Circulating Tumor Cells Prediction in Hormone Receptor Positive HER2-Negative Advanced Breast Cancer: A Retrospective Analysis of the MONARCH 2 Trial.

BACKGROUND: The MONARCH 2 trial (NCT02107703) showed the efficacy of abemaciclib, a cyclin-dependent kinase 4 & 6 inhibitor (CDK4/6i), in combination with fulvestrant for hormone receptor-positive, HER2-negative metastatic breast cancer (MBC). The aim of this analysis was to explore the prediction of circulating tumor cells (CTCs) stratification using machine learning for hypothesis generation of biomarker-driven clinical trials. PATIENTS AND METHODS: Predicted CTCs were computed in the MONARCH 2 trial through a K nearest neighbor (KNN) classifier trained on a dataset comprising 2436 patients with MBC. Patients were categorized into predicted Stage IVaggressive (pStage IVaggressive, ≥5 predicted CTCs) or predicted Stage IVindolent (pStage IVindolent, <5 predicted CTCs). Prognosis was tested in terms of progression-free-survival (PFS) and overall survival (OS) through Cox regression. RESULTS: Patients classified as predicted pStage IVaggressive and predicted pStage Stage IVindolent were, respectively, 183 (28%) and 461 (72%). After multivariable Cox regression, predicted CTCs were confirmed as independently associated with prognosis in terms of OS, together with ECOG performance status, liver involvement, bone-only disease, and treatment arm. Patients in the pStage Stage IVindolent subgroup treated with abemaciclib experienced the best prognosis both in terms of PFS and OS. The treatment effect of abemaciclib on OS was then explored through subgroup analysis, showing a consistent benefit across all subgroups. CONCLUSION: This study is the first analysis of CTCs modeling for stage IV disease stratification. These results show the need to expand biomarker profiling in combination with CTCs stratification for improved biomarker-driven drug development.

Interplay between ESR1/PIK3CA codon variants, oncogenic pathway alterations and clinical phenotype in patients with metastatic breast cancer (MBC): comprehensive circulating tumor DNA (ctDNA) analysis.

BACKGROUND: although being central for the biology and druggability of hormone-receptor positive, HER2 negative metastatic breast cancer (MBC), ESR1 and PIK3CA mutations are simplistically dichotomized as mutated or wild type in current clinical practice. METHODS: The study analyzed a multi-institutional cohort comprising 703 patients with luminal-like MBC characterized for circulating tumor DNA through next generation sequencing (NGS). Pathway classification was defined based on previous work (i.e., RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Single nucleotide variations (SNVs) were annotated for their oncogenicity through OncoKB. Only pathogenic variants were included in the models. Associations among clinical characteristics, pathway classification, and ESR1/PIK3CA codon variants were explored. RESULTS: The results showed a differential pattern of associations for ESR1 and PIK3CA codon variants in terms of co-occurring pathway alterations patterns of metastatic dissemination, and prognosis. ESR1 537 was associated with SNVs in the ER and RAF pathways, CNVs in the MYC pathway and bone metastases, while ESR1 538 with SNVs in the cell cycle pathway and liver metastases. PIK3CA 1047 and 542 were associated with CNVs in the PI3K pathway and with bone metastases. CONCLUSIONS: The study demonstrated how ESR1 and PIK3CA codon variants, together with alterations in specific oncogenic pathways, can differentially impact the biology and clinical phenotype of luminal-like MBC. As novel endocrine therapy agents such as selective estrogen receptor degraders (SERDS) and PI3K inhibitors are being developed, these results highlight the pivotal role of ctDNA NGS to describe tumor evolution and optimize clinical decision making.

Trajectories of participation in daily life among individuals newly diagnosed with cancer: A 5-month longitudinal study.

PURPOSE: To determine how participation in daily life is impacted during the first six months following a new cancer diagnosis and to identify risk factors for participation restrictions. Patient-reported outcomes (PROs) were used to suggest referrals to rehabilitation services. METHODS: Participants (n = 123) were adults (> 18 years) with the newly diagnosed primary brain, breast, colorectal, or lung cancer. PROs were collected at baseline (within 30 days of diagnosis/treatment initiation), two and five months post baseline. Daily life participation was assessed through the community participation indicators (CPI) (score range: 0-1) and patient-reported outcome measurement information system (PROMIS) ability to participate, (score range: 20-80; mean: 50, SD: 10). PROMIS-43 profile was also completed. Linear mixed-effect models with random intercept evaluated change in participation over time. RESULTS: The baseline total sample mean CPI score was 0.56; patients reported mildly impaired participation based on PROMIS scores (baseline: 46.19, 2-month follow-up: 44.81, 5 months: 44.84). However, no statistically significant changes in participation were observed over the study period. Risk factors for lower participation included receiving chemotherapy, lower physical function, higher anxiety and fatigue, and reduction in employment, p < 0.05. PROs indicated that roughly half of the participants may benefit from physical or occupational therapy or mental health support, but only 20-36% were referred by their medical team. CONCLUSION: People newly diagnosed with cancer experience impaired participation, but they are infrequently referred to supportive services such as rehabilitation. The use of PROs to assess participation, physical function, and mental health can promote access to supportive care services by identifying patients who may benefit from rehabilitation beyond those identified through routine clinical care.

Modeling the Prognostic Impact of Circulating Tumor Cells Enumeration in Metastatic Breast Cancer for Clinical Trial Design Simulation.

Despite the strong prognostic stratification of circulating tumor cells (CTCs) enumeration in metastatic breast cancer (MBC), current clinical trials usually do not include a baseline CTCs in their design. This study aimed to generate a classifier for CTCs prognostic simulation in existing datasets for hypothesis generation in patients with MBC. A K-nearest neighbor machine learning algorithm was trained on a pooled dataset comprising 2436 individual MBC patients from the European Pooled Analysis Consortium and the MD Anderson Cancer Center to identify patients likely to have CTCs ≥ 5/7 mL blood (StageIVaggressive vs StageIVindolent). The model had a 65.1% accuracy and its prognostic impact resulted in a hazard ratio (HR) of 1.89 (Simulatedaggressive vs SimulatedindolentP < .001), similar to patients with actual CTCs enumeration (HR 2.76; P < .001). The classifier's performance was then tested on an independent retrospective database comprising 446 consecutive hormone receptor (HR)-positive HER2-negative MBC patients. The model further stratified clinical subgroups usually considered prognostically homogeneous such as patients with bone-only or liver metastases. Bone-only disease classified as Simulatedaggressive had a significantly worse overall survival (OS; P < .0001), while patients with liver metastases classified as Simulatedindolent had a significantly better prognosis (P < .0001). Consistent results were observed for patients who had undergone CTCs enumeration in the pooled population. The differential prognostic impact of endocrine- (ET) and chemotherapy (CT) was explored across the simulated subgroups. No significant differences were observed between ET and CT in the overall population, both in terms of progression-free survival (PFS) and OS. In contrast, a statistically significant difference, favoring CT over ET was observed among Simulatedaggressive patients (HR: 0.62; P = .030 and HR: 0.60; P = .037, respectively, for PFS and OS).

Association of Financial Conflicts of Interest With Academic Productivity Among Junior Faculty in Hematology and Oncology

INTRODUCTION: Financial conflicts of interest (COIs) represent a common and complex issue in hematology and oncology. However, little is known about the timing of when COIs begin to develop during a career trajectory. We evaluated self-reported COIs for junior faculty members at top cancer centers to determine how these financial relationships correlated with measures of academic career productivity. METHODS: We analyzed data from 230 assistant professors at 10 academic cancer centers. Financial COIs were identified from the CMS Open Payments (Sunshine Act dollars) database. Self-reported COIs were obtained from American Society of Clinical Oncology (ASCO) and American Society of Hematology (ASH) disclosures, and from disclosures in recent publications. Number of publications and h-index (defined as the largest number of publications [h] such that h publications each have at least h citations) were used as measures of academic productivity. Scatter plots and Spearman correlation coefficients were used to assess the relationship between COIs or Sunshine Act dollars with number of publications and h-index. Linear regression modeling was used to analyze the relationships between COIs or Sunshine Act dollars with number of publications and h-index, adjusting for years of experience since completing fellowship (YSF). RESULTS: A total of 46% of junior faculty had at least 1 COI. Number of COIs reported to ASCO/ASH was positively correlated with total Sunshine Act dollars (Spearman correlation, 0.53; P <.01). The number of COIs and the number of Sunshine Act dollars increased with years in practice (Spearman correlation, 0.38 and 0.25, respectively; P <.01 for both). COIs and Sunshine Act dollars correlated with h-index (Spearman correlation, 0.41 and 0.37, respectively; both P <.01). After adjusting for YSF, linear regression demonstrated that log-transformed h-index and number of publications were associated with Sunshine Act dollars (both P <.01) and COIs (ASCO/ASH) (both P = .01). CONCLUSIONS: Financial COIs increased with number of YSF. Measures of academic productivity were positively correlated with COIs (ASCO/ASH) and Sunshine Act dollars. These data suggest that the cultivation of industry relationships is associated with the early academic productivity of junior faculty.

Circulating tumor cells, circulating tumor DNA, and disease characteristics in young women with metastatic breast cancer.

BACKGROUND: Clinical and genomic data from patients with early-stage breast cancer suggest more aggressive disease in premenopausal women. However, the association between age, disease course, and molecular profile from liquid biopsy in metastatic breast cancer (MBC) is not well characterized. METHODS: Patients were classified as premenopausal (< 45 years), perimenopausal (45-55 years), or postmenopausal (> 55 years). Cohort 1 consisted of patients with MBC who consented for prospective serial evaluation of circulating tumor cells (CTCs) using CellSearch™. Cohort 2 included patients who, as part of routine care, had circulating tumor DNA (ctDNA) sequenced by the Guardant360™ assay. Clinicopathologic data were collected from retrospective review to compare disease features between premenopausal and postmenopausal women. RESULTS: Premenopausal women represented 26% of 138 patients in Cohort 1 and 21% of 253 patients in Cohort 2. In Cohort 1, younger patients had a shorter time to metastases and a higher prevalence of lung and brain metastases. Overall, there were similar rates of ≥ 5 CTCs/7.5 mL, HER2 + CTC expression, and CTC clusters between pre- and postmenopausal women. However, for those with triple negative breast cancer, premenopausal women had a higher proportion of ≥ 5 CTCs/7.5 mL. In Cohort 2, premenopausal women had a higher incidence of FGFR1 (OR 2.75, p = 0.022) and CCND2 (OR 6.91, p = 0.024) alterations. There was no difference in the ctDNA mutant allele frequency or the number of detected alterations between these age groups. CONCLUSIONS: Our data reveal that premenopausal women diagnosed with MBC have unique clinical, pathologic, and molecular features when compared to their postmenopausal counterparts. Our results highlight FGFR1 inhibitors as potential therapeutics of particular interest among premenopausal women.

Cell-free DNA comparative analysis of the genomic landscape of first-line hormone receptor-positive metastatic breast cancer from the US and China.

PURPOSE: Meaningful comparison of mutational landscapes across ethnic groups requires the use of standardized platform technology. We have used a harmonized NGS-based liquid biopsy assay to explore the differential genomic landscape of patients with initially hormone receptor-positive (HR+), HER2-negative MBC of first line metastasis or primary Stage IV at diagnosis from the United States (US) and China (CN). METHODS: Plasma circulating tumor DNA (ctDNA) from 27 US patients and 65 CN patients was sequenced using the harmonized CLIA-certified, 152-gene PredicineCare™ liquid biopsy assay. Kaplan-Meier survival analysis was performed to analyze the correlation between genomic alterations and progression-free survival (PFS), and p-values were calculated using the log-rank test. RESULTS: All patients in the CN cohort received chemotherapy and/or hormonal therapy, while 85.2% (23/27) patients in the US cohort received hormonal therapy plus CDK4/6 inhibitors. Mutations were detected in 23 of 27 (85%) US patients and 54 of 65 (83%) CN patients. The prevalence of AKT1 (P = 0.008) and CDH1 (P = 0.021) alterations were both higher in the US vs. CN cohort. In addition, FGFR1 amplification were more frequent in the CN vs. US cohort (P = 0.048). PTEN deletions (P = 0.03) and ESR1 alterations (P = 0.02) were associated with shorter PFS in the CN cohort, neither of these associations were observed in the US cohort. Interestingly, a reduced association between PTEN deletion and PFS was observed in patients receiving CDK4/6 inhibitor treatment. CONCLUSION: The differential prevalence of ctDNA-based alterations such as FGFR1, AKT1, and CDH1 was observed in initially HR+/HER2- MBC patients in the US vs. CN. In addition, the association of PTEN deletions with shorter PFS was found in the CN but not the US cohort. The differential genomic landscapes across the two ethnic groups may reflect biologic differences and clinical implications.

Novel Neoadjuvant Treatment Strategies for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), which accounts for approximately 10% to 15% of breast cancers, remains the most aggressive subtype and is characterized by early disease relapse for a subset of patients. TNBC remains a clinical challenge, given the lack of effective targeted treatments such as endocrine therapy for hormone receptor-positive (HR+) tumors or therapies against HER2.

Association of a novel circulating tumor DNA next-generating sequencing platform with circulating tumor cells (CTCs) and CTC clusters in metastatic breast cancer.

PURPOSE: Liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), can be used to understand disease prognosis, tumor heterogeneity, and dynamic response to treatment in metastatic breast cancer (MBC). We explored a novel, 180-gene ctDNA panel and the association of this platform with CTCs and CTC clusters. METHODS: A total of 40 samples from 22 patients with MBC were included in the study. For the primary analysis, all patients had ctDNA sequencing using the PredicinePLUS™ platform. CTCs and CTC clusters were examined using the CellSearch™ System. Clinical and pathological variables were reported using descriptive analyses. Associations between CTC count and specific genomic alterations were tested using the Mann-Whitney U test. RESULTS: Of 43 sequenced patients, 40 (93%) had at least one detectable genomic alteration with a median of 6 (range 1-22). Fifty-seven different genes were altered, and the landscape of genomic alterations was representative of MBC, including the commonly encountered alterations TP53, PTEN, PIK3CA, ATM, BRCA1, CCND1, ESR1, and MYC. In patients with predominantly hormone-receptor-positive MBC, the number of CTCs was significantly associated with alterations in ESR1 (P < 0.005), GATA3 (P < 0.05), CDH1 (P < 0.0005), and CCND1 (P < 0.05) (Mann-Whitney U test). Thirty-six percent of patients had CTC clusters, which were associated with alterations in CDH1, CCND1, and BRCA1 (all P < 0.05, Mann-Whitney U test). In an independent validation cohort, CTC enumeration confirmed significant associations with ESR1 and GATA3, while CTC clusters were significantly associated with CDH1. CONCLUSIONS: We report on a novel ctDNA platform that detected genomic alterations in the vast majority of tested patients, further indicating potential clinical utility for capturing disease heterogeneity and for disease monitoring. Detection of CTCs and CTC clusters was associated with particular genomic profiles.

Clinical Implications of Circulating Tumor DNA Tumor Mutational Burden (ctDNA TMB) in Non-Small Cell Lung Cancer.

BACKGROUND: Tissue tumor mutational burden (TMB) has emerged as a potential biomarker predicting response to anti-programmed cell death-1 protein receptor (PD-1)/programmed cell death-1 protein ligand (PD-L1) therapy, but few studies have explored using circulating tumor DNA (ctDNA) TMB in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: A total of 136 patients with NSCLC with ctDNA testing were retrospectively evaluated from a single institution, along with a validation cohort from a second institution. ctDNA TMB was derived using the number of detected mutations over the DNA sequencing length. RESULTS: Higher ctDNA TMB was significantly correlated with smoking history (p < .05, chi-squared test). Among patients treated with immune checkpoint inhibitors (n = 20), higher ctDNA TMB was significantly correlated with shorter progressive free survival (PFS) and overall survival (OS; 45 vs. 355 days; hazard ratio [HR], 5.6; 95% confidence interval [CI], 1.3-24.6; p < .01, and OS 106 days vs. not reached; HR, 6.0; 95% CI, 1.3-27.1; p < .01, respectively). In a small independent validation cohort (n = 12), there was a nonsignificant numerical difference for higher ctDNA TMB predicting shorter OS but not PFS. ctDNA TMB was not correlated with RECIST tumor burden estimation in the subset of patients treated with immune checkpoint blockade. CONCLUSION: The findings indicate that higher ctDNA TMB, at the current commercial sequencing length, reflects worse clinical outcomes. IMPLICATIONS FOR PRACTICE: Biomarkers to identify patients who will respond to immune checkpoint blockade are critical. Tissue tumor mutational burden (TMB) has emerged as a viable biomarker to predict response to anti-PD-1/PD-L1 therapy, but few studies have explored the meaning and potential clinical significance of noninvasive, blood-based TMB. Here, we investigated circulating tumor DNA (ctDNA) TMB and present data demonstrating that current ctDNA TMB may reflect tumor burden and that ctDNA panels with a greater number of mutations may be necessary to more accurately reflect tissue TMB.

Emerging Role of Genomics and Cell-Free DNA in Breast Cancer.

OPINION STATEMENT: Precision Medicine is gaining momentum as the future gold standard healthcare strategy as it enables treatment optimization and consequently a potential improvement for quality of life and survival. This paradigm shift was possible thanks to new high-throughput genomics technologies, which provide prognostic and predictive information on tumor biology and potential treatment options, as standard pathological procedures are unable to capture both spatial and temporal tumor heterogeneity. As a result of decreasing costs, both solid and liquid-based genomics have an increasingly important role in clinical trials' screening procedures and are gradually being incorporated into clinical practice. Notwithstanding the great potential, its clinical utility is still a matter of debate and clinicians need to be aware of caveats in interpreting resulting data.

Characteristics of individuals with breast cancer rearrangements in BRCA1 and BRCA2.

BACKGROUND: Large rearrangements in BRCA1 and BRCA2 occur in a small percentage (< 1%) of patients tested for hereditary breast (BC) and ovarian cancer. It is unclear what factors predict BRACAnalysis Large Rearrangement Test (BART) positivity. METHODS: Data from 6 centers were included in this analysis. Individuals with negative Comprehensive BRACAnalysis tested for BART were included. RESULTS: From 1300 individuals, 42 (3.2%) were BART positivity. Factors positively associated with BART positivity were Myriad score, first-degree relatives with BC, infiltrating BC with ductal carcinoma in situ, younger age at BC diagnosis, estrogen receptor-negative BC for both the first and second BC, and Latin American/Caribbean ethnicity. Presence of unilateral BC was inversely associated with BART positivity. Several analyses were performed on the variables available to find the model that best predicts for BART positivity. The BART predictive model, including first BC, ovarian cancer, primary maternal ancestry being Latin America/Caribbean, number of first-degree relatives with BC of 1 or more versus 0, and family history of prostate and pancreatic cancer, had good predictive ability with an area under the curve of 0.77. CONCLUSIONS: Several factors are significantly associated with BART positivity. Among them we have found that Latin American/Caribbean ancestry, Myriad score, first degree relatives with BC, younger age at BC diagnosis, estrogen receptor-negative status of BC, and infiltrating ductal carcinoma with ductal carcinoma in situ features are significantly associated with BART positivity. A BART predictive model may help in a clinical setting.

Early evaluation of risk stratification and clinical outcomes for patients with advanced breast cancer through combined monitoring of baseline circulating tumor cells and DNA.

PURPOSE: Early evaluation of tumor heterogeneity related to metastasis and outcomes is a major challenge in the management of advanced BCa in the clinic. Here we introduce the value of baseline CTCs and ctDNA to early differentiate clinical stages, tumor heterogeneity, and prognosis. EXPERIMENTAL DESIGN: We enrolled 254 stage IV and 38 stage III BCa patients and examined the baseline levels of CTCs, CTC-clusters, and plasma ctDNA before initiating therapies. Outcome including PFS, and OS were evaluated. RESULTS: The baseline CTCs for stage IV patients were approximately 9.5 times higher than those detected in stage III patients. Baseline CTC counts with a cutoff of 5 were significantly associated with prognosis. Within each stage, patients with <5 CTCs had longer PFS. Stage III patients with no CTCs exhibited the longest survival compared to patients with ≥1 CTC. CTC-clusters were only found in stage IV patients, among whom 15 stage IV patients with ≥5 CTC-clusters had the worst PFS compared to the 239 stage IV patients with <5 CTC-clusters. Similar outcomes were observed in 28 out of 254 stage IV patients who had at least 1 CTC-cluster detected, as these patients had shorter PFS. The major differences in ctDNA mutations between stage III and stage IV BCa were in PIK3CA and ESR1, which were associated with specific organ metastasis and worse outcomes. CONCLUSIONS: Assessing the baseline levels of CTCs, CTC-clusters, and mutational ctDNA profile could reliably aid in differentiation of clinical stage and early prediction of metastasis and outcomes in advanced BCa.

ECOG-ACRIN EAZ171: Prospective Validation Trial of Germline Predictors of Taxane-Induced Peripheral Neuropathy in Black Women With Early-Stage Breast Cancer.

PURPOSE: Black women experience higher rates of taxane-induced peripheral neuropathy (TIPN) compared with White women when receiving adjuvant once weekly paclitaxel for early-stage breast cancer, leading to more dose reductions and higher recurrence rates. EAZ171 aimed to prospectively validate germline predictors of TIPN and compare rates of TIPN and dose reductions in Black women receiving (neo)adjuvant once weekly paclitaxel and once every 3 weeks docetaxel for early-stage breast cancer. METHODS: Women with early-stage breast cancer who self-identified as Black and had intended to receive (neo)adjuvant once weekly paclitaxel or once every 3 weeks docetaxel were eligible, with planned accrual to 120 patients in each arm. Genotyping was performed to determine germline neuropathy risk. Grade 2-4 TIPN by Common Terminology Criteria for Adverse Events (CTCAE) v5.0 was compared between high- versus low-risk genotypes and between once weekly paclitaxel versus once every 3 weeks docetaxel within 1 year. Patient-rated TIPN and patient-reported outcomes were compared using patient-reported outcome (PRO)-CTCAE and Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity. RESULTS: Two hundred and forty of 249 enrolled patients had genotype data, and 91 of 117 (77.8%) receiving once weekly paclitaxel and 87 of 118 (73.7%) receiving once every 3 weeks docetaxel were classified as high-risk. Physician-reported grade 2-4 TIPN was not significantly different in high- versus low-risk genotype groups with once weekly paclitaxel (47% v 35%; P = .27) or with once every 3 weeks docetaxel (28% v 19%; P = .47). Grade 2-4 TIPN was significantly higher in the once weekly paclitaxel versus once every 3 weeks docetaxel arm by both physician-rated CTCAE (45% v 29%; P = .02) and PRO-CTCAE (40% v 24%; P = .03). Patients receiving once weekly paclitaxel required more dose reductions because of TIPN (28% v 9%; P < .001) or any cause (39% v 25%; P = .02). CONCLUSION: Germline variation did not predict risk of TIPN in Black women receiving (neo)adjuvant once weekly paclitaxel or once every 3 weeks docetaxel. Once weekly paclitaxel was associated with significantly more grade 2-4 TIPN and required more dose reductions than once every 3 weeks docetaxel.

Optoporation and genetic manipulation of cells using femtosecond laser pulses.

Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irradiation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell transfection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggesting that plasmids may enter the cell by adhering to the membrane and then being translocated.

Cyclin-Dependent Kinase 4/6 Inhibitors Beyond Progression in Metastatic Breast Cancer: A Retrospective Real-World Biomarker Analysis.

PURPOSE: As the continuation beyond progression (BP) of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) is becoming increasingly attractive for the treatment of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC), the definition of resistance factors is crucial. The aim of the study was to investigate the impact of CDK 4/6i BP and to explore potential genomic stratification factors. MATERIALS AND METHODS: We retrospectively analyzed a multi-institutional cohort of patients with HR-positive HER2-negative MBC characterized for circulating tumor DNA through next-generation sequencing before treatment start. Differences across subgroups were analyzed by chi-square test, and survival was tested by univariable and multivariable Cox regression. Further correction was applied by propensity score matching. RESULTS: Among the 214 patients previously exposed to CDK4/6i, 172 were treated with non-CDK4/6i-based treatment (non-CDK) and 42 with CDK4/6i BP. Multivariable analysis showed a significant impact of CDK4/6i BP, TP53 single-nucleotide variants, liver involvement, and treatment line on both progression-free survival (PFS) and overall survival (OS). Propensity score matching confirmed the prognostic role of CDK4/6i BP both for PFS and OS. The favorable impact of CDK4/6i BP was consistent across all subgroups, and a differential benefit was suggested for ESR1-mutated patients. ESR1 and RB1 mutations were more represented in the CDK4/6i BP subgroup with respect to CDK4/6i upfront. CONCLUSION: The study highlighted a significant prognostic impact of the CDK4/6i BP strategy with a potential added benefit in patients with ESR1 mutations suggesting the need for an extensive biomarker characterization.

Genomic Complexity Predicts Resistance to Endocrine Therapy and CDK4/6 Inhibition in Hormone Receptor-Positive (HR+)/HER2-Negative Metastatic Breast Cancer.

PURPOSE: Clinical biomarkers to identify patients unlikely to benefit from CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) are lacking. We implemented a comprehensive circulating tumor DNA (ctDNA) analysis to identify genomic features for predicting and monitoring treatment resistance. EXPERIMENTAL DESIGN: ctDNA was isolated from 216 plasma samples collected from 51 patients with hormone receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (MBC) on a phase II trial of palbociclib combined with letrozole or fulvestrant (NCT03007979). Boosted whole-exome sequencing (WES) was performed at baseline and clinical progression to evaluate genomic alterations, mutational signatures, and blood tumor mutational burden (bTMB). Low-pass whole-genome sequencing was performed at baseline and serial timepoints to assess blood copy-number burden (bCNB). RESULTS: High bTMB and bCNB were associated with lack of clinical benefit and significantly shorter progression-free survival (PFS) compared with patients with low bTMB or low bCNB (all P < 0.05). Dominant APOBEC signatures were detected at baseline exclusively in cases with high bTMB (5/13, 38.5%) versus low bTMB (0/37, 0%; P = 0.0006). Alterations in ESR1 were enriched in samples with high bTMB (P = 0.0005). There was a high correlation between bTMB determined by WES and bTMB determined using a 600-gene panel (R = 0.98). During serial monitoring, an increase in bCNB score preceded radiographic progression in 12 of 18 (66.7%) patients. CONCLUSIONS: Genomic complexity detected by noninvasive profiling of bTMB and bCNB predicted poor outcomes in patients treated with ET and CDK4/6i and identified early disease progression before imaging. Novel treatment strategies including immunotherapy-based combinations should be investigated in this population.

Genetic Alterations Detected by Circulating Tumor DNA in HER2-Low Metastatic Breast Cancer.

PURPOSE: About 50% of breast cancers are defined as HER2-low and may benefit from HER2-directed antibody-drug conjugates. While tissue sequencing has evaluated potential differences in genomic profiles for patients with HER2-low breast cancer, genetic alterations in circulating tumor DNA (ctDNA) have not been well described. EXPERIMENTAL DESIGN: We retrospectively analyzed 749 patients with metastatic breast cancer (MBC) and ctDNA evaluation by Guardant360 from three academic medical centers. Tumors were classified as HER2-low, HER2-0 (IHC 0) or HER2-positive. Single-nucleotide variants, copy-number variants, and oncogenic pathways were compared across the spectrum of HER2 expression. Overall survival (OS) was evaluated by HER2 status and according to oncogenic pathways. RESULTS: Patients with HER2-low had higher rates of PIK3CA mutations [relative risk ratio (RRR), 1.57; P = 0.024] compared with HER2-0 MBC. There were no differences in ERBB2 alterations or oncogenic pathways between HER2-low and HER2-0 MBC. Patients with HER2-positive MBC had more ERBB2 alterations (RRR, 12.43; P = 0.002 for amplification; RRR, 3.22; P = 0.047 for mutations, in the hormone receptor-positive cohort), fewer ERS1 mutations (RRR, 0.458; P = 0.029), and fewer ER pathway alterations (RRR, 0.321; P < 0.001). There was no difference in OS for HER2-low and HER2-0 MBC [HR, 1.01; 95% confidence interval (CI), 0.79-1.29], while OS was improved in HER2-positive MBC (HR, 0.32; 95% CI, 0.21-0.49; P < 0.001). CONCLUSIONS: We observed a higher rate of PIK3CA mutations, but no significant difference in ERBB2 alterations, oncogenic pathways, or prognosis, between patients with HER2-low and HER2-0 MBC. If validated, our findings support the conclusion that HER2-low MBC does not represent a unique biological subtype.

Targeted Treatment for High-Risk Early-Stage Triple-Negative Breast Cancer: Spotlight on Pembrolizumab.

Triple-negative breast cancer (TNBC) is a biologically aggressive yet heterogeneous disease that disproportionately affects younger women and women of color compared to other breast cancer subtypes. The paucity of effective targeted therapies and the prevalence of chemotherapeutic resistance in high-risk, early-stage TNBC pose significant clinical challenges. Deeper insights into the genomic and immune landscape have revealed key features of TNBC, including intrinsic genomic instability, DNA repair deficiency, and potentially an immunogenic tumor microenvironment. These advances led to landmark trials with immune checkpoint inhibitors in the advanced-stage setting, which subsequently translated into immunotherapy-based clinical trials in the early-stage setting and recent promising results. Pembrolizumab, an anti-programmed death 1 (PD-1) monoclonal antibody, was investigated in combination with platinum-, taxane- and anthracycline-based neoadjuvant chemotherapy followed by adjuvant pembrolizumab monotherapy for patients with high-risk, early-stage TNBC in the randomized, double-blind, placebo-controlled phase 3 KEYNOTE-522 trial. In July 2021, the US Food and Drug Administration (FDA) granted approval for pembrolizumab based on marked improvement in pathologic complete response rate and 3-year event-free survival compared to neoadjuvant chemotherapy alone. This advance immediately altered the longstanding treatment paradigm. Here, we review the impact of pembrolizumab plus chemotherapy for the treatment of patients with high-risk, early-stage TNBC, and discuss immunotherapy-related toxicity considerations, key immunomodulatory biomarkers under active investigation, and remaining clinical questions for future research directions.

Positive predictive value of ERBB2 copy number gain by tissue or circulating tumor DNA next-generation sequencing across advanced cancers.

BACKGROUND: The correlation of ERBB2 copy number gain (CNG) from tissue or circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) with standard HER2 tissue evaluation is not well understood. MATERIALS AND METHODS: We retrospectively identified patients with ERBB2 CNG on commercial NGS. We described their clinical-pathologic features and calculated the positive predictive value (PPV) of ERBB2 CNG by NGS for HER2-positivity by IHC and FISH testing. RESULTS: 176 patients had NGS revealing an ERBB2 CNG (112 by tumor tissue and 91 by ctDNA). The cancer subtypes with the most cases with ERBB2 CNG by NGS were breast (n = 67), non-small cell lung (n = 25), colorectal (n = 18), gastroesophageal (n = 17), pancreatic (n = 11), and uterine (n = 11). The PPV of ERBB2 CNG in determining HER2 positivity by standard IHC/FISH definitions was 88% for tissue NGS (n = 57) and 80% for ctDNA (n = 47). The PPV among breast cancer patients for tissue NGS was 97% (n = 35) and ctDNA was 93% (n = 39). However, for non-breast cancer cases, the PPV of ERBB2 amplification by tissue NGS dropped to 76% (n = 22) and by ctDNA to 44% (n = 7). CONCLUSIONS: ERBB2 CNG by NGS is detected in numerous malignancies for which HER2 testing is not standard. Detection of ERBB2 CNG by tissue NGS and ctDNA has a high PPV for true HER2-positivity by standard IHC and/or FISH testing in breast cancer.