Commercial Next-generation Sequencing Research Articles

Pan-cancer microsatellite instability testing using a multiplex assay including long mononucleotide repeats.

290 Background: Microsatellite instability (MSI)/mismatch repair deficiency (dMMR) testing is a critical component of molecular testing for gastrointestinal cancers, among others. These analyses are important for the identification of patients with lynch syndrome and eligibility for treatment with immunotherapy. The current standard of care testing can identify most cases, however improved identification of MSI-H status is needed for non-colorectal cancers and for cancers with alterations outside MLH1/PMS2. A recent analysis indicated the potential for improved MSI-H detection with a multiplex assay including long mononucleotide repeats (LMR). Methods: Patients were selected based on MSI status, MMR gene variants, or the presence of a high tumor mutation burden and consented for participation as part of an IRB-approved protocol (UW15068). Formalin-fixed tissue slides were annotated by a pathologist for tumor and normal tissue. Slides were scraped and DNA isolated. The LMR MSI Analysis System (Promega, Madison, WI) and the OncoMate MSI Dx Analysis System (Promega) were performed on cancer DNA and normal tissue DNA when available per the manufacturer’s protocols. Instability of microsatellites and the MSI score were reported and correlated with clinical MMR immunohistochemistry (IHC) and commercial next-generation sequencing (NGS). Results: A total of 96 cancer samples were tested across 94 patients with over 20 different cancer types, including lung, colon, rectal, gynecologic, and esophagogastric cancers. Tumors from 20 patients were known to have microsatellite instability based on clinical NGS testing. 27 cases were found to be MSI-H based on the OncoMate assay. All of these cases were also MSI-H per the LMR assay and an additional 3 cases were found to be MSI-H using only the LMR assay. Of these 3 cases all were microsatellite stable by NGS. This number includes 1 colorectal and 2 esophagogastric cancer cases. One of the cases was found to have loss of MSH6 by IHC with an MSH6 mutation (MSH6 L1061fs). One of the cases was MMR intact by IHC, though did have a MSH6 exon 7 splice acceptor alteration that failed NGS calling quality control. The last case did not have MMR IHC performed, no detected MMR gene alterations, and a tumor mutation burden of 6.3 mutations per megabase. Conclusions: In this limited dataset, the utility of various methods for determination of MSI, dMMR, and TMB including PCR, IHC, and NGS were demonstrated using a patient cohort spanning solid tumor types. Promising results were observed with the LMR MSI Analysis System. These findings need to be further validated in a larger dataset and correlated with the response to immunotherapeutics.

Comparison of commercial next-generation sequencing assays to conventional culture methods for bacterial identification and antimicrobial susceptibility of samples obtained from clinical cases of canine superficial bacterial folliculitis.

Bacterial identification and antimicrobial susceptibility testing is an important step in timely therapeutic decisions for canine superficial bacterial folliculitis (SBF), commonly caused by Staphylococcus pseudintermedius. Next-generation sequencing (NGS) offers the appeal of potentially expedited results with complete detection of bacterial organisms and associated resistance genes compared to culture. Limited studies exist comparing the two methodologies for clinical samples. To compare and contrast genotypic and phenotypic methods for bacterial identification and antimicrobial susceptibility from cases of canine SBF. Twenty-four client-owned dogs with lesions consistent with SBF were enrolled. A sterile culturette swab was used to sample dogs with SBF lesions. The swab was rinsed in 0.9 mL of sterile phosphate-buffered saline and vortexed to create a homogenous solution. Two swabs for NGS laboratories (Labs) and one swab for culture (Culture Lab) were randomly sampled from this solution and submitted for bacterial identification and antimicrobial susceptibility. No statistical difference regarding turnaround time for NGS Labs compared to Culture Lab was found. NGS Lab 1 identified more organisms than NGS Lab 2 and Culture Lab, which were both statistically significant. There was no statistical difference in detection frequency for Staphylococcus spp. among all laboratories. There was poor agreement for the presence of meticillin resistance and most antimicrobials among all laboratories. Utilisation of NGS as a replacement for traditional culture when sampling canine SBF lesions is not supported at this time.

Simultaneous sequencing of 102 Y-STRs on Ion Torrent ™ GeneStudio ™ S5 System

The Precision ID NGS System from Thermo Fisher Scientific is a mainstream next-generation sequencing (NGS) platform used in forensic laboratories to detect almost all commonly used forensic markers, except for Y-chromosomal short tandem repeats (Y-STRs). This study aimed to: 1) develop a Y-STR panel compatible with the automatic workflow of the NGS system using Ion AmpliSeq Technology, 2) evaluate the panel performance following the SWGDAM guidelines, and 3) explore the possibility of using a combination workflow to detect autosomal STRs and Y-STRs (AY-STR NGS workflow). The GrandFiler Y-STR Panel was successfully designed using the ‘separating’ and ‘merging’ strategies, including 102 Y-STRs and Amelogenin with an average amplicon length of 133 bp. It is a mega Y-STR multiplex system in which up to 16 samples can be sequenced simultaneously on an Ion 530 ™ Chip. Developmental validation studies of the performance of the NGS platform, species specificity, reproducibility, concordance, sensitivity, degraded samples, case-type samples, and mixtures were conducted to unequivocally determine whether the GrandFiler Y-STR Panel is suitable for real scenarios. The newly developed Y-STR panel showed compelling run metrics and NGS performance, including 92.47% bases with ≥ Q20, 91.80% effective reads, 2106 × depth of coverage (DoC), and 97.09% inter-locus balance. Additionally, it showed high specificity for human males and 99.40% methodological and bioinformatical concordance, generated complete profiles at ≥ 0.1 ng input DNA, and recovered more genetic information from severely degraded and diverse case samples. Although the outcome when used on mixtures was not as expected, more genetic information was obtained compared to that from capillary electrophoresis (CE) methods. The AY-STR NGS workflow was established by combining the GrandFiler Y-STR Panel with the Precision ID GlobalFiler ™ NGS STR Panel v2 at a 2:1 concentration ratio. The combination workflow on NGS performance, reproducibility, concordance, and sensitivity was as stable as the single Y-STR NGS workflow, providing more options for forensic scientists when dealing with different case scenarios. Overall, the GrandFiler Y-STR Panel was confirmed as the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System, which is compatible with 51 Y-STRs in commercial CE kits and 51 Y-STRs in commercial NGS kits and the STRBase. The panel is as robust, reliable, and sensitive as current CE/NGS kits, and is suitable for solving real cases, especially for severely degraded samples (degradation index > 10).

Abstract B034: A phase 2 basket study of the oral MDM2 inhibitor milademetan for MDM2-amplified advanced solid tumors (MANTRA-2)

Abstract Milademetan is an oral, selective, small molecular inhibitor of the MDM2-p53 complex that reactivates p53 to induce cancer cell apoptosis and tumor regressions in nonclinical models. Clinically, MDM2 gene amplification (amp) occurs in 1–2% of advanced solid tumors and is associated with a poor prognosis. Preliminary antitumor activity was seen in patients with MDM2 amp and TP53 wild-type (WT) tumors in a phase 1 clinical trial [Tirunagaru et al. AACR-NCI-EORTC 2021]. We conducted a phase 2 basket study to evaluate the efficacy of milademetan in patients with advanced or metastatic solid tumors (except liposarcoma, intimal sarcoma, and primary CNS tumors) with MDM2 copy number (CN) ≥8 and WT TP53 status based on local next-generation sequencing (NGS) (NCT05012397). Milademetan was administered at 260 mg on days 1–3 and 15–17 of a 28-day cycle. Tumor assessments were performed every 8 weeks (RECIST v1.1). MDM2 CN was retrospectively analyzed centrally using a commercial NGS assay, which also allowed correlation with other co-occurring alterations. As of June 23, 2023, 39 patients were enrolled with 13 distinct tumor types. The most common histologies were biliary tract cancers (8, 20.5%), sarcomas (7, 17.9%), and breast cancer (6, 15.3%). Median number of prior systemic therapies was 3 (range 0–12). Thirty-eight patients were evaluable for safety. The most common hematologic grade 3/4 adverse events (AEs) related to milademetan were thrombocytopenia (26.3%), neutropenia (15.8%), and anemia (10.5%). All grade treatment-related AEs (>15% incidence) included thrombocytopenia (44.7%), nausea (42.1%), fatigue (36.8%), vomiting (31.6%), anemia (23.7%), diarrhea (23.7%) and neutropenia (18.4%). No bleeding or neutropenic sepsis events were reported, and no grade 5 events were observed. The dose reduction rate was 13.2%, and 7.9% of patients discontinued milademetan due to AEs. Central tumor testing was available on 30 (76.9%) patients and only 2 (6.7%) patients demonstrated MDM2 CN below the threshold of 8. Thirty-two patients were efficacy evaluable and had confirmed CN ≥8 or pending central analysis. Five partial responses (15.6%) were observed (4 unconfirmed, 1 confirmed) with one each in lung adenocarcinoma (co-alteration of EGFR + KRAS), pancreatic (KRAS), bladder (pending), gastric cancer (DNAB1-PRKACA) and endometrial sarcoma (CCND2). No clear correlation between MDM2 CN and tumor regression was observed. The median progression-free survival was 3.3 months (95% CI, 2.5–6.1). The longest patient on milademetan therapy was 41 weeks (biliary, best RECIST 1.1 response SD with –29% tumor reduction). Ten patients are currently ongoing. Milademetan monotherapy demonstrated clinically meaningful antitumor responses with a manageable toxicity profile in a variety of MDM2 amp, TP53 WT solid tumors, including those with other oncogenic drivers such as KRAS. Rational combination strategies with milademetan should be considered in tumors with WT TP53 to further enhance durable anti-tumor activity. Citation Format: Ecaterina E. Dumbrava, Christopher T. Chen, Gregory M. Cote, Glenn J. Hanna, Thomas E. Stinchcombe, Bradley Sumrall, Trisha Wise-Draper, Mohammed Kanaan, Steven Duffy, Christopher Sumey, Patrick Cobb, Naisargee Shah, Bo Zhang, Nora Ku, Robert C. Doebele, Mrinal Gounder. A phase 2 basket study of the oral MDM2 inhibitor milademetan for MDM2-amplified advanced solid tumors (MANTRA-2) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B034.

Impact of Pre-Transplant Molecular and Cytogenetic Remission on Outcomes of Allogeneic Stem Cell Transplant in Patients with Myelodysplastic Syndrome

Background: Allogeneic stem cell transplant (allo-SCT) carries a high relapse rate (~35% within 2 years) in patients (pts) with myelodysplastic syndrome (MDS). Pre-transplant measurable residual disease (MRD) increases the risk of relapse and death in acute myeloid leukemia (Thol et al, Blood 2018). However, the impact of pre-transplant MRD on post-transplant outcomes is less clear in MDS. Prior studies have compared outcomes between myeloablative (MAC) and reduced intensity conditioning (RIC) in pts with pre-transplant MRD persistence based on either cytogenetic remission alone or ultra-deep genomic sequencing with a limited 10-gene panel, suggesting reduced risk of relapse in pts receiving MAC (Festuccia et al, Biol Blood Marrow Transplant 2016; Dillon et al, JCO PO 2021). However, pre-transplant MRD assessment using a commercial next-generation sequencing (NGS) panel in addition to cytogenetic testing to assess post-transplant outcomes has not been evaluated in MDS. Methods: We conducted a multicenter retrospective review of MDS pts who underwent allo-SCT from 2015-2022. Pts with FISH/cytogenetic (FC) and/or molecular abnormalities at diagnosis and MRD assessment by commercial myeloid-panel NGS (with a variant-allele frequency detection limit up to 5%) and FC performed on peripheral blood (PB) or bone marrow (BM) aspirate within 3 months prior to transplant were included for analysis. The Kaplan-Meier method and log-rank tests were used to estimate overall survival (OS). Gray's test/cumulative incidence functions were used to estimate relapse-free survival (RFS) with non-relapse mortality (NRM) as competing risk. Results: Table 1 includes baseline pt characteristics. Of 81 pts, 69 pts (85.2%) had residual FC and/or molecular abnormalities (MRD+) at pre-transplant PB/BM assessment. Twelve (14.8%) pts were in both FC and molecular remission (MRD-) prior to transplant. With a median follow-up of 13.9 months [IQR 6.8 - 38.9], 43 (53%) died and 24 (30%) pts experienced a relapse. Twenty-three of 69 (33%) MRD+ pts had relapsed disease with a 38% cumulative incidence of relapse (CIR) at 2 years. In contrast, only 1/12 (8.3%) MRD- pts experienced a relapse after 7 years, with chromosomal loss of TP53 not present at initial diagnosis. CIR at 2 years was 33% and 41% (p=0.207) for MRD+ pts who received MAC vs RIC, respectively with NRM of 34.5% associated with MAC vs 28.2% for RIC (p=0.61). TP53 mutations were most frequently associated with relapse in 8/24 (33%) pts with a median OS of 9.8 months [8.2 - NR]. Besides the previously defined 10-gene panel (Dillon et al, JCO PO 2021), relapses were also driven by SRSF2, U2AF1, KRAS, SETBP1 and NF1 mutations present in the original clone. Six pts had DNMT3A, TET2 and/or ASXL1 (DTA) mutations only on pre-transplant NGS, with 2/6 pts relapsing without the re-emergence of DTA mutations. Median OS was not reached for MRD- pts and was 14.1 months [11.35-NR] for MRD+ pts, p = 0.061 [Fig.1]. OS probability at 2 years was 35% for MRD+ pts vs 75% for MRD- pts. Type of conditioning regimen, RIC vs MAC, did not impact OS (p= 0.6). An equal number of deaths, 35% each, were attributable to GvHD and relapse, respectively. Thirteen (30%) pts died from other causes, primarily infections and bleeding complications. NRM was 25% and 36% for MRD- and MRD+ cohorts, respectively. Pts received a median of 6 cycles [range, 2-36] of hypomethylating agent (HMA) +/- venetoclax prior to transplant with no association between the number of cycles and MRD status (p=0.80). The incidence of grade 3-4 acute GvHD and chronic GvHD requiring systemic therapy did not significantly differ, at 25% and 18.8% (p= 0.696) and 33.3% and 31.8% (p= 1.00) in MRD+ and MRD- pts, respectively. Conclusions: MRD assessment using commercially available NGS panels and cytogenetic testing that are routinely performed in MDS pts in real-world practice can predict risk of relapse. Pre-transplant MRD is associated with worse OS irrespective of the intensity of conditioning regimen. Although limited in statistical power due to a small sample size of MRD- pts in our cohort, further studies with a larger cohort are underway to better clarify the role of MRD on transplant outcomes in MDS. Our data also highlights that only a small minority of MDS pts (<15%) achieve complete molecular and cytogenetic remission at the time of transplant; strategies to eliminate the pre-transplant MDS clone are urgently needed.

Confirmed Pathogenic Germline Variants in Cancer Predisposition Genes Incidentally Detected in Somatic Genomic Profiling of Multiple Myeloma Patients

Introduction: We previously showed that ~10% of patients with multiple myeloma (MM) carry pathogenic or likely-pathogenic (P/LP) germline variants (PGVs) in known cancer predisposition genes, and that moderate/high-penetrance PGVs in DNA repair genes may be associated with MM risk and favorable response to alkylating chemotherapy. Increasingly applied in MM, genomic profiling by next-generation sequencing (NGS) can identify prognostically-relevant and therapeutically-actionable somatic mutations, but it may also incidentally unveil PGVs, which can have significant implications for patients and their families. We here test the diagnostic accuracy of a novel referral tool designed to flag likely PGVs warranting confirmatory germline testing among mutations identified in somatic NGS studies of 1161 MM patients from our institution. Methods: We retrospectively reviewed 1715 commercial NGS reports of 1161 MM patients treated at the Icahn School of Medicine at Mount Sinai (ISMMS) between 2015 & 2022 (targeted 448-gene somatic profiling panels performed by a single genomic testing company for clinical purposes on bone marrow aspirate samples). In collaboration with certified genetic counselors at our institution, we applied evidence-based criteria to develop a tool to flag P/LP variants of likely germline origin warranting a referral for confirmatory germline testing (“Likely PGVs”). The resulting referral tool flags variants detected in 35 well-established cancer predisposition genes at a variant allele frequency (VAF) of >10%, as well as 24 cancer predisposition founder variants at any VAF. Next, we tested the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of our approach for the detection of true PGVs by analyzing paired germline whole-exome sequencing (WES) in 382/1161 (33%) MM patients in the study cohort. Germline WES data were generated for research purposes using a clinically-validated pipeline for PGV detection and annotation. Results: 385 P/LP variants were reported in somatic NGS studies of 382 MM patients who had paired somatic/germline sequencing data. Of these, 38/385 (9.9%) were found to be true PGVs in confirmatory germline testing. True PGVs detected in more than one gene included nine APC, seven CHEK2, five BRCA1, three BRCA2, three ATM and three MUTYH variants. The referral tool flagged 41/385 (10.6%) of the cohort's somatic variants as likely PGVs, identifying 32/38 (84.2%) true PGVs. The six PGVs missed by the referral tool were detected in the following genes: one APC, one CHEK2, one MITF, one MUTYH, one NF1 and one SHDA variant. In contrast, the nine variants that were mislabeled as likely PGVs but were in fact somatic were: two ATM, two BRCA2, two TSC1, one CHEK2, one BRIP1, and one RET. With a variant allele threshold of >10%, the tool had a PPV of 78.0% and a NPV of 98.3% for the detection of true PGVs. The sensitivity and specificity were 84.2% and 97.5%, respectively. Among the 692/1161 MM patients who did not have confirmatory germline testing (67%), the proportion of variants flagged as likely PGVs was very similar to that of the paired somatic/germline group (77/696, 11.1%), and variants were detected in similar genes. Assuming a PPV of 78%, 60 of these variants could represent true PGVs. Conclusion: A novel tool designed to flag likely PGVs among mutations identified in somatic genomic profiling of MM patients had high sensitivity and specificity for the detection of true PGVs. 9.9% of all MM patients with paired somatic/germline testing were found to have a true PGV, validating the results of our prior study and once again highlighting the need to implement screening strategies in high-risk subgroups of MM patients, such as those with strong family history of cancer or prior personal history of cancer.

Analysis of ASXL1 Mutated T Cells in Patients with Myeloid Malignancies

Background: Myeloid malignancies, including myelodysplastic neoplasms (MDS), chronic myelomonocytic leukemia (CMML), and myeloproliferative neoplasms (MPNs), pose significant morbidity and mortality for affected patients and can progress to acute myeloid leukemia (AML). Additional sex combs-like protein 1 ( ASXL1) is an essential epigenetic regulator with important biological functions. Damaging ASXL1 mutations are prevalent in myeloid neoplasms and are associated with poor prognosis and lower response rates with hypomethylating agents (HMA). In a phase I-II trial published by O'Connell et al. in 2022, patients with MDS and CMML previously exposed to HMA were treated with a combination of atezolizumab, an immune checkpoint inhibitor (ICI), and guadecitabine, an HMA. Bone marrow (BM) CD3+ T cells and CD34+ myeloid cells were tested for a panel of recurrent myeloid mutations. The two patients who achieved complete remission carried the ASXL1 mutation in both cell compartments. Furthermore, the ASXL1 mutation in T cells correlated with a significantly longer overall survival among the 27 patients tested, suggesting that ASXL1 mutations may prime T cells for response to immune checkpoint blockade. In this pilot study, we aim to determine the frequency of ASXL1 mutated T cells and the impact on T cells methylation profiles in patients with untreated, ASXL1 mutated myeloid neoplasms. Methods:We included eight patients with known ASXL1 mutations detected by next-generation sequencing (NGS) in BM mononuclear cells . CD3+ T cells were isolated from the peripheral blood and separated by magnetic bead flow cytometry into CD8+ and CD4+ fractions. Sequencing libraries were generated using ClearSeq (Agilent) panel and were sequenced on MiSeq(Illumina). CD8+ T cell samples were aligned using a BWA aligner on Partek Flow (Partek) pipeline. Somatic calling was performed using the Strelka algorithm on Partek Flow. Each significant variant was confirmed by manual review using Integrative Genomics Viewer (IGV), and here we report the mutations present at a variant allele frequency (VAF) of at least 0.05 that correspond to those detected by NGS in the whole blood. In addition, DNA methylation studies were performed in the sorted T cells. We removed the loci with inadequate quality, and those associated with single nucleotide polymorphisms/age/sex chromatin. We performed methylation studies to identify differentially methylated locus and copy number variations. Results: We obtained peripheral blood samples from eight patients with ASXL1 mutated myeloid neoplasms detected by commercial NGS testing; aspirate was also obtained from a diagnostic BM biopsy in one patient. Table 1 shows patient demographics, characteristics and mutational analyses in both T and myeloid cells. Most patients were treatment-naïve, but one had been treated with HMA until 6 months prior to sample collection, and one patient had received cyclosporine for over 20 years for an undiagnosed autoimmune condition. T cells generated a mean sequencing coverage of 388x (min=341x, max=475x). ASXL1 mutated T cells were present in 50% (4/8) of the patients with myeloid ASXL1 mutations (Table 1). The median ASXL1VAF detected in T cells was 0.112. In 1 patient who also had a BM sample, the ASXL1 VAF in the T cells within the BM for this patient was 0.142, but only 0.082 in the peripheral blood. Methylation studies of peripheral blood T cell DNA did not delineate a clear difference between ASXL1 mutated and wild-type T cells (Figure 1). Discussion: Approximately 50% of patients with ASXL1 mutated myeloid malignancies carry the same mutation in their T lymphocytes, which confirms the findings from a published report in a different cohort of patients, all of whom had been previously exposed to HMA. In that study the T cells were collected from BM aspirate whereas in this study they were from the peripheral blood. Unsurprisingly, the ASXL1 VAFis lower in T cells than in myeloid cells and may be lower in the peripheral blood than in the marrow, but this needs confirmation. Methylation studies did not detect unique profiles in samples with mutated T cells, but we were not able to isolate ASXL1 mutated T cells, so these data are less likely to represent the affected clones. Given the potential benefit of treatment with ICI in these patients, further characterization of ASXL1 mutated T cells including functional studies and receptor profiling is warranted.

SAT603 A Heterogeneous Genetic Background Underlies Familial And Young-onset Pituitary Tumors

Abstract Disclosure: J.M. Zuarth-Vazquez: None. R.G. Rebollar-Vega: None. C. Ramírez-Rentería: None. W.E. Hernández-Núñez: None. M. Torres-Morán: None. A.L. Franco-Álvarez: None. J. Eseiza-Acevedo: None. M. Aguilar-Soto: None. D. Cuevas-Ramos: None. E. Sosa-Eroza: None. M. Mercado: None. A. Gamboa-Dominguez: None. L.C. Hernández-Ramírez: None. Introduction: Inherited pituitary neuroendocrine tumors (PitNETs) are considered rare and may present either as isolated lesions or in association with other endocrine tumors. Despite significant progress in the understanding of their molecular basis, the precise pathophysiological mechanisms in many patients with familial and sporadic pituitary tumors are unknown. Although the clinical presentation may point towards a specific genetic cause, a combination of careful clinical assessment and state-of-the-art genetic testing is required. Objective: To describe 4 cases of PitNETs with heterogeneous clinical presentations and genetic causes from a cohort of Mexican patients with neuroendocrine tumors. Methodology: Patients were recruited under informed consent from two different reference hospitals in Mexico City. Germline genetic testing was performed using either a custom-made or a commercial next-generation sequencing (NGS) panel. When available, relevant variants were confirmed in tissue samples. Genetic results were correlated with clinical, biochemical, and imaging characteristics. Results: Case 1 is an 18-year-old male with gigantism due to a 3-cm GH/prolactin-secreting PitNET, that was not cured after transsphenoidal surgery (TSS). Genetic testing showed a CDKN1B (NM_004064.5) c.356T>C, p.I119T variant of uncertain significance. No relevant family history was informed. Currently, the patient is treated with monthly octreotide LAR. Case 2 is a patient with young-onset acromegaly (15y) due to a GH-secreting PitNET. She was initially treated with bromocriptine and then underwent TSS without achieving remission. Radiotherapy was therefore indicated, resulting in panhypopituitarism. The likely pathogenic variant of AIP (NM_003977.4) c.872_877del, p.V291_L292del, was identified. Case 3 was diagnosed at the age of 15 with gigantism, treated with TSS and radiotherapy, subsequently demonstrating biochemical cure. Whole-exome sequencing showed the AIP pathogenic variant c.910C.T, p.R304*, indicative of familial isolated pituitary adenoma, confirmed by the history of two paternal first cousins with gigantism. Finally, a 54-year-old man (case 4), with a personal and family history of neurofibromatosis type 1, was incidentally diagnosed with a 16 mm, apparently non-functional PitNET, for which he is awaiting surgery. The pathogenic NF1 (NM_001042492.3) variant c.147C>A, p.Y49* was identified. Conclusions: Our data confirm that inherited PitNETs encompass a variety of clinical phenotypes. Modern NGS-based genetic testing is an effective method for identifying such cases. Precise molecular testing has an impact on the timely diagnosis of these lesions, as well as on their prognosis and the need for genetic counseling. Presentation: Saturday, June 17, 2023

Commercial next generation sequencing in a real-world cohort of patients with metastatic breast cancer.

e13082 Background: Clinicians increasingly order next generation sequencing (NGS) of tissue or liquid (ctDNA) biopsies from patients with metastatic breast cancer. Although some genetic information is clinically actionable, some, particularly non-ERBB2 gene amplifications, is not. We reviewed NGS data from a real-world cohort of metastatic breast cancer patients to evaluate the prevalence of somatic abnormalities using commercial NGS technology. Methods: We evaluated Foundation One NGS data from 44 tissue and 17 liquid biopsies from patients seen in a medical oncology practice between 2016 and 2022. We used KM Plotter to measure overall survival in early-stage patients by relative gene expression. Results: 76% of patients had ductal and 24% had lobular carcinomas. 59% of patients had luminal (ER-positive) and 32% of patients had basal breast cancer (ER-negative). Only 9% of patients had HER2-positive disease. 48% of the tumors had a p53 abnormality, either a point (59%) or frameshift mutation (36%). 50% tumors had a somatic mutation in a tumor suppressor gene such as PTEN, RB or APC. One patient had a germline BRCA1 mutation and 5 patients had germline BRCA2 mutations. 39% of patients carried a somatic mutation in PIK3CA, predominantly H1047R (45%) in the kinase domain or E545X (20%) in the helicase domain. KM plotter showed worse OS in early-stage patients with low p53 gene expression or high PIK3CA expression. 10/11 patients with lobular carcinoma had somatic mutations in CDH1, 70% of which were frameshift mutations. 8/27 patients with luminal cancer had an ESR1 mutation, predominantly Y537 or D538. 9 patients, predominantly luminal, had amplifications of the 11q13 (CCND1). 7 patients had amplifications of 8q24 (MYC). 20q13 (AURKA) amplifications were only seen in luminal cancers. 12p13 (CCND2) amplifications were only seen in basal cancers. 27% of lobular cancers (vs. 6-17% of ductal cancers) demonstrated amplification of the 11q13, 8q24 and/or 8p11 amplicons. 20q13 and 12p13 amplifications were only seen in ductal cancers. Liquid biopsies were more likely to detect point mutations than tissue biopsies. p53 mutations were seen in 65% of liquid vs. 48% of tissue biopsies. 47% of liquid biopsies showed two p53 mutations. PIK3CA mutations were seen in 71% of liquid vs. 39% of tissue biopsies. 18% of liquid biopsies showed two PIK3CA mutations. 93% of basal cancers had at least one p53 mutation. 44% of luminal cancers had a PIK3CA mutation. ESR1, GATA3 and AKT1 mutations were only seen in luminal cancers. While ESR1 mutations were seen in ductal and lobular cancers, GATA3 and AKT1 mutations were only seen in ductal cancers. Conclusions: Commercial NGS testing of a real-world cohort yields actionable mutations that can impact clinical management and outcome of metastatic breast cancer patients. NGS testing demonstrates the range of gene amplifications and corresponds closely with what has been reported in the published literature.

Turnaround time of tissue next generation sequencing in cancer patients at a safety net hospital: A quality improvement project.

e18707 Background: Tissue Next Generation Sequencing (NGS) testing can identify targetable genetic alterations, and has become an essential component of cancer care. Prolonged turnaround time (TAT) for tissue NGS testing (time from ordering until report arrival) can be a barrier for optimal treatment. Reducing the TAT can significantly aid the decision making process and improve the time to treatment initiation. As a quality improvement project, we explored a strategy to improve the TAT at our safety net hospital. Methods: We studied the TAT of commercial NGS test (FoundationOne CDx) ordered between January and December of 2022 by the oncology service at Bellevue Hospital Center (BHC), an affiliated public hospital. Tissue NGS testing was ordered for solid tumors regardless of tumor origin at the discretion of the provider. During the first half of the year, specimens prepared at BHC lab were manually dropped off at a central location for shipping to Foundation Medicine lab (testing site). The workflow was changed on 7/7/2022 to introduce a courier system to pick up the specimens from pathology lab at BHC. We analyzed the data reported by Foundation Medicine. Results: As of 2/1/2023, all specimens ordered between 1/1/2022 to 12/29/2022 were reported. There were 123 reports, of which 122 were used for the analysis. 1 report was excluded due to mislabelling of the specimen at the testing site that led to significant delay in TAT. The mean TAT of all reported specimens for the year 2022 was 23 days. Introduction of the courier reduced the mean TAT from 25 days to 20 days. After introducing the courier system, the mean time for specimen analysis was similar, but the mean time from specimen ordering to arrival at the testing site was reduced from 14 days to 10 days. Conclusions: The TAT for tissue NGS testing in underserved cancer patients at a safety net hospital can be successfully improved by implementing a courier system. The improvement was achieved regardless of tumor origin or patients’ financial ability to pay and can aid decision making for treatment initiation. This strategy can be adopted by community hospitals without access to on-site NGS testing. We are exploring additional interventions to optimize the workflow to further reduce the TAT. [Table: see text]

Abstract 921: Identification of NRG1 fusions in patients with solid tumors: analysis from a real-world community oncology network

Abstract Neuregulin-1 (encoded by NRG1) serves as a ligand for ERBB3 and can lead to dysregulated cellular proliferation in cases of NRG1 fusions. The reported incidence of NRG1 fusions in solid tumors is ~0.1-0.3% with enrichment in invasive mucinous adenocarcinoma of the lung and KRAS wildtype pancreatic adenocarcinoma. However, the real-world incidence of NRG1 fusions across diverse tumor types continues to evolve with the clinical implementation of comprehensive next-generation sequencing (NGS) methods including RNA based NGS. The true diversity and prognostic significance of specific NRG1 fusion partners has yet to be elucidated. We report on NRG1 fusions from a large clinico-genomic database of patients (pts) in community-based oncology settings where NGS is ordered as standard clinical practice. We conducted a retrospective, records-based analysis of pts across Sarah Cannon’s network of over 299 partner community-based oncology clinics to identify pts with NRG1 fusions detected by commercial NGS testing ordered as a part of standard of care from 1/1/2017 - 7/7/2022. NRG1 fusions were reported in 0.05% (19 of 40,857) of pts with commercial NGS test results across a range of tumor types including lung (59%; n=11/19), pancreas (11%; n=2), breast (5%; n=1), colorectal (5%), esophageal (5%), endometrial (5%), soft tissue liposarcoma (5%), and carcinoma of unknown primary (5%). NGS identifying an NRG1 fusion was performed in 5 (26%) pts with early stage disease while 11 (58%) pts had NGS testing after initial diagnosis of or progression to advanced/metastatic disease. A majority of pts harboring NRG1 fusions were female (68%; n=13). NRG1 fusions were detected by DNA (58%; n=11) and RNA based (42%; n=8) NGS from liquid (5%; n=1) and tissue biopsies (95%; n=18). A total of 11 different fusion partners were reported. CD74 was the most prevalent fusion partner and was primarily detected by DNA NGS including in one pt from liquid biopsy. Five novel, as yet to be described, fusion partners (CDK13, IL1RL2, FUT10, PPP2R2A, PIM3) were detected - 3 by RNA sequencing and 2 by DNA sequencing of NRG1 (versus sequencing of the fusion partner). TP53 (42%; n=8), CDKN2A (32%; n=6), and MTAP (16%; n=3) were the most frequently co-altered genes. Other notable co-altered genes included mutations in PIK3CA (11%; n=2), BRAF (5%; n=1), EGFR (5%), ALK (5%), and NRAS (5%). Immune biomarkers were largely negative, with 16% (n=3) PD-L1 positive, 0% MSI-high, and 5% (n=1) TMB-high cases. These data highlight the importance of comprehensive molecular profiling for pts with solid tumors, as NRG1 fusions occur across tumor types and stage of disease. Appropriate test selection is paramount as novel NRG1 fusions are more robustly detected by RNA over DNA NGS and are more routinely detected in tissue over liquid biopsies. Clinical trials investigating therapies to target NRG1 fusions are ongoing [CRESTONE (NCT04383210); eNRGy (NCT02912949)]. Citation Format: Emma G. Sturgill, Jaya Srivastava, Jessica Correia, Cooper Schumacher, Daniel Luckett, Cesar A. Perez, Judy S. Wang, Stephen G. Divers, Babar Bashir, Jennifer Johnson, Valerie M. Jansen, Andrew J. McKenzie, David R. Spigel. Identification of NRG1 fusions in patients with solid tumors: analysis from a real-world community oncology network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 921.

Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis.

To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK). Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n=11 and n=4, respectively) and IUK patients (n=22 and n=8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined. The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples. A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.

Clinical utility of plasma cell-free DNA in gliomas.

Noninvasive molecular profiling of tumors using plasma-based next-generation sequencing (NGS) is increasingly used to aid in diagnosis, treatment selection, and disease monitoring in oncology. In patients with glioma, however, the plasma cell-free DNA (cfDNA) tumor fraction, defined as the fractional proportion of circulating tumor-derived DNA (ctDNA) relative to total cfDNA, is especially low, in large part due to the blood-brain barrier. As a result, commercial plasma-based NGS assays, designed to screen for a small number of actionable genomic alterations, are not sensitive enough to guide the management of patients with glioma. As this has been long recognized in neuro-oncology, significant research efforts have been undertaken to improve the sensitivity of plasma ctDNA detection in patients with glioma and to understand the biology and clinical relevance of non-tumor-derived cfDNA, which makes up most of the total cfDNA pool. Here, we review key recent advances in the field of plasma cfDNA analysis in patients with glioma, including (1) the prognostic impact of pre-treatment and on-treatment total plasma cfDNA concentrations, (2) use of tumor-guided sequencing approaches to improve the sensitivity of ctDNA detection in the plasma, and (3) the emergence of plasma cfDNA methylomics for detection and discrimination of glioma from other primary intracranial tumors.

Make it "SNPPY" - Updates to SRM 2391d: PCR-Based DNA Profiling Standard

Standard Reference Material (SRM) 2391d: PCR-Based DNA Profiling Standard was released to the forensic community in 2019. Next Generation Sequencing (NGS) was used as the primary method of certification, where certified values were assigned when a high coverage sequence string was available for a marker. Using NGS to assign values has allowed for additional marker sets beyond short tandem repeat (STR) loci, including single nucleotide polymorphisms (SNPs) and mitochondrial DNA (mtDNA) whole genome sequences, to be included in the Certificate of Analysis (COA). Since the 2019 release, several commercial NGS panels have become available including the Verogen ForenSeq mtDNA Control Region, mtDNA Whole Genome, MainstAY, and Kintelligence Kits. In addition, three community Ion AmpliSeq panels from Thermo Fisher (MH-74 Plex, VISAGE, and Y-SNP) are now available. While the mtDNA whole genome sequence for the components are already included and no new STR markers are introduced by MainstAY, the other recently released panels allow for the inclusion of > 11,000 additional SNPs (e.g., identity, ancestry, phenotype, kinship, and X- and Y-SNPs) and 74 microhaplotypes to the COA for SRM 2391d in an update completed by fall of 2022.

Tumor Mutational Burden by Whole-Genome Sequencing in Resected NSCLC of Never Smokers.

Data are scarce about tumor mutational burden (TMB) as a biomarker in never smokers with non-small cell lung cancer (NSCLC). TMB was assessed by whole-genome sequencing (WGS) and compared with in silico reduced whole-exome sequencing (WES) and targeted commercial next-generation sequencing (NGS) gene panels in 92 paired tumor-normal samples from never smokers who underwent NSCLC resection with curative intent. Analyses were performed to test for association with survival after surgery and to identify the optimal prognostic TMB cutoff. Tumors of never smokers with NSCLC had low TMB scores (median 1.57 mutations/Mb; range, 0.13-17.94). A TMB cutoff of 1.70 mutations/Mb was associated with a 5-year overall survival of 58% in the high-TMB (42% of cases) compared with 86% in low-TMB patients (Wald P = 0.0029). TMB scores from WGS and WES were highly correlated (Spearman ρ = 0.93, P < 2.2e-16). TMB scores from NGS panels demonstrated high intraindividual fluctuations and identified high-TMB patients with 65% concordance in average compared with WGS. In resected NSCLC of never smokers, high TMB was associated with worse prognosis. WES provided a good estimate of TMB while targeted NGS panels seem to lack adequate depth and resolution in the setting of low mutation burden. TMB is a prognostic indicator of survival in resected NSCLC from individuals who never smoked. In this setting of low mutation counts, TMB can be accurately measured by WGS or WES, but not NGS panels.

Establishment of NGS-based HLA 9-locus haplotypes in the Eastern Han Chinese population highlights the role of HLA-DP in donor selection for transplantation.

We collected HLA typing data from 653 families in the Eastern Han Chinese population. HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, and DPB1 (HLA-11 loci) typing of 1781 subjects was performed using a commercial next-generation sequencing (NGS) method in our laboratory. The phasing of haplotypes in each family was determined by Mendelian segregation. Haplotype analysis revealed 1634 different haplotypes among a total of 2230 haplotypes. The predominant haplotype was A*30:01-C*06:02-B*13:02-DRB1*07:01-DRB4*01:03-DQA1*02:01-DQB1*02:02-DPA1*02:01-DPB1*17:01 (HF=4.04%), followed by A*02:07-C*01:02-B*46:01-DRB1*09:01-DRB4*01:03-DQA1*03:02-DQB1*03:03-DPA1*02:02-DPB1*05:01 (HF=1.84%) and A*33:03-C*03:02-B*58:01-DRB1*03:01-DRB3*02:02-DQA1*05:01-DQB1*02:01-DPA1*01:03-DPB1*04:01 (HF = 1.48%), accounting for 7.35% of the total. Meanwhile 76.41% of all haplotypes were observed only once or twice (HF < 0.1%). Different from HLA-DRB3/4/5 and DQA1 loci, DP linkage markedly increased haplotype variation by 34.82% based on the 5-locus haplotype. The much weaker linkage disequilibrium (LD) of DQB1-DPB1 indicated the reason. We observed 10 analyzable recombination events, most of which occurred at DP loci. Even with the same common 5-locus haplotype, HLA-DP linkage alters the haplotype diversity and frequency. Analysis of related haplotype assignment and unrelated recipient-donor pairs matching at the 9-locus haplotype revealed that HLA-DP affects the donor selection strategy. Haplotype study of a large sample size using NGS identified linkage haplotypes beyond the 5 loci. LD, recombination events, and haplotype variation caused by DP loci emphasized that HLA 9-locus haplotype matching should be considered in donor selection, particularly the effect of DP loci. The finding lays the foundation for further studies on the effect of HLA-DP mismatch on transplantation.

Efficacy and potential resistance mechanisms of afatinib in advanced non-small cell lung cancer patients with EGFR G719X/L861Q/S768I.

Afatinib is the only currently approved EGFR-tyrosine kinase inhibitors for advanced non-small cell lung cancer (NSCLC) patients with EGFR G719X/L861Q/S768I. However, there are limited real-world data concerning the benefits and resistance mechanisms of afatinib in patients with these nonclassical mutations. To fill this gap, the present study was conducted. All NSCLC patients treated with afatinib were screened, and patients with EGFR G719X/L861Q/S768I were enrolled into the analysis. Either tumor tissue or blood specimens were detected by the commercial next-generation sequencing (NGS) panels or amplification-refractory mutation system (ARMS)-polymerase chain reaction (PCR) to figure out the mutation genotype. A total of 106 advanced NSCLC patients with EGFR G719X/L861Q/S768I received afatinib treatment. The benefits of afatinib exhibited heterogeneity in different mutation genotypes. Notably, at baseline, NGS testing was performed in 59 patients, and TP53 was the most frequently coexisting mutation. Patients with TP53 mutations obtained fewer survival benefits than those with TP53 wild-type. A total of 68 patients ultimately experienced progression, and 27 patients received NGS testing to clarify the potential resistance mechanisms. EGFR-T790M, CDK4 amplification, FGFR1 amplification, PIK3CA, MET amplification, RET fusions, HER2, and BRAF mutations were identified in three (11.1%), three (11.1%), three (11.1%), three (11.1%), three (11.1%), one (3.7%), one (3.7%), and one (3.7%) of the cases, respectively. Five patients underwent ARMS-PCR testing for detecting EGFR-T790M mutation, and only one patient was T790M-positive. The present study elucidated the differential benefits of afatinib within different mutation genotypes and first revealed the spectrum of potential resistance mechanisms in patients with EGFR G719X/L861Q/S768I. The results of this study may provide practical clinical information that can guide optimal treatment in this setting.

P876: INCIDENTAL DISCOVERY OF PROBABLE PATHOGENIC GERMLINE VARIANTS IN MULTIPLE MYELOMA PATIENTS UNDERGOING SOMATIC GENOMIC PROFILING

Background: Next-generation sequencing (NGS) is increasingly used to characterize the somatic mutational landscape of patients with multiple myeloma (MM) and guide precision medicine approaches. Somatic genomic profiling (SGP) in cancer patients can lead to the incidental discovery of pathogenic or likely-pathogenic germline variants (PGVs) in ~4% of cases (PMID 26787237). Our previous analysis of whole exome germline data from 895 MM patients in the MMRF CoMMpass dataset showed ~8% of MM patients carry a PGV in a hereditary cancer gene (HCG), and identified shared characteristics among PGV carriers including younger age at diagnosis, higher likelihood of having family history of hematologic malignancy, higher likelihood of t(11;14) MM, and favorable outcomes (Thibaud et al, ASH 2021). Aims: The present study aims to assess the rate of detection of probable PGVs (PPGVs) in MM patients undergoing targeted SGP. Methods: We retrospectively reviewed results of 1,614 commercial NGS panels performed by a single genetic testing laboratory on bone marrow aspirates of 1,095 MM patients treated at our institution since 2015 who were not enrolled in the MMRF CoMMpass study. We considered individual variants to be PPGVs when all of the following criteria were met: 1) variant found in a panel of 106 well-established HCGs (same panel used in our prior study, but excluding TP53 given high rate of somatic mutations in MM); 2) variant unambiguously listed as pathogenic or likely pathogenic (P/LP) in ClinVar, or variant classified as “conflicting interpretations of pathogenicity” but reported by ≥2 CLIA-certified labs as P/LP; 3) variant allele frequency (VAF) >30% (per PMID 33236764); 4) variant detected in all available test reports for a given patient (and VAF >30% in all available test reports). Well known founder variants (FV) in specific populations were labeled as PPGV-FVs. Results: 95 MM patients (8.7%) had a genetic variant meeting criteria to be classified as a PPGV. Two patients had 2 distinct PPGV. PPGV were most commonly detected in the following HCGs (Table 1): APC (n=22), CHEK2 (n=20), ATM (n=7), BRCA1 (n=6), MUTYH (n=6). Of all PPGV carriers, 47 (49%) carried a PPGV-FV. Median VAF for PPGVs was 48.2% (range 30-85%). 34 PPGV carriers had serial NGS tests available (range 2-5 reports); in these patients, VAF standard deviation across serial measurements was <5% in 89% of cases. Taken together, these findings indicate a high likelihood that PPGV detected in these MM patients while undergoing tumor sequencing are indeed incidentally-discovered PGV warranting referral to a genetic counselor for consideration of confirmatory testing. Image:Summary/Conclusion: SGP of bone marrow aspirate in MM patients incidentally detected PPGV in well-established HCG in 8.7% of cases. These findings are consistent with those of our prior study, which found an 8.6% prevalence of PGV in newly-diagnosed MM patients. In MM patients undergoing SGP, detection of a mutation involving a HCG and with a VAF >30% should prompt referral to genetic counseling for confirmatory testing, as PGV detection can have clear implications for patients and their families.

Discordance in Tumor Mutation Burden from Blood and Tissue Affects Association with Response to Immune Checkpoint Inhibition in Real-World Settings.

BackgroundTumor mutation burden (TMB), a biomarker for immune checkpoint inhibitor (CPI) response, is reported by both blood- and tissue-based next-generation sequencing (NGS) vendors. However, the agreement between TMB from blood (bTMB) and tissue (tTMB) in real-world settings, both in absolute value and association with CPI response, is not known.Materials and MethodsThis study utilizes Sarah Cannon’s precision medicine platform, Genospace, to harmonize clinico-genomic data from 17 206 patients with cancer with NGS results from September 2015 to August 2021. A subset of patients have both bTMB and tTMB results. Statistical analyses are performed in R and include (1) correlation (r) and concordance (ρ) between patient-matched bTMB-tTMB pairs, (2) distribution of total bTMB and tTMB values, and (3) association of bTMB and tTMB with time to CPI therapy failure.ResultsIn 410 patient-matched bTMB-tTMB pairs, the median bTMB (m = 10.5 mut/Mb) was significantly higher than the median tTMB (m = 6.0 mut/Mb, P < .001) leading to conflicting “high” and “low” statuses in over one-third of cases at a threshold of 10 mut/Mb (n = 410). Significant differences were observed in the distribution of bTMB values from blood-NGS vendors, with guardant health (GH) reporting higher (m = 10.5 mut/Mb, n = 2183) than Foundation Medicine (FMI, m = 3.8 mut/Mb, n = 462, P < .001). bTMB from GH required a higher threshold (≥40 mut/Mb) than bTMB from FMI (≥12 mut/Mb) in order to be associated with CPI response.ConclusionsThis study uncovers variability in bTMB reporting among commercial NGS platforms, thereby evidencing a need for assay-specific thresholds in identifying patients who may respond to CPI therapy.

Abstract P2-01-13: Longitudinal circulating tumor DNA (ctDNA) monitoring by digital droplet PCR (ddPCR) in metastatic breast cancer

Abstract Background: A common challenge in treating patients with metastatic breast cancer (MBC) is ensuring adequate therapeutic efficacy, maintenance of quality of life, and avoidance of drug toxicity by preventing exposure to ineffective treatment. Critical to balancing these goals is the ability to quickly identify progression in order to minimize exposure to ineffective therapies. Imaging is the current standard-of-care for disease response evaluation, but high cost, relative low sensitivity, and tumor flare phenomenon remain significant issues. Circulating tumor DNA (ctDNA) testing based on next generation sequencing (NGS) is emerging as an alternative to imaging-based response assessment, but remains an expensive proposition. Here we evaluate a hybrid monitoring strategy using an initial baseline NGS-based ctDNA analysis to identify mutant alleles, followed by response monitoring with a low-cost, personalized digital droplet PCR (ddPCR) assay. Methods: Blood for ddPCR ctDNA analysis was collected at the same timepoint as a sample for commercial ctDNA analysis by NGS. PCR primer and probe sets were designed against clinically relevant mutations identified from each patient’s commercial ctDNA NGS report. ddPCR was then performed. Variant allele fractions (VAFs) detected by ddPCR were compared to allelic fractions reported by commercial NGS using Bland-Altman analysis and significance was determined by paired t-test. To demonstrate the potential for longitudinal ddPCR-based ctDNA monitoring, serial samples from the same patient were analyzed. Results: In our pilot validation set, 10 paired patient samples were analyzed by NGS and ddPCR. NGS-reported VAFs ranged from 0.30% to 16.20%. High comparative performance was observed between NGS and ddPCR platforms over expected mutant fractional abundance ranges. There was no significant quantitative difference between the two approaches (p=0.45). Longitudinal monitoring of ctDNA by ddPCR was performed on a metastatic hormone-positive breast cancer patient and revealed a significant rise in the clinically-relevant PIK3CA H1047L mutation that preceded imaging-based documentation of progression by 3 months. A final timepoint was taken several months before the patient’s death with a further significant increase in ctDNA burden. Conclusions: Here we demonstrate that highly-sensitive ddPCR-based ctDNA assays provide comparable results to next generation sequencing, but at considerably lower cost. These results indicate that bespoke tracking of select alleles by ddPCR offers an inexpensive and rapid approach to serial ctDNA monitoring in metastatic breast cancer patients. A larger prospective study has been designed and is underway. Citation Format: Nicole Higashiyama, LaTerrica Williams, Bryant McCue, Alphi Kuriakose, Tiffaney Tran, Stephanie Gonzalez, Mayra Licerio, Carol Chenault, Heidi Dowst, Susan Hilsenbeck, Matthew Ellis, Bora Lim, George Miles. Longitudinal circulating tumor DNA (ctDNA) monitoring by digital droplet PCR (ddPCR) in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-13.