own native urinary microbiome that in healthy individuals may be modulated by local bladder innate immunological mechanisms. In patients with rUTI those deficient mucosal–based mechanisms may be enhanced by an effective vaccine. This possible concept–shift regarding the cause of rUTI in women would have profound management implications. Source of Funding: Inmunotek, MicroGenDx © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e15 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information J. Curtis Nickel More articles by this author R. Christopher Doiron More articles by this author Kerri-Lynn Kelly More articles by this author Nicolle Diaz More articles by this author Caleb D. Phillips More articles by this author Craig D. Tipton More articles by this author Expand All Advertisement PDF DownloadLoading ...

Guidelines, quality control and standardization of NGS testing

Guidelines, quality control and standardization of NGS testing

Next generation sequencing in neonatology: what does it mean for the next generation?

Rapid whole genome sequencing (WGS) and whole exome sequencing (WES), sometimes referred to as "next generation sequencing" (NGS) are now recommended by some experts as a first-line diagnostic test to diagnose infants with suspected monogenic conditions. Estimates of how often NGS leads to diagnoses or changes in management vary widely depending on the population being studied and the indications for testing. Finding a genetic variant that is classified as pathogenic may not necessarily equate with being able to predict the resultant phenotype or to give a reliable prognosis. Molecular diagnoses do not usually lead to changes in clinical management but they often end a family's diagnostic Odyssey and allow informed decisions about future reproductive choices. The likelihood that NGS will be beneficial for patients and families in the NICU remains uncertain. The goal of this paper is to highlight the implications of these ambiguities in interpreting the results of NGS. To do that, we will first review the types of cases that are admitted to NICUs and show why, at least in theory, NGS is unlikely to be useful for most NICU patients and families and may even be harmful for some, although it can help families in some cases. We then present a number of real cases in which NGS results were obtained and show that they often lead to unforeseen and unpredictable consequences. Finally, we will suggest ways to communicate with families about NGS testing and results in order to help them understand the meaning of NGS results and the uncertainty that surrounds them.

EP452: Diagnostic yield and outcomes of NGS testing in pregnancies with a fetal structural anomaly

Exome sequencing has been shown to be an excellent diagnostic tool for infants, children and adults. It’s diagnostic yield in the prenatal setting, however, is not as well understood. Small studies of the diagnostic yield for fetal structural anomalies ranges from 10-50% depending on the inclusion criteria. In many studies, the inclusion criteria focused on a pattern of structural anomalies that is highly consistent with a genetic etiology, for example shortened long bones concerning for skeletal dysplasias.

EP064: Consumer-initiated genetic testing identifies individuals predisposed to multiple hereditary cancer syndromes

Cancer is a leading cause of death and suffering worldwide, affecting millions each year with the impact reverberating beyond just the person with the diagnosis. Historically, relevant personal and family histories of cancer have been necessary prerequisites for the recommendation of genetic testing to uncover the diagnosis of a hereditary cancer syndrome. However, there is mounting evidence that many people without relevant histories harbor pathogenic or likely pathogenic variants in hereditary cancer genes that put them at high risk of developing cancer during their lifetime. Recently, there has also been an increase in genetic testing in healthy populations through at-home consumer-initiated genetic testing (CIT), which allows for a genotype-first approach to testing, rather than a traditional route of undergoing genetic testing following a cancer diagnosis. Based on this influx in testing in previously unstudied healthy adult populations, and the prevalence of pathogenic variants in genes associated with hereditary cancer genes at a population level, there is potential for the occurrence of biallelic events to be higher than previously understood. While there is some disparate information in the literature describing occurrences of biallelic pathogenic variants in disease and high risk cohorts, we aim to expand on that limited pool, and examined a population of healthy adults seeking CIT who are identified to have multiple pathogenic variants associated with multiple hereditary cancer syndromes. A retrospective case review of individuals who received results for a CIT healthy adult panel over the course of 6 months from Nov 2020-June 2021 was conducted to determine the number of cases that tested positive for more than one pathogenic or likely pathogenic variant in two different cancer genes. The CIT NGS testing panel included the following genes associated with hereditary cancer: ATM, BRCA1, BRCA2, PALB2, APC, BMPR1A, MLH1, MSH2, MSH6, PMS2, MUTYH, POLD1, POLE, SMAD4, CHEK2, and STK11. Individuals who tested positive for two variants within the same gene or for two variants causing the same hereditary cancer syndrome were excluded from this analysis. All individuals reside in the U.S. and are at least 18 years of age. Only pathogenic and likely pathogenic variants were reported. Personal and family history information was self-reported at the time that the test was ordered and further clarification of those histories was provided during post-test genetic counseling sessions. While proactive outreach, including emails and one phone call was completed for each individual to encourage them to schedule a genetic counseling consultation, not all individuals opted to have genetic counseling. There were a total of 100,436 individuals from Nov 2020-June 2021 that underwent CIT NGS panel testing which included all high risk cancer genes listed in the methods. Of those, 2,977 (3%) individuals were identified to have at least one positive finding in a high risk cancer gene. There were 43 individuals (0.04% of total population and 1% of positive cases) who tested positive for two distinct hereditary cancer syndromes, meaning a pathogenic or likely pathogenic variant in two different genes associated with increased cancer risk. In this cohort of 43 individuals, ages ranged from 18-years-old through 66-years-old, with a reported sex breakdown of 18 males and 25 females. Only 13 out of the 43 cases (30%) identified would have met NCCN criteria to qualify them for clinical testing. Therefore, 70% of individuals would not have qualified for clinical genetic testing. This study demonstrates that healthy individuals without relevant histories can harbor pathogenic variants in genes associated with multiple hereditary cancer syndromes, and that the majority of these individuals would not have qualified for clinical testing based on current NCCN criteria. This study sets the framework for future research into the occurrence of biallelic pathogenic variants conferring risk for multiple cancer syndromes at a population level. Future characterisation of this group may be able to help determine more precise risk figures, a better understanding of the effect of gene-gene interaction on risks, and further the discussion of increased access to genetic testing to ensure timely preventative medical decisions can take place.

A phase I study of pharmacokinetic (PK)-driven sequential dosing of rucaparib (RUB) with irinotecan liposome (nal-IRI) and fluorouracil (5FU) in metastatic gastrointestinal (mGI) and pancreas (PANC) cancers.

563 Background: RUB is an oral PARP1,2,3 inhibitor that demonstrated efficacy in patients (pts) with ovarian and prostate cancers harboring deleterious BRCA mutations. RUB exerts synergistic anti-tumor effect with IRI preclinically though the combination has overlapping toxicities. We previously published on the population PK of nal-IRI (Adiwijaya, Ma et al, Clin Pharm Ther 2017). We conducted a phase I study to evaluate a novel sequential dosing of RUB with nal-IRI/5FU in mGI cancer pts. Methods: Eligible pts had incurable mGI cancer previously received &gt; 1 line of therapy (rx), ECOG PS 0-1, had RECIST measurable disease, adequate organ reserves and not received IRI for metastatic disease. Previous PARPi rx was excluded. The endpoints included dose limiting toxicity (DLT), maximum tolerated dose (MTD) and toxicity profile. The dose escalation utilized the 3+3 design. RUB was given oral bid on Day 4 to 13 and 18 to 27 with nal-IRI i.v. and 5FU i.v. 2400 mg/m2 over 46 hr on Day 1 and 15, every 28 day. Planned dose levels were RUB 400 mg/nal-IRI 50 mg/m2 (DL1), 400 mg/70 mg/m2 (DL2) and 600 mg/70 mg/m2 (DL3). Adverse events (AEs) were scored per CTCAE v4.03. Molecular profile was evaluated by CLIA-certified NGS testing. Results: Eighteen pts including 11 colorectal (CRC), 6 PANC, 1 gastroesophageal (GE) were enrolled and 12 were evaluable for DLTs. DL2 was not tolerable (DLT: G3 diarrhea, nausea and vomiting) and DL2A was added (RUB 600 mg/nal-IRI 50 mg/m2). DL2A enrolled 6 pts with no DLT and was determined as the MTD. Of DLT-evaluable pts, G3 and worse treatment-related AEs from all cycles were diarrhea (33%), fatigue (25%), leukopenia (25%), neutropenia (25%), anemia (8%) and nausea (8%). Four of 12 response evaluable pts had partial response: 2 CRC (1 had ATM mut), 1 PANC ( ATM mut), 1 GE ( BRCA2 mut) whilst 3 responders previously had platinum (PLA). Five pts had stable disease beyond 16 weeks (range 18.9 to 100.7 weeks), and all had prior PLA. Conclusions: The study successfully determined the MTD of RUB in combination with nal-IRI and 5FU. Encouraging efficacy was observed in PLA-treated mGI cancers including responses in those harboring ATM and BRCA alterations. The study is proceeding to evaluate the efficacy of the combination in metastatic pancreas cancer pts with and without BRCA1/2 or PALB2 alterations. Clinical trial information: NCT03337087.