Mutant Allele Frequency Research Articles

Abstract 2297: Sensitive mutation detection by simultaneous targeted sequencing of the Watson and Crick strands

Abstract Background Detecting mutations in circulating cell-free DNA (ccfDNA) allows for non-invasive monitoring of tumors, however, is hampered by low fractions of tumor DNA and low efficiencies of current technologies. Methods We describe a new and simple method for simultaneous multiplex targeted next-generation sequencing (NGS) of the Watson and Crick strands. We also develop a custom bin-based algorithm and unique molecule identifier (UMI)-aware data algorithm to segregate and suppress background noises. The performance of the method was evaluated with a reference standard DNA that contains 44 mutations, and experimented with dilutions for low mutant allele frequencies (MAFs) and with various amounts of DNA inputs. We validated our methods using blood samples collected from lung cancer patients with various stages and at follow-ups. A commercial droplet digital PCR (ddPCR) for a seven-plex KRAS mutation detection assay was used as an orthogonal method to validate the sensitivity and specificity of our NGS methods. Results Using merely 1 ng of DNA (300 genome equivalent) containing 44 known mutations at 1% minor allele frequencies (MAFs) across 19 genes, our method detected 43 known and 0 false mutations, demonstrating a superior enrichment efficiency (> 99%) approaching the theoretical limit that misses target molecules owning to random sampling alone and high specificity. In the analysis of cfDNA obtained from plasma (1 mL per patient) of 77 lung cancer cases, mutations were identified in 47 (61%) of the cancer patients. Using the equal-volume (1 mL) aliquot plasma available from 70 patients (7 cases had insufficient samples), the ddPCR assay showed 6 samples were positive and 64 samples were negative. Among the 6 ddPCR-positive samples, all were tested positive by our NGS method, demonstrating similar sensitivity as the ddPCR assay while having the advantage of the capacity to test a panel of genes. Among the 64 ddPCR-negative samples, our method showed 63 were KRAS-negative and 1 was positive, indicating a specificity of 98.4%. With a detailed examination of the “false-positive” sample when using the ddPCR as the gold standard, the image of ddPCR result in fact showed two dots in the ‘grey zone’, indicating a possibility of a false-negative ddPCR result for the sample, rather than a false-positive in our NGS method. Using 1 mL of plasma, our method can detect mutation at 0.05% MAF and predict lung cancer relapse prior to current clinical methods. Conclusion Our new NGS method enables sensitive detection of mutations when only a trace amount of material is available and may be useful for cancer minimal residual disease monitoring. Citation Format: Firaol T. Kebede, Weiwei Bian, William C. Cho, Zongli Zheng. Sensitive mutation detection by simultaneous targeted sequencing of the Watson and Crick strands [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 2297.

Abstract 939: Genomic characterization of PMBCL, cHL and DLBCL utilizing tissue and liquid biopsies

Abstract Background: Primary mediastinal large B-cell lymphoma (PMBCL), arising from thymic medullary B cells, shares molecular features with classic Hodgkin lymphoma (cHL), including activation of JAK/STAT and Nuclear Factor Kappa B (NF-ƙB) pathways, as well as PD-L1 mediated immune evasion. Much of the molecular characterization relied on availability of tissue samples. Cell-free DNA (cfDNA) has emerged as a promising non-invasive approach for molecular profiling in lymphomas. In this study, we characterized the molecular features of PMBCL and cHL in the Chinese population utilizing both tissue-based and liquid biopsies. Methods: Tissue and/or plasma samples from a total of 35 PMBCL patients, 52 cHL patients and 81 DLBCL patients that were subjected to targeted next-generation sequencing (NGS) using a 475 cancer-related gene panel were included in the analysis. Results: Analysis of matched tumor and plasma samples from 38 cHL patients revealed 152 (28.10%) overlapping genetic alterations, with a higher number of mutations detected in the plasma compared to tissue (Total=472 vs. 221; Median=11 vs. 3 per patient, P<0.01). Of these, 320 (59.15%) were unique to the plasma, whereas 69 (12.15%) were only found in the tissue. Mutant allele frequencies (MAFs) were also lower in the tissue than in the plasma, with a median of 1.71% vs 2.99% (P<0.01). In 18 PMBCL patients, who had paired plasma and tumor samples, similar number of genetic alterations were detected (Total=347 vs. 411; Median=19.5 vs. 21 per patient, P=0.09). Of these, 273 (56.29%) alterations were shared variants, 74 (15.28%) were unique to the plasma sample and 138 (28.45%) were unique to the tissue. MAFs were higher in the tissue compared with the plasma (23.35% vs. 8.78%, P<0.01). Comparisons of mutational profiles among PMBCL, cHL, and DLBCL showed similar mutational profiles between PMBCL and cHL. The most frequently detected genetic alterations in PMBCL and cHL were STAT6 (68.57% vs. 40.38%), SOCS1 (57.14% vs. 57.69%), ACTB (48.57% vs. 23.08%), B2M (48.57% vs. 42.31%), and TNFAIP3 (34.29% vs. 40.38%). On the contrary, the frequencies of STAT6 (68.57% vs. 6.17%, P <0.01), SOCS1 (57.14% vs. 18.52%, P<0.05), and ACTB (48.57% vs. 9.88%, P<0.05) were markedly different comparing PMBCL and DLBCL. Meanwhile, the more common genetic alterations in DLBCL, such as MYD88 (33.33% vs. 0.00%, P<0.01), BCL6 (33.33% vs. 11.43%, P=0.06), BCL2 (20.99% vs. 2.86%, P<0.05), and CDKN2A (24.69% vs. 2.86%, P<0.05) were rarely detected in PMBCL patients. Conclusion: Our findings support that ctDNA in PMBCL and cHL may be an attractive source of genetic materials to assess tumor genomics and guide treatment decisions, especially in cHL with only 1% Hodgkin and Reed/Sternberg (HRS) cells. We also demonstrate for the first time in the Chinese population that PMBCL and cHL shared comparable mutational profiles, which were different from that of DLBCL. Citation Format: Xiaonan Wang, Lu Shen, Liuqing Zhu, Jiani C. Yin, Haimeng Tang, Yang Shao. Genomic characterization of PMBCL, cHL and DLBCL utilizing tissue and liquid biopsies [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 939.

Abstract 5710: Detection and monitoring of t(11;14) in liquid biopsies from patients with relapsed/refractory multiple myeloma treated with venetoclax-based regimens

Abstract Venetoclax (Ven) is a selective B-cell lymphoma 2 inhibitor being studied in t(11;14)+ relapsed/refractory multiple myeloma (MM) Detection of t(11;14) in MM requires bone marrow (BM) aspiration and evaluation of CD138+ plasma cells by fluorescence in situ hybridization (FISH) Innovative techniques may provide less invasive detection of t(11;14) in liquid biopsies Here we present results of the SAGAsign® integrated approach combining low-coverage whole-genome sequencing (WGS) to characterize t(11;14) breakpoints together with personalized digital polymerase chain reaction (dPCR) assays to efficiently detect and monitor the genomic rearrangements in circulating tumor DNA (ctDNA) Baseline BM aspirates were collected from 270 patients (pts) from Ven clinical trials (NCT02755597, NCT01794520, NCT03314181, NCT02899052) Previously generated WGS to an average coverage ~22 × was used Paired samples of peripheral blood mononuclear cell (PBMC) DNA and plasma circulating cell-free DNA (cfDNA) were analyzed by dPCR at timepoints after Ven-based treatment Of the 90 t(11;14)+ pts by FISH, 160 t(11;14) breakpoints were identified by WGS in 74 pts (concordance in Table) At the time of data cutoff, dPCR assays were designed and evaluated in 8 t(11;14)+ pts; 7/8 (88%) and 6/8 (75%) pts had detectable t(11;14) in cfDNA or PBMCs, respectively Higher levels of t(11;14) mutant allele frequency (MAF) were observed in cfDNA compared with PBMCs After Ven-based treatment, t(11;14) MAF in cfDNA became undetectable in pts with a complete response In conclusion, this approach has the capability to reconstruct t(11;14) breakpoints from WGS data that is highly concordant with FISH; translocations appear more readily detectable in cfDNA than PBMC samples from pts with MM SAGAsign assays detected and monitored t(11;14) in liquid biopsies thus highlighting its potential utility for identifying pts with t(11;14) for targeted therapies Concordance between WGS and FISH testing for t(11;14) Samples, n FISH+ FISH− Total WGS+ 74 11 83 WGS− 16 169 187 Total 90 180 270 Concordance, % n/N 95% CI t(11;14) positive 82.2 74/90 72.7-89.5 t(11;14) negative 93.9 169/180 89.3-96.9 Overall testing 90.0 243/270 85.8-93.3 Citation Format: Xiaotong Li, Lao H. Saal, Christian Brueffer, Yilun Chen, Johanna Asklin, Saman Alvi, Srinivas Venkatram, Jeremy A. Ross. Detection and monitoring of t(11;14) in liquid biopsies from patients with relapsed/refractory multiple myeloma treated with venetoclax-based regimens. [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 5710.

Abstract 5714: Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer

Abstract BACKGROUND: Cell-free circulating tumor DNA (ctDNA) assays have been adopted to monitor therapeutic response in both early- and late-stage cancer. However, tests currently available require deep-targeted sequencing to detect cancer-specific mutations at low mutant allele frequency (MAF) levels in the circulation. Recently, we developed a tumor-agnostic approach called DELFI Tumor Fraction (DELFI-TF), a Bayesian probabilistic model designed to predict plasma tumor fractions based on genome-wide fragmentation-related features. METHODS: Overall, 692 longitudinal plasma samples collected from 153 patients with initially unresectable colorectal cancer (CRC) liver-only metastases participating in the phase III study CAIRO5 (NCT02162563) were split across a training (RAS/BRAF mutant) and a validation cohort (RAS/BRAF wildtype). For all cell-free DNA (cfDNA) samples of patients within the training cohort (n=312), the tumor burden was quantified as the MAF of the tumor-tissue-proven RAS/BRAF variant measured by digital droplet PCR (ddPCR). Using cfDNA fragment-sequencing statistics from low-coverage WGS data, a Bayesian regression model was trained against the MAF of the tumor-specific driver RAS/BRAF variant in all longitudinal timepoints in the training cohort. Changes in DELFI-TF scores during first-line FOLFOX/FOLFIRI and bevacizumab therapy were assessed to predict disease response. A tissue-informed approach was applied to detect minimal residual disease in the subgroup of patients treated with curative intent (complete resection of the primary tumor and liver metastases). RESULTS: In the training cohort, DELFI-TF scores strongly correlated with RAS/BRAF MAF measured by ddPCR (r=0.85, p<0.001). DELFI-TF captured plasma tumor fractions associated with copy number changes when ddPCR failed to detect RAS/BRAF mutations. Before treatment initiation, DELFI-TF correlated with the sum of the largest diameters on CT scans according to RECIST1.1 (r=0.49, p<0.001). Patients with negative and positive changes in DELFI-TF more often exhibited response and progressive disease based on consecutive RECIST assessments, respectively. Median progression-free survival was 9.9 and 12.8 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.007). Overall survival was 28.8 and 58.9 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.001). In patients treated with curative intent, tissue-informed focal and arm-level copy number changes were detected 4-12 weeks after metastasectomy. Most patients presenting molecular relapse were later diagnosed with clinical recurrence by conventional CT imaging. CONCLUSIONS: DELFI-TF demonstrates the ability to use cfDNA fragmentomes to estimate cfDNA tumor burden with a performance comparable to standard approaches for treatment response monitoring and clinical outcome prediction. Citation Format: Bahar Alipanahi, Iris van 't Erve, Keith Lumbard, Laurel K. Millberg, Zach Skidmore, Lorenzo Rinaldi, Jacob Carey, Jennifer Tom, Cornelis J. A. Punt, Nicholas C. Dracopoli, Gerrit A. Meijer, Robert B. Scharpf, Victor E. Velculescu, Remond Fijneman, Alessandro Leal. Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer. [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 5714.

Abstract 4354: An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples

Abstract Introduction: Hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) breast cancer is the most common type of breast cancer. Patients with HR+/HER2- metastatic breast cancer (mBC) often become resistant to aromatase inhibitors commonly used in endocrine therapy (ET). Estrogen receptor (ESR1) ligand binding domain mutations are frequently detected in HR+ mBC and have been reported to be associated with ET resistance, noting up to 60% patients with mBC will develop resistance to treatment via acquired resistance in ESR1. Recent studies have shown that monitoring of ESR1 mutations in plasma may serve as a predictive biomarker of acquired resistance to ET, showcasing a strong need for sensitive nucleic acid-based assays. We describe a comprehensive methodology for targeted clinical RT-qPCR monitoring of ESR1 mutations in plasma that utilizes both exosomal nucleic acids and cfDNA to report multiple mutations, provide a streamlined workflow, and accommodate a range of inputs from clinically relevant samples. Methods: Exosomal nucleic acids (exosomal DNA and RNA) and circulating cell-free DNA (cfDNA) were co-isolated using the ExoLution Plus Isolation Kit (Exosome Diagnostics). RT-qPCR-based target enrichment was performed using modified QuantideX reagents (Asuragen) and mutations were confirmed on the QuantStudio qPCR Platform (ThermoFisher). Bioinformatic analyses were conducted using custom software. Results: We developed novel technologies and tested greater than samples across key ET resistance mutations, utilizing contrived samples as needed due to availability. Preliminary studies demonstrated that ESR1 exosomal RNA + cfDNA ranged from approximately 9,500 copies to 64,000 copies in a set of 2mL female presumed normal plasma samples; these results suggest that even 2mL plasma could provide sufficient exosomal RNA/DNA + cfDNA for mutation detection at variant frequencies less than or equal to 0.01%. The ESR1 RT-qPCR showed greater than 90% analytical sensitivity (at less than or equal to 1% mutant allele frequency) and greater than90% analytical specificity. Further, the technology detected less than 10 mutant copies for most tested mutants, showing an LOD of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Conclusion: A fast, efficient, and sensitive ESR1 RT-qPCR panel approach was developed and evaluated, demonstrating the reliable and specific detection of rare variants in liquid biopsy specimens. Importantly, this prototype technology has potential to address several challenges associated with mutation monitoring in liquid biopsies by expanding detection of mutant analytes (exosomal RNA and DNA), improving analytical sensitivity (novel reagents and software), and increasing accessibility (qPCR instrument install base). Citation Format: Sarah Statt, Julie Thibert, Kurt Franzen, Aquiles Sanchez, Liangjing Chen, Megan Yociss, Elliot Hallmark, Melissa Church, Stela Filipovic-Sadic, Gary Latham, Johan Skog. An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples. [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 4354.

Abstract 782: Circulating tumour DNA-based molecular response predicts treatment outcome in multiple myeloma

Abstract Background: Multiple myeloma (MM) is an incurable and multi-focal plasma cell malignancy manifesting predominantly within the bone marrow (BM). BM-based minimal residual disease (MRD) assessment utilising next generation flow (NGF - EuroFlowTM) has become an important measure of treatment response and a validated predictor of patient outcome. However, MRD is a single-site BM-derived assay and is conceptually limited due to the spatially heterogenous nature of MM and response to treatment. Objective: We investigated if the evaluation of circulating tumour DNA (ctDNA) can be utilised as an adjunct to MRD analysis in predicting patient outcome. Methods: Peripheral blood plasma was obtained at baseline (B), cycle 3 day 1 (C3D1) and end of study (EOS)/relapse from a phase II, multicentre single arm study of carfilzomib-thalidomide-dexamethasone (KTd) in 50 transplant-eligible newly diagnosed MM patients who were refractory to or had demonstrated a suboptimal response to first-line therapy. Extracted ctDNA was assessed utilising high-sensitivity targeted amplicon sequencing (TAS) of 22 MM-relevant genes. The variant allele frequency (VAF) of mutations at B was utilised to calculate a fold change in VAF at C3D1 and/or EOS. A negative fold change was defined as a decrease in VAF (ctDNA-) and a positive fold change an increase in VAF (ctDNA+) with the average fold change across all mutations calculated for each patient. EuroflowTM was performed on BM obtained pre and post-autologous stem cell transplant (ASCT) and at EOS and the ‘best MRD response’ achieved (MRD+ or MRD-) was recorded. The ctDNA and MRD responses were combined and correlated with progression-free survival (PFS) calculated using Kaplan-Meier estimates. Results: A total of 45 patients had one or more mutations identified at B that could be tracked at C3D1 and/or EOS and 40 patients had one or more mutations that emerged at C3D1, EOS and/or relapse. Of these, 33 patients were evaluable for both MRD and ctDNA response. Patients that were MRD- and ctDNA- as best response for B mutations had a significantly longer PFS compared to those who were MRD+/ctDNA- or MRD-/ctDNA+ (p<0.0001, Log-rank tests; 23.9 months vs not reached for MRD-ctDNA-, Log rank-tests). Patients that were MRD- and ctDNA- at C3D1 (as early response for B mutations) had a significantly longer PFS compared to those that were MRD+/ctDNA- or MRD-/ctDNA+ (p=0.0011, 26.4 months vs not reached in MRD-ctDNA-, Log-rank tests). Conclusions: Our results demonstrate that treated MM patients manifesting a molecular response, as defined by a reduction in ctDNA mutational burden, and who are also MRD- have a superior outcome to those patients who are MRD- but without a molecular response. These results for the first time confirm the utility of ctDNA-based early molecular response as a predictor of patient outcome in MM. Citation Format: Sridurga Mithraprabhu, Tiffany Khong, Flora Yuen, John Reynolds, Brian Durie, Andrew Spencer. Circulating tumour DNA-based molecular response predicts treatment outcome in multiple myeloma [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 782.

Abstract 130: Distinct mutational drivers and evolutionary pathways in stage IA non-myoinvasive endometrioid endometrial cancer

Abstract Background: Endometrioid endometrial cancer (EC), the most common histologic type of EC, is underpinned by somatic genetic alterations of genes in the PI3K and MAPK pathways, and ARID1A. Here we sought to evaluate the genomic landscape of stage IA, low-volume, non-myometrial invasive ECs to define the earliest drivers and tumor evolution of endometrioid EC. Methods: Early-stage, non-myoinvasive EC of patients who underwent surgery at our institution and were subjected to clinical tumor-normal targeted sequencing of up to 505 cancer-related genes. Tumor volume was determined based on the gross and histologic measurements, and the tumors were dichotomized into low- and high- volume disease using a cutoff of 1 cm3. Genomic data was extracted and cancer cell fractions (CCF) of the somatic mutations were determined using ABSOLUTE integrating the mutational variant allele frequency and copy number alterations. Results: A total of 160 non-invasive, stage I ECs were identified, of which the majority (n=128; 80%) were FIGO grade 1 (15.6% grade 2, 3.1% grade 3). The median calculated volume of disease was 1.3 cm3, and 18 cases did not have gross disease with only microscopic disease identified on pathologic evaluation. The tumors on average had 6.7 somatic mutations affecting known EC cancer-related genes, including PTEN (n=127; 79%), ARID1A (n=84; 52.5%), PIK3CA (n=81; 50.6%), CTNNB1 (n=61; 38.1%), PIK3R1 (n=59; 36.8%) and KRAS (n=48; 30%). None of the alterations showed a significant correlation with tumor volume. Analysis of the CCF revealed that in 43% of ECs (69/160)[WB1] , PTEN mutations were clonal and had the highest CCF, indicating that PTEN mutation is most likely the initiating carcinogenic event. In other subsets of the early-stage ECs studied, the highest CCFs were mutations affecting PIK3CA/PIK3R1 (n=26), ARID1A (n=17), and KRAS (n=16). We also found that 109 (68%) ECs harbored activating mutations involving at least two genes in the PI3K/AKT/mTOR pathway, with the most common combination being PTEN/PIK3CA or PTEN/PIK3R1. AKT1 mutations occurred in absence of other PI3K/AKT/mTOR pathway alterations. Unsupervised Louvain clustering based on somatic mutations and gene copy number alterations revealed 5 distinct clusters of ECs with unique mutational drivers. Conclusion: Stage IA non-myoinvasive ECs show considerable genomic heterogeneity suggestive of multiple evolutionary pathways. Further studies are warranted to define whether this heterogeneity is a sign of early genomic drift (i.e. neutral tumor evolution) or whether the various evolutionary pathways all confer similar selective advantages. Citation Format: Sara Moufarrij, Carol Aghajanian, Nadeem Abu Rustum, Lora Ellenson, Britta Weigelt, Amir Momeni. Distinct mutational drivers and evolutionary pathways in stage IA non-myoinvasive endometrioid endometrial cancer [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 130.

Abstract 6612: Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid leukemia

Abstract The cancer hallmark concept defines biological properties that play a key role in cancer development and progression. The hallmarks reflect generic properties across all cancer types, and are not directly quantifiable nor applicable in cancer diagnostics. Here, we applied a data-driven approach to identify quantifiable hallmarks to be used as a basis for precision cancer medicine (PCM) in Acute Myeloid Leukemia (AML). We applied deep exome, transcriptome, DNA methylation and proteome profiling as well as ex-vivo functional testing of 525 drugs to 118 AML patient samples. Unsupervised multi-omic dimensionality reduction defined 11 independent axes of biological variability that we interpreted to reflect data-driven hallmarks (DDHM) of AML. Each DDHM integrates and ranks different data types and features, pinpointing those molecular features that were most informative for each hallmark. We calculated values for the 11 DDHM for each patient, constructing a DDHM-based precision medicine approach for AML diagnosis and therapy assignment. Most DDHMs were driven by other data types than genomics. For several DDHMs, different cytogenetic and mutational drivers converge on the same hallmarks and specific drug vulnerabilities. We also see how DDHM predictors of poor prognosis and high-risk AML are distinct from those that dictate specific drug response vulnerabilities. DDHM 2 reflected the cell differentiation path leading towards resistance to BH3 mimetics, ACK-inhibitors, and anthracyclines, and sensitivity to MEK inhibitors and TLR8 agonists. DDHM 2 derived a strong impact from protein expression of GATA2, MLLT11, DNMT3B and from RNA expression of WT1 and SOX4 as well as from multiple markers of different monocytic subtypes. DDHM 5 depicted cell cycle regulation and the Megakaryocytic-erythrocyte progenitor cell state that was enriched in AML patients with antecedent hematological disease. Interestingly, DDHM5 was linked to the sensitivity to purine analogs and vinca alkaloids. Moreover, DDHMs 1 and 8 captured clinical risk groups, prognosis, and responsiveness to hypomethylating agents. These DDHMs also revealed the phenotypic and functional biology that distinguished patients with high versus low FLT3-ITD mutant allele frequency. Validation of the DDHMs is realized through profiling and analysis of prospective AML samples, AML cell lines and previously published datasets. In summary, we present a data-driven approach for defining hallmarks in AML. The application of the DDHMs in AML provides a new paradigm for PCM and an opportunity for combinatorial therapeutic targeting. Each patient is characterized by a combination of independent and potentially druggable hallmarks, as opposed to traditional stratification in PCM, where each patient is assigned to one specific subgroup defined by genetic or other biomarkers. Citation Format: Tom Erkers, Nona Struyf, Tojo James, Francesco Marabita, Mattias Vesterlund, Nghia Vu, Cornelia Arnroth, Albin Österroos, Anna Bohlin, Sofia Bengtzén, Matthias Stahl, Rozbeh Jafari, Lukas Orre, Yudi Pawitan, Brinton Seashore-Ludlow, Janne Lehtiö, Sören Lehmann, Päivi Östling, Olli Kallioniemi. Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid leukemia. [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 6612.

Detection of BRAF V600E mutation in radiological Langerhans cell histiocytosis-associated neurodegenerative disease using droplet digital PCR analysis.

Langerhans cell histiocytosis-associated neurodegenerative disease (LCH-ND) is the most serious late complication secondary to LCH and is gradually progressive, destructive, and irreversible. Detection of the BRAF V600E mutation in peripheral blood mononuclear cells (PBMCs), even in the absence of active LCH lesions, is considered a sign of clinical LCH-ND, presenting with both abnormal imaging findings and neurological symptoms. However, the detection of the BRAF V600E mutation in PBMCs of patients with asymptomatic radiological LCH-ND (rLCH-ND) without active LCH lesions who present only with abnormal imaging findings is unknown. In this study, we analyzed the BRAF V600E mutations in PBMCs and cell-free DNA (cfDNA) of patients with rLCH-ND without active LCH lesions (n = 5) using a droplet digital polymerase chain reaction (ddPCR) assay. The BRAF V600E mutation in PBMCs was detected in three out of five (60%) cases. The mutant allele frequencies in the three positive cases were 0.049%, 0.027%, and 0.015%, respectively. However, the cfDNA BRAF V600E mutation remained undetected in all patients. Detection of the BRAF V600E mutant allele in PBMCs may be helpful in identifying asymptomatic rLCH-ND in patients at high risk for developing LCH-ND, including those with relapses at CNS risk sites or central diabetes insipidus.

PD46-03 UTILITY OF URINARY TUMOR DNA DETECTION IN TREATMENT-NAÏVE UTUC

You have accessJournal of UrologyCME1 Apr 2023PD46-03 UTILITY OF URINARY TUMOR DNA DETECTION IN TREATMENT-NA&IUML;VE UTUC Heather Huelster, Facundo Davaro, Kyle Rose, Billie Gould, Tiantian Zheng, Prithvi Murthy, Shreyas Naidu, Lucia Camperlengo, Pan Du, Shidong Jia, Aram Vosoughi, and Roger Li Heather HuelsterHeather Huelster More articles by this author , Facundo DavaroFacundo Davaro More articles by this author , Kyle RoseKyle Rose More articles by this author , Billie GouldBillie Gould More articles by this author , Tiantian ZhengTiantian Zheng More articles by this author , Prithvi MurthyPrithvi Murthy More articles by this author , Shreyas NaiduShreyas Naidu More articles by this author , Lucia CamperlengoLucia Camperlengo More articles by this author , Pan DuPan Du More articles by this author , Shidong JiaShidong Jia More articles by this author , Aram VosoughiAram Vosoughi More articles by this author , and Roger LiRoger Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003359.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urine-based molecular biomarkers correlate with tumor variants in urothelial carcinoma of the bladder, showing promise for noninvasive detection of newly diagnosed tumors. However, the utility of next-generation sequencing (NGS) to detect urinary tumor DNA (utDNA) in upper tract urothelial carcinoma (UTUC) remains to be explored. Herein, we prospectively sequenced tumors and utDNA at the time of and serially after radical nephroureterectomy (RNU) to examine the feasibility of using utDNA in detection and surveillance of UTUC. METHODS: Fourteen systemic therapy naïve patients with high-grade, clinically-localized UTUC undergoing RNU were prospectively accrued. Urine was collected preoperatively on the day of surgery and urinary supernatant and cell pellets (UCP) processed for extraction of utDNA. NGS used shallow whole-genome sequencing (WGS) to detect genome-wide copy number changes and a 152-pancancer panel (PredicineCARE) to profile single nucleotide variants, small insertions/deletions, and gene-level copy number changes. Variants with mutation allele frequency ≥0.25% in supernatant, ≥0.3% in UCP, and cancer hotspot variants >0.1% were reported. RESULTS: Prior to RNU, utDNA variants were detected in 11/11 (100%) of urinary supernatants and 9/14 (64%) of UCPs using NGS. The most common alterations detected in the urine were in TP53 (81%), TERT promoter (55%), FGFR3 (55%), and ERBB2 (45%). More tumor variants were detected in urine supernatant than UCP (22.9 vs 2.4, p=0.004). Use of a bespoke tumor-informed approach to adjust MAF thresholds for calling variants increased utDNA detection by a mean 0.5 (range 0-3) variants in urinary supernatant and mean 2.3 (range 0-7) variants in UCP. There was no significant difference in the number of variants detected in urine of patients with UTUC staged as <pT2 compared to ≥pT2 or pN+. CONCLUSIONS: UTUC tumor variants are detectable in the urine of treatment-naïve patients undergoing RNU for UTUC using both a panel-based approach and WGS. utDNA detection and dynamics may present an opportunity for personalized monitoring of UTUC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1170 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Heather Huelster More articles by this author Facundo Davaro More articles by this author Kyle Rose More articles by this author Billie Gould More articles by this author Tiantian Zheng More articles by this author Prithvi Murthy More articles by this author Shreyas Naidu More articles by this author Lucia Camperlengo More articles by this author Pan Du More articles by this author Shidong Jia More articles by this author Aram Vosoughi More articles by this author Roger Li More articles by this author Expand All Advertisement PDF downloadLoading ...

8MO Adagrasib (MRTX849) in patients with advanced/metastatic KRAS G12C-mutated non-small cell lung cancer (NSCLC): Preliminary analysis of mutation allele frequency

8MO Adagrasib (MRTX849) in patients with advanced/metastatic KRAS G12C-mutated non-small cell lung cancer (NSCLC): Preliminary analysis of mutation allele frequency

Development of Multiplex Drop-Off Digital PCR Assays for Hotspot Mutation Detection of KRAS, NRAS, BRAF, and PIK3CA in the Plasma of Colorectal Cancer Patients

The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. We assessed the analytical performance of the assays using synthetic oligonucleotides, which were further validated on plasma cfDNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cfDNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring.

Frequent PIK3CA mutation in normal endometrial gland drives spheroid formation and may be involved in stem cell propagation.

Recent studies reported the presence of oncogenic mutations in normal endometrial glands, but the biological significance remains unclear. The present study investigated the status of KRAS/PIK3CA driver mutations in normal endometrial glands as well as spheroids derived from single glands. The normal endometria of surgically removed uteri (n=3) were divided into nine regions, and 40 endometrial single glands were isolated from each region. The DNAs of 10 glands in each region were extracted and subjected to Sanger sequencing for KRAS or PIK3CA driver mutations, while the remaining 30 glands were conferred to a long-term spheroid culture, followed by Sanger sequencing. Immunohistochemical analyses of stem cell (Axin2, ALDH1A1, SOX9) markers were undertaken for spheroids. Sanger sequencing successfully detected oncogenic mutations of KRAS or PIK3CA in a single gland. Twenty-five of the 270 glands (9.3%) had mutations in either KRAS or PIK3CA, and the mutation frequency in each endometrial region varied from 0% to 50%. The droplet digital PCR showed high mutation allele frequency (MAF) of PIK3CA mutation, suggestive of clonal expansion of mutated cells within a gland. Over 60% of the collected spheroids had PIK3CA mutations, but no KRAS mutations were detected. Immunohistochemically, spheroids were mainly composed of cells with stem cell marker expressions. High MAF of PIK3CA mutation in a single gland as well as frequent PIK3CA mutation in stem cell-rich spheroids that originated from a single gland suggest the role of PIK3CA mutation in stem cell propagation. This information could improve our understanding of endometrial physiology as well as stem cell-oriented endometrial regeneration and carcinogenesis.

Development of Highly Sensitive Digital Droplet PCR for Detection of cKIT Mutations in Circulating Free DNA That Mediate Resistance to TKI Treatment for Gastrointestinal Stromal Tumor (GIST).

Mutations in cKIT or PDGFRA are found in up to 90% of patients with gastrointestinal stromal tumors (GISTs). Previously, we described the design, validation, and clinical performance of a digital droplet (dd)PCR assay panel for the detection of imatinib-sensitive cKIT and PDFGRA mutations in circulating tumor (ct)DNA. In this study, we developed and validated a set of ddPCR assays for the detection of cKIT mutations mediating resistance to cKIT kinase inhibitors in ctDNA. In addition, we cross-validated these assays using next generation sequencing (NGS). We designed and validated five new ddPCR assays to cover the most frequent cKIT mutations mediating imatinib resistance in GISTs. For the most abundant imatinib-resistance-mediating mutations in exon 17, a drop-off, probe-based assay was designed. Dilution series (of decreasing mutant (MUT) allele frequency spiked into wildtype DNA) were conducted to determine the limit of detection (LoD). Empty controls, single wildtype controls, and samples from healthy individuals were tested to assess specificity and limit of blank (LoB). For clinical validation, we measured cKIT mutations in three patients and validated results using NGS. Technical validation demonstrated good analytical sensitivity, with a LoD ranging between 0.006% and 0.16% and a LoB ranging from 2.5 to 6.7 MUT fragments/mL. When the ddPCR assays were applied to three patients, the abundance of ctDNA in serial plasma samples reflected the individual disease course, detected disease activity, and indicated resistance mutations before imaging indicated progression. Digital droplet PCR showed good correlation to NGS for individual mutations, with a higher sensitivity of detection. This set of ddPCR assays, together with our previous set of cKIT and PDGFRA mutations assays, allows for dynamic monitoring of cKIT and PDGFRA mutations during treatment. Together with NGS, the GIST ddPCR panel will complement imaging of GISTs for early response evaluation and early detection of relapse, and thus it might facilitate personalized decision-making.

Paired Comparison of Routine Molecular Screening of Patient Samples with Advanced Non-Small Cell Lung Cancer in Circulating Cell-Free DNA Using Three Targeted Assays.

Progressive advanced non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor targetable activating mutations, such as in-frame deletions in Exon 19 (Ex19del) of EGFR. Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of tyrosine kinase inhibitors. Plasma was collected from patients with NSCLC. We carried out targeted NGS using the Plasma-SeqSensei™ SOLID CANCER IVD kit on cfDNA (circulating free DNA). Clinical concordance for plasma detection of known oncogenic drivers was reported. In a subset of cases, validation was carried out using an orthogonal OncoBEAMTM EGFR V2 assay, as well as with our custom validated NGS assay. Somatic alterations were filtered, removing somatic mutations attributable to clonal hematopoiesis for our custom validated NGS assay. In the plasma samples, driver targetable mutations were studied, with a mutant allele frequency (MAF) ranging from 0.00% (negative detection) to 82.25%, using the targeted next-generation sequencing Plasma-SeqSensei™ SOLID CANCER IVD Kit. In comparison with the OncoBEAMTM EGFR V2 kit, the EGFR concordance is 89.16% (based on the common genomic regions). The sensitivity and specificity rates based on the genomic regions (EGFR exons 18, 19, 20, and 21) were 84.62% and 94.67%. Furthermore, the observed clinical genomic discordances were present in 25% of the samples: 5% in those linked to the lower of coverage of the OncoBEAMTM EGFR V2 kit, 7% in those induced by the sensitivity limit on the EGFR with the Plasma-SeqSensei™ SOLID CANCER IVD Kit, and 13% in the samples linked to the larger KRAS, PIK3CA, BRAF coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit. Most of these somatic alterations were cross validated in our orthogonal custom validated NGS assay, used in the routine management of patients. The concordance is 82.19% in the common genomic regions (EGFR exons 18, 19, 20, 21; KRAS exons 2, 3, 4; BRAF exons 11, 15; and PIK3CA exons 10, 21). The sensitivity and specificity rates were 89.38% and 76.12%, respectively. The 32% of genomic discordances were composed of 5% caused by the limit of coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit, 11% induced by the sensitivity limit of our custom validated NGS assay, and 16% linked to the additional oncodriver analysis, which is only covered by our custom validated NGS assay. The Plasma-SeqSensei™ SOLID CANCER IVD kit resulted in de novo detection of targetable oncogenic drivers and resistance alterations, with a high sensitivity and accuracy for low and high cfDNA inputs. Thus, this assay is a sensitive, robust, and accurate test.

Abstract P5-05-04: Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene or fulvestrant in the ELAINE 1 study

Abstract Introduction: Acquired ESR1 mutations (mutESR1) after long-term endocrine therapy drive treatment resistance, metastasis, and poor prognosis for patients (pts) with ER+/HER2- metastatic breast cancer (mBC). Lasofoxifene (LAS), a selective estrogen receptor modulator, alone or with a CDK4/6 inhibitor (CDK4/6i) reduced tumor growth better than fulvestrant (Fulv) in mutESR1 BC xenograft models. ELAINE 1 is a randomized trial of LAS vs Fulv in pts with mutESR1 and prior progression on aromatase inhibitor and CDK4/6i. Preliminary results (ESMO 2022) showed that LAS prolonged median progression-free survival (mPFS) compared with Fulv with a favorable safety profile. Here, we report changes in ESR1 ctDNA mutant allele frequency (MAF) from baseline to 8 wks and their associations with clinical benefit (CB) and mPFS. Methods: ELAINE 1 pts were randomized to oral LAS 5 mg daily or IM Fulv 500 mg on days 1, 15, and 29, then every 4 wks, until disease progression or severe toxicity. ctDNA mutESR1 mutations (baseline and 8 wks) were assessed using the Sysmex Inostics OncoBeam or SafeSeq assays—which detect mutESR1 at low allele fractions. MAF changes from baseline to wk 8 were characterized as decreased (decrease in ESR1 MAF or fully cleared), increased (increase in MAF), or equivocal (polyclonal patients [&amp;gt;1 mutESR1] with some increasing and decreasing MAF trends); correlations with PFS and CB were explored. Efficacy measures included objective response rate (ORR), PFS, and CB at 24 wks (CB defined as response or stable disease ≥24 wks). Results: 103 pts received LAS (n=52) or Fulv (n=51). Most common baseline ESR1 variants detected were D538G (56%), Y537S (39%), Y537N (29%), E380Q (22%); 56 (54%) pts were polyclonal. Of the 61 pts with evaluable baseline and wk 8 ctDNA, LAS decreased mutESR1 MAF in 29/35 pts (83% [11 complete clearance]) while Fulv decreased mutESR1 MAF in 16/26 pts (61.5% [6 complete clearance]) (Table). mPFS with LAS was 8 and 4 mos for pts with decreased/cleared MAF and increased MAF, respectively, and with Fulv was 4.5 and 2.8 mos, respectively (Table). LAS decreased the common mutESR1 variants more frequently than Fulv (median relative change -87.1% vs -14.7%). In pts with decreased MAF, CB was observed in 16/29 LAS pts (55%) and 4/16 Fulv pts (25%). The predictiveness of ESR1 MAF clearance for CB was also explored. Of 11 pts with ESR1 MAF clearance taking LAS, 10 achieved CB, yielding a positive predictive value (PPV) of 90.9%. In contrast, 2/6 pts with ESR1 MAF clearance taking Fulv had CB for a PPV of 33.3%. Sensitivity for predicting CB based on direction of ESR1 MAF change was 94% with LAS and 80% with Fulv. In pts with Y537S MAF (n=33), LAS decreased Y537S in 13/15 (87%), with a median relative MAF decrease of 89%. In marked contrast, Fulv increased Y537S MAF in 11/18 pts (61%), corresponding to an MAF relative increase of 82%. LAS and Fulv resulted in complete clearance of Y537S MAF in 33% and 6% of pts, respectively. Conclusion: Our data demonstrate that LAS more effectively decreased or cleared mutESR1 than Fulv. Further, mutESR1 clearance was associated with prolonged PFS and more CB in LAS but not Fulv pts, suggesting that LAS results in robust mutESR1 target engagement. Taken together, our data suggest mutESR1 as a potential liquid biomarker for predicting response to LAS in mutESR1, endocrine-resistant mBC pts. Table. Change from baseline to week 8 in ESR1 MAF and clinical benefit at 24 weeks. CI, confidence interval; MAF, mutant allele fraction; ND, none detected; PFS, progression-free survival. Citation Format: Matthew P. Goetz, Einav Gal-Yam, Daniel Stover, Sarah L. Sammons, Stephanie L. Graff, Grace Wang, Massimo Cristofanilli, Gary Riordan, Hillary S. Sloane, Dominic Carroll, Paul V. Plourde, David J Portman. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene or fulvestrant in the ELAINE 1 study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-04.

Abstract P5-05-02: Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene plus abemaciclib in the ELAINE 2 study

Abstract Introduction: Use of long-term endocrine therapy (ET) for ER+ breast cancer often leads to acquired ESR1 mutations (mutESR1), causing endocrine resistance, tumor progression, and poor prognosis. An unmet clinical need exists for treating ER+ mBC patients with mutESR1, particularly after progression on CDK4/6 inhibitors (CDK4/6i). ELAINE 2 is an open-label, phase 2, multicenter trial evaluating safety and efficacy of lasofoxifene (LAS [selective estrogen receptor modulator]) plus abemaciclib (Abema [CDK4/6i], provided by Eli Lilly) in patients with ER+/HER2- and mutESR1 mBC who progressed after prior ET. Preliminary data with LAS plus Abema showed median progression-free survival of 55.7 wks, objective response rate of 50%, and 24-wk clinical benefit (CB) rate of 69%, with an acceptable safety and tolerability profile. Here, we report ESR1 ctDNA mutant allele frequency (MAF) and correlations of ESR1 MAF changes with CB. Methods: ELAINE 2 patients with detectable ctDNA mutESR1 at baseline (BL) were analyzed. Oral LAS 5 mg/day and Abema 150 mg BID were taken until disease progression, death, unacceptable toxicity, or withdrawal from the study. ctDNA was assessed by the Sysmex-Inostics SafeSeq assay—which detects mutESR1 at low allele fractions—at BL, every 4 wks, and end of treatment. MAF changes from BL to wk 4 were characterized as decreased (decrease in ESR1 MAF or none detected [ND]), increased (increase in MAF), or equivocal (in polyclonal patients [&amp;gt;1 mutESR1] with some MAF increasing and decreasing trends). Correlations of MAF change at 4 wks with CB at 24 wks were explored. Results: 29 patients (median of 2 prior metastatic therapies: 97% CDK4/6i, 79% fulvestrant, 48% chemotherapy) had BL mutESR1 of Y537S (66%), D538G (45%), Y537N (28%), and other less frequently detected mutations; 14 (48.3%) patients were polyclonal. 26 of 29 patients had evaluable BL and wk-4 ctDNA results: 21 patients had decreased MAF (81% [54% with ND]), 3 (12%) had increased, and 2 (8%) had equivocal ESR1 MAF changes (Table). CB at 24 wks was seen in 17 patients with a decrease, 2 with an increase, and 1 with equivocal MAF change. A sensitivity of 89.5% and specificity of 20% were calculated for predicting CB based on direction of ESR1 MAF change. The positive predictive value (PPV) for CB with decreased MAF was 81% and the negative predictive value (NPV) for an increased MAF was 33%. Of the 14 (54%) patients with ND ESR1 MAF, 13 had CB resulting in 87% sensitivity, 50% specificity, 93% PPV, and 33% NPV. Conclusion: In ELAINE 2, 81% of patients had decrease/cleared (ND) mutESR1 after 4 wks of LAS plus Abema, which correlated with clinical benefit. All mutESR1 detected appear targeted with this therapy. High sensitivity and favorable PPV were observed in patients with decreased MAF, and even more so in those with ND MAF; however, increased MAF was less specific and not as predictive of treatment failure. Our results indicate that ESR1 liquid biopsy evaluation may be an adequate non-invasive surrogate marker for monitoring patients on treatment. Further study in a larger population of women with endocrine-resistant mBC and acquired mutESR1 is warranted to explore this potential for monitoring treatment response or resistance to this novel LAS-Abema combination. Table. Change from baseline to week 4 in ESR1 MAF and clinical benefit at 24 weeks. CI, confidence interval; MAF, mutant allele fraction; ND, none detected; NPV, negative predictive value; PPV, positive predictive value. *Sensitivity and specificity analyses do not include equivocal results. Citation Format: Senthil Damodaran, Halle Moore, Ciara C. O’Sullivan, Paul V. Plourde, Gary Riordan, Hillary S. Sloane, Debu Tripathy, Dominic Carroll, David J Portman. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene plus abemaciclib in the ELAINE 2 study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-02.

Abstract P2-26-06: Association between CK+/CD45+ circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) alterations in advanced breast cancer patients

Abstract Background: Circulating tumor cells (CTCs) in breast cancer (BC) are commonly defined as epithelial cells (EPCAM and cytokeratin (CK) positive), lacking the universal blood cell marker CD45. Nonetheless, CTCs expressing both CK and CD45 (= dual-positive, DP cells) can be observed in the blood of cancer patients. Early evidence suggests that DP cells might derive from the fusion of tumor cells and macrophages, and we have previously demonstrated that they present aberrant genomes and are associated with worse prognosis in BC [1,2]. Here, to further investigate the mechanisms/pathways underlying their presence, we analyzed the association between DP cells and circulating tumor DNA (ctDNA) alterations. Methods: Blood samples were collected from patients with advanced BC (aBC), before starting a new line of therapy. All patients were enrolled in a prospective clinical trial. For CTC and DP cells analysis, 7.5 ml of blood collected in CellSave® tubes was processed with the FDA-approved CellSearch® platform (positivity cutoffs were ≥1 cell for DPcells and ≥5 cells for CTCs). For ctDNA analysis, plasma was collected from Streck stabilizing tubes and analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs), insertions/deletions (indels), gene fusions/rearrangements and copy number variations (CNVs), which were then classified into pathways based on previously defined profiles generated on the Cancer Genome Atlas database (RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Associations between ctDNA-detected gene alterations and circulating cell types were analyzed through chi square test, while mutant allele frequency (MAF) and number of detected alterations (NDA) were tested by Mann Whitney test. Results: We analyzed blood samples from 169 patients with luminal-like (n=80), HER2+ (n=34) and triple-negative (n=52) aBC. DPcells were detected in 85 patients (50.3 %, range 0-53), of which 40 (47 %) were CTC-positive and 45 (53%) CTC-negative. Somatic ctDNA alterations were detected in all analyzed samples. In the overall population, the presence of ≥1 DPcell was associated with SNVs in the cell cycle pathway (p = 0.043), a numerically higher incidence was also observed for CNVs in this pathway. SNVs and CNVs in the cell cycle pathway were associated with CTCs ≥ 5 as well (p = 0.005 and p = 0.003, respectively). Moreover, associations with CTCs ≥ 5 were observed for RTK SNVs and CNVs (p = 0.041 and p = 0.046, respectively), PI3K SNVs and CNVs (p = 0.006 and p = 0.007, respectively), MYC SNVs and CNVs (p = 0.042). No associations were observed in terms of MAF and NDA. In the luminal-like subgroup an association was highlighted for CNVs in the cell cycle pathway, p = 0.038. CTCs ≥ 5 were associated with PI3K SNVs (p = 0.031). In the triple-negative subgroup DPcells were associated with SNVs in the RAF pathway (p = 0.041), whereas CTCs ≥ 5 were associated with PI3K SNVs and CNVs (p = 0.044 and p = 0.024, respectively) and RTK SNVs (p = 0.008). In the HER2 positive subgroup, a higher MAF and number of detected SNVs was observed for samples with ≥1 DPcell (p = 0.0286 and p = 0.0099, respectively). Conclusions: The study analyzed ctDNA features associated with canonical and CK+/CD45+ CTCs, showing differential gene alteration profiles. Cell cycle pathway SNVs were common in both CTC populations, while other pathways (RTK, PI3K, MYC and RAF) were significantly altered in a mutually exclusive pattern. These results suggest that DPcells might have a different biological meaning compared to canonical CTCs. More studies need to be conducted to better characterize this understudied CTC subpopulation and understand their specific contribution to cancer progression.

Abstract OT1-11-01: Phase II study of talazoparib, a PARP inhibitor, in somatic BRCA1/2 mutant metastatic breast cancer identified by cell-free DNA or tumor tissue genotyping

Abstract Background: PARP inhibitors are currently approved for the treatment of germline BRCA1/2 mutant metastatic breast cancer (MBC), which accounts for 5-10% of breast cancer. We hypothesize that a PARP inhibitor may also have efficacy in somatic BRCA1/2 mutant MBC, expanding the potential clinical applicability of PARP inhibitors. We previously demonstrated that somatic BRCA1/2 mutations can be identified by both cell-free DNA and tumor tissue genotyping in a subset of patients with MBC who are not germline BRCA1/2 carriers. Furthermore, a PARP inhibitor was demonstrated to induce cell growth inhibition in a circulating tumor cell culture model generated from a patient with pathogenic somatic BRCA1 mutant MBC (Vidula, Dubash, CCR, 2020). In this trial, we are evaluating the efficacy of a PARP inhibitor in somatic BRCA1/2 mutant MBC. Trial Design: This phase II investigator-initiated clinical trial is enrolling 30 patients with somatic BRCA1/2 mutant MBC identified via cell-free DNA or tumor tissue genotyping. Patients are treated with talazoparib, a PARP inhibitor, until disease progression. At baseline and every 3 months, patients undergo CT chest, abdomen, and pelvis, and a bone scan for disease assessment. Patients undergo blood collection at baseline for the Cancer Risk B (CR-B) assay, a novel flow variant assay to assess double-strand break repair mutations in circulating blood cells (Syeda, 2017) and monthly blood collection for cell-free DNA analysis to evaluate changes in the genomic environment. Eligibility Criteria: Patients with MBC with a pathogenic somatic BRCA1/2 mutation identified by cell-free DNA or tumor tissue genotyping are eligible. Both patients with triple-negative breast cancer (≥ 1 prior chemotherapy) or hormone receptor positive/HER2- MBC (≥ 1 prior hormone therapy) are eligible. Patients should not be known germline BRCA1/2 carriers. There is no limit on prior therapies including receipt of a prior platinum (in the absence of disease progression on prior platinum). A prior PARP inhibitor is not allowed. Adequate performance status and organ function are needed. Specific Aims: Primary aim is progression-free survival (PFS) assessed by RECIST 1.1. Secondary aims include objective response rate and toxicity assessed by NCI CTCAE v 5.0. Exploratory aims include assessing impact of BRCA1/2 reversion mutations in cell-free DNA, studying serial changes in BRCA1/2 mutant allelic frequency in cell-free DNA, comparing pre- and post-treatment cell-free DNA results to identify changes in the genomic environment, assessing the CR-B assay positivity rate, and correlating these biomarker analyses with patient response. Statistical Methods: This study uses a two-stage design with 80% power to demonstrate that talazoparib is associated with “success” (PFS &amp;gt; 12 weeks) in 53% patients (4% alpha). Accrual: This study (NCT03990896) is open at Massachusetts General Hospital, MD Anderson, University of California San Francisco, and Emory, with pending activation at Northwestern, Cornell, and Vanderbilt. Five patients are enrolled as of 7/2022. Funding: This study is supported by a Pfizer ASPIRE award and Conquer Cancer Foundation of ASCO–Breast Cancer Research Foundation- Career Development Award. Contact information: Neelima Vidula, MD, Massachusetts General Hospital, nvidula@mgh.harvard.edu Citation Format: Neelima Vidula, Senthil Damodaran, Erica L. Blouch, Nora Horick, Nathan Royce Ruffle-Deignan, Manali Bhave, Ami N. Shah, Leticia Varella, Vandana Abramson, Joseph Sparano, Leif Ellisen, Ishraq Alim, Harry Ostrer, Hope Rugo, Aditya Bardia. Phase II study of talazoparib, a PARP inhibitor, in somatic BRCA1/2 mutant metastatic breast cancer identified by cell-free DNA or tumor tissue genotyping [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-11-01.

Abstract P2-03-14: Tumor-agnostic measurement of ctDNA levels in first line ER+, HER2- metastatic breast cancer patients as a marker for survival

Abstract INTRODUCTION: Circulating tumor cell (CTC) count and levels of circulating tumor DNA (ctDNA) have proven their individual prognostic value in metastatic breast cancer (MBC). Whereas CTCs can be quantified by enumeration, levels of ctDNA are often expressed as the variant allele frequency of the dominant mutation. The modified fast aneuploidy screening test-sequencing system (mFAST-SeqS) estimates ctDNA-load by measuring chromosomal aneuploidy instead of mutations. The mFAST-SeqS method is cheap and provides a comprehensive overview of the tumor genome without prior knowledge on genomic tumor aberrations. Previously, we showed in a small MBC cohort (n=50), heterogeneous with regard to subtype and line of treatment, that the mFAST-SeqS-derived genome-wide aneuploidy score (GWA-score) was an independent prognostic biomarker (1). Our current aim was to provide evidence for the prognostic value of GWA-score in a homogeneous cohort of estrogen receptor positive (ER+), HER2 negative (HER2-) MBC patients, starting first line treatment with an aromatase inhibitor. MATERIALS &amp; METHODS: CTCs were enumerated using the CellSearch-system (Menarini) and screened for ctDNA fraction using mFAST-SeqS in ER+, HER2- MBC patients, starting with first line aromatase inhibitors. CTC-count was divided into high (CTChigh) and low (CTClow) using the validated cut-off of 5 CTCs/7.5 ml blood; similarly GWA-score was divided in high (GWAhigh)and low (GWAlow) based on the previously described cut-off of 5 (2). PFS and OS were estimated using Kaplan-Meier. Cox regression was used for uni- and multivariable analysis. RESULTS: One hundred and thirty-four patients were included in this analysis. Mean age was 68 (range 40-90). Median follow up was 26 months. Sixty-five samples had both scores low and 30 both high, 25 samples were CTChigh/GWAlow and 14 were CTClow/GWAhigh. PFS and OS were not significantly different between CTChigh versus CTClow groups (p=0.07 and p=0.26, median PFS 15 vs 24 months, median OS 45 vs 49 months). However, PFS and OS were significantly different between GWAhigh versus GWAlow groups (p &amp;lt; 0.01 for both, median PFS 11 vs 24 months, median OS 31 vs 51 months). In multivariate analysis, GWA-score was a significant prognostic factor for both PFS and OS while presence of visceral disease was also prognostic for OS (table 1). CONCLUSIONS: In patients treated with aromatase inhibitors in the first line of treatment, GWA-score was an independent prognostic marker for a worse PFS and OS and may have added value beyond CTC-count. 1. Verschoor, N., et al., Abstract P2-01-17: Circulating tumor cell count and levels of circulating tumor DNA are complementary prognostic biomarkers in metastatic breast cancer - A pilot study. Cancer Research, 2022. 82(4_Supplement): p. P2-01-17-P2-01-17. 2. Belic, J., et al., mFast-SeqS as a Monitoring and Pre-screening Tool for Tumor-Specific Aneuploidy in Plasma DNA. Adv Exp Med Biol, 2016. 924: p. 147-155. Table 1: Uni- and multivariate Cox regression for PFS and OS Citation Format: Noortje Verschoor, Jaco Kraan, Vanja de Weerd, Ngoc M Van, Agnes Jager, Stefan Sleijfer, John WM Martens, Saskia M Wilting. Tumor-agnostic measurement of ctDNA levels in first line ER+, HER2- metastatic breast cancer patients as a marker for survival [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-14.