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Background: Cyclin E1 (CCNE1) plays a critical role in cell cycle regulation. CCNE1 overexpression and/or gene amplification (amp) have been associated with poor outcome in several tumors, including breast cancer (BC). CCNE1 amp has recently been identified as a potential therapeutic target for novel synthetic lethality-based therapies. Here we sought to define the clinical and genomic features of BCs carrying CCNE1 amp. Methods: Genomic and clinical data from all consecutive BCs, which had been subjected to targeted sequencing using the FDA-authorized MSK-IMPACT assay from April 2014 to December 2021, were retrieved. Allele-specific copy number and fraction genome altered (FGA) were assessed using FACETS. Whole genome doubling (WGD) status was inferred from MSK-IMPACT sequencing data. Samples were categorized as CCNE1 amp or non-amp based on copy number profile assessed by FACETS. Mutual exclusivity and co-occurrence analyses between CCNE1 amp and other genetic alterations were performed using CoMEt. Multiple testing correction using the Benjamini–Hochberg procedure was applied to control for the false discovery rate. Progression-free survival (PFS) was assessed by Kaplan Meier method and Cox proportional-hazards models. Survival analyses were restricted to only patients with available pre-treatment samples. Results: Of 3,753 BCs with full clinical and genomic data, 125 (3.3%) harbored CCNE1 amp. A significant difference in the proportion of CCNE1 amp between treatment-naïve and post-treatment/metastatic samples was observed (2.4% vs 4%, p=0.007). CCNE1 amp was significantly less frequently detected in hormone receptor (HR)+/HER2- BCs than in HR-/HER2+ and HR-/HER2- subtypes (2% vs 7.6% and 7.2%, respectively, p< 0.001), and was particularly rare in invasive lobular BCs (1/452 cases). BCs with CCNE1 amp displayed a higher frequency of WGD (p< 0.001) and higher median FGA (p< 0.001) than non-amp tumors, overall and in different subtypes, suggesting increased genomic instability. No difference in tumor mutational burden (TMB) between CCNE1 amp and non-amp was found. In primary BC (n=1,385), a higher proportion of TP53 alterations was found in cases with CCNE1 amp (odds ratio [OR] 6.0, 95% confidence interval [CI] 2.5-16.6, q< 0.001). Conversely, CCNE1 amp was mutually exclusive with CDH1 alterations (q< 0.001). Comparable results were found in the analysis of post-treatment/metastatic samples (n=2,368). A subset analysis on HR+/HER2- BCs confirmed that TP53 (OR 4.2, 95%CI 2.28-8.11, q< 0.001) and CDH1 (OR 0.09, 95%CI 0.002-0.57, q< 0.1) alterations co-occurred and were mutually exclusive, respectively, with CCNE1 amp. ARID2 alterations were also enriched in HR+/HER2- tumors harboring CCNE1 amp (OR 10.6, 95%CI 2.54-33.93, q< 0.1). CCNE1 amp was significantly associated with reduced median PFS (8.8 vs 15.2 months in CCNE1 amp [n=9] vs CCNE1 non-amp [n=402]; hazard ratio [HR] 2.82, 95% CI 1.38-5.75, p=0.004) on first line treatment with CDK4/6 inhibitor plus endocrine therapy (ET) in HR+/HER2- metastatic BCs, regardless of the ET partner, FGA and TMB. CCNE1 amp was also associated with numerically inferior median PFS (7.3 vs 20.8 months in CCNE1 amp [n=5] vs CCNE1 non-amp [n=106]; HR 3.1, 95% CI 1.24-7.87, p=0.01) on first line trastuzumab/pertuzumab/taxane treatment in HER2+ metastatic BCs, with a trend toward significance after adjusting for FGA and TMB (p=0.09). Conclusions: CCNE1 amp is associated with specific clinicopathological and genomic features in BCs and linked to an increased genomic instability. CCNE1 amp defines a subset of metastatic BCs with marked poor clinical response to available standard-of-care treatments. Further studies testing novel therapeutic approaches, including synthetic lethality-based strategies targeting CCNE1 amp and CDK2-selective inhibition, are warranted. Citation Format: Antonio Marra, Pier Selenica, Yingjie Zhu, Pedram Razavi, Anton Safonov, Emanuela Ferraro, Sarat Chandarlapaty, Jorge Reis-Filho. Clinical and Genomic Landscape of Breast Cancers Carrying CCNE1 Amplification [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 P4-08-12.

Background: Varying levels of HER2 expression without ERBB2 gene amplification can be detected by immunohistochemistry (IHC) in approximately 60% of all invasive breast cancers (BCs). HER2-low-expressing BCs have recently been shown to respond to novel anti-HER2 antibody drug conjugates. Several studies have demonstrated that HER2-low BCs do not seem to constitute a distinct clinical and transcriptomic subtype as compared to HER2-0 BCs. Here we sought to define the clinicopathologic features and repertoire of somatic genetic alterations in HER2-low BCs. Methods: We retrieved clinical, pathological, and genomic data of BCs that were subjected to targeted sequencing using the FDA-cleared MSK-IMPACT assay from April 2014 to December 2021. After removing cases where any available biopsy had HER2 3+ and/or positive ERBB2 fluorescence in situ hybridization (FISH), 3608 samples (primary=1347; post-treatment/metastatic=2261) were included. Tumors were classified as HER2-low if they had an HER2 IHC score of 1+ or 2+ with a non-amplified FISH assay and HER2-0 if they had an HER2 IHC score of 0. Somatic mutations and DNA copy number alterations from MSK-IMPACT were analyzed. Multiple testing correction using the Benjamini-Hochberg method was applied to control for the false discovery rate (q). Q values < 0.1 were considered significant. Results: Among 3608 HER2- BCs, 1460 (40%) and 2148 (60%) were HER2-0 and HER2-low, respectively. Hormone receptor (HR) expression was significantly higher in HER2-low than HER2-0 tumors in both primary (781 [68.3%] vs 362 [31.7%]; p< 0.001) and metastatic (1031 [60.5%] vs 673 [39.5%]; p< 0.001) samples. A higher proportion of HER2-low tumors was found in metastatic than primary samples (59% vs 41%; p< 0.001) in this cohort. No difference in histology subtype, tumor grade, disease stage (among primary tumors), mutational signatures, and tumor mutational burden was found overall and when cases were stratified by HR expression. In HR-positive BCs, HER2-0 BCs harbored higher frequency of TP53 (33% vs 25%; odds ratio [OR] 1.49, 95% confidence interval [CI] 1.25-1.78, q< 0.001) and CDKN1A (1% vs 0%; OR 17.47, 95% CI 2.48-756.37, q=0.02) alterations than HER2-low BCs. Similar findings were observed in metastatic but not in primary HR-positive BCs. No differences were detected in HR-negative BCs stratified into HER2-low and HER2-0. Given the potential misclassification that exists between IHC HER2-0 and HER2-1+, we then conducted an exploratory analysis splitting the HER2-low group into HER2 1+ and 2+. TP53 alterations remained significantly enriched in HER2-0 compared to HER2-1+ HR-positive tumors (33% vs 24%; OR 1.55, 95% CI 1.28-1.87, q< 0.001). In HR-positive BCs, HER2-2+ displayed a higher frequency of genetic alterations in genes encoding for transcription factors, such as MYC (14.2% vs 7.3%; OR 2.09, 95% CI 1.44-3.04, q=0.02) and YAP1 (2% vs 0.3%; OR 6.86, 95% CI 1.7-39.57, q=0.1), and DNA damage response, such as FAM175A (1.6% vs 0%; OR 18.23, 95% CI 2.43-807.73, q=0.03) and BRCA2 (4% vs 1%; OR 3.09, 95% CI 1.49-6.55, q=0.1), than HER2-0 tumors. In HR-negative HER2-2+ tumors, a higher frequency of PIK3CA mutations was observed in comparison to HER2-0 (36.9% vs 19.5%; OR 2.41, 1.4-4.1, q=0.1), overall and in the metastatic setting. Conclusions: HER2-low BCs seem not to represent a distinct pathologic subtype. At the genomic level, however, some differences were identified and these became more conspicuous upon subclassification of HER2 IHC expression into 1+ and 2+. Further investigation into methods that more accurately detect and quantify low levels of HER2 expression in BC samples as well as better characterize the biology behind the HER2-low/ultralow expression is warranted. Citation Format: Antonio Marra, Anton Safonov, Joshua Drago, Emanuela Ferraro, Pier Selenica, Andrea Gazzo, Giuseppe Curigliano, Shanu Modi, Pedram Razavi, Jorge Reis-Filho, Sarat Chandarlapaty. HER2-07 Genomic Characterization of Primary and Metastatic HER2-low Breast Cancers [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 HER2-07.

Accurate ancestry inference is critical for identifying genetic contributors of cancer disparities among populations. Although methods to infer genetic ancestry have historically relied upon genome-wide markers, the adaptation to targeted clinical sequencing panels presents an opportunity to incorporate ancestry inference into routine diagnostic workflows. We show that global ancestral contributions and admixture of continental populations can be quantitatively inferred using markers captured by the MSK-IMPACT clinical panel. In a pan-cancer cohort of 45,157 patients, we observed differences by ancestry in the frequency of somatic alterations, recapitulating known associations and revealing novel associations. Despite the comparable overall prevalence of driver alterations by ancestry group, the proportion of patients with clinically actionable alterations was lower for African (30%) compared with European (33%) ancestry. Although this result is largely explained by population-specific cancer subtype differences, it reveals an inequity in the degree to which different populations are served by existing precision oncology interventions. Significance: We performed a comprehensive analysis of ancestral associations with somatic mutations in a real-world pan-cancer cohort, including >5,000 non-European individuals. Using an FDA-authorized tumor sequencing panel and an FDA-recognized oncology knowledge base, we detected differences in the prevalence of clinically actionable alterations, potentially contributing to health care disparities affecting underrepresented populations. This article is highlighted in the In This Issue feature, p. 2483

A myriad of organ-specific complications have been observed with COVID-19. While racial/ethnic minorities have been disproportionately burdened by this disease, our understanding of the unique risk factors for complications among a diverse population of cancer patients remains limited. This is a multi-institutional, multi-ethnic cohort study evaluating COVID-19 complications among cancer patients. Patients with an invasive cancer diagnosis and confirmed SARS-CoV-2 infection were identified from March to November 2020. Demographic and clinical data were obtained and a multivariate logistic regression was employed to evaluate the impact of demographic and clinical factors on COVID-19 complications. The study endpoints were evaluated independently and included any complication, sepsis, pulmonary complications and cardiac complications. A total of 303 patients were evaluated, of whom 48% were male, 79% had solid tumors, and 42% were Hispanic/Latinx (Hispanic). Malignant hematologic cancers were associated with a higher risk of sepsis (OR 3.93 (95% CI 1.58–9.81)). Male patients had a higher risk of sepsis (OR 4.42 (95% CI 1.63–11.96)) and cardiac complications (OR 2.02 (95% CI 1.05–3.89)). Hispanic patients had a higher odds of any complication (OR 2.31 (95% CI 1.18–4.51)) and other race was associated with a higher odds of cardiac complications (OR 2.41 (95% CI 1.01–5.73)). Clinically, fever, cough, and ≥2 co-morbidities were independently significantly associated with any complication. This analysis evaluated covariates that can significantly predict a myriad of complications among a multi-ethnic cohort of cancer patients. The conclusions drawn from this analysis elucidate a mechanistic understanding of differential illness severity from COVID-19.

Purpose: Oncogenic alterations of the PI3K/AKT pathway occur in >40% of patients with metastatic castration-resistant prostate cancer, predominantly via PTEN loss. The significance of other PI3K pathway components in prostate cancer is largely unknown. Experimental Design: Patients in this study underwent tumor sequencing using the MSK-IMPACT clinical assay to capture single-nucleotide variants, insertions, and deletions; copy-number alterations; and structural rearrangements, or were profiled through The Cancer Genome Atlas. The association between PIK3R1 alteration/expression and survival was evaluated using univariable and multivariable Cox proportional-hazards regression models. We used the siRNA-based knockdown of PIK3R1 for functional studies. FDG-PET/CT examinations were performed with a hybrid positron emission tomography (PET)/CT scanner for some prostate cancer patients in the MSK-IMPACT cohort. Results: Analyzing 1,417 human prostate cancers, we found a significant enrichment of PIK3R1 alterations in metastatic cancers compared with primary cancers. PIK3R1 alterations or reduced mRNA expression tended to be associated with worse clinical outcomes in prostate cancer, particularly in primary disease, as well as in breast, gastric, and several other cancers. In prostate cancer cell lines, PIK3R1 knockdown resulted in increased cell proliferation and AKT activity, including insulin-stimulated AKT activity. In cell lines and organoids, PIK3R1 loss/mutation was associated with increased sensitivity to AKT inhibitors. PIK3R1-altered patient prostate tumors had increased uptake of the glucose analogue 18F-fluorodeoxyglucose in PET imaging, suggesting increased glycolysis. Conclusions: Our findings describe a novel genomic feature in metastatic prostate cancer and suggest that PIK3R1 alteration may be a key event for insulin–PI3K–glycolytic pathway regulation in prostate cancer.

Supplementary Figure from Genetic Ancestry Correlates with Somatic Differences in a Real-World Clinical Cancer Sequencing Cohort

Patients with B-lymphoid malignancies have been consistently identified as a population at high risk of severe COVID-19. Whether this is exclusively due to cancer-related deficits in humoral and cellular immunity, or whether risk of severe COVID-19 is increased by anticancer therapy, is uncertain. Using data derived from the COVID-19 and Cancer Consortium (CCC19), we show that patients treated for B-lymphoid malignancies have an increased risk of severe COVID-19 compared with control populations of patients with non–B-lymphoid malignancies. Among patients with B-lymphoid malignancies, those who received anticancer therapy within 12 months of COVID-19 diagnosis experienced increased COVID-19 severity compared with patients with non–recently treated B-lymphoid malignancies, after adjustment for cancer status and several other prognostic factors. Our findings suggest that patients recently treated for a B-lymphoid malignancy are at uniquely high risk for severe COVID-19.Significance:Our study suggests that recent therapy for a B-lymphoid malignancy is an independent risk factor for COVID-19 severity. These findings provide rationale to develop mitigation strategies targeted at the uniquely high-risk population of patients with recently treated B-lymphoid malignancies.This article is highlighted in the In This Issue feature, p. 171

Accurate ancestry inference is critical for identifying genetic contributors of cancer disparities among populations. Although methods to infer genetic ancestry have historically relied upon genome-wide markers, the adaptation to targeted clinical sequencing panels presents an opportunity to incorporate ancestry inference into routine diagnostic workflows. We show that global ancestral contributions and admixture of continental populations can be quantitatively inferred using markers captured by the MSK-IMPACT clinical panel. In a pan-cancer cohort of 45,157 patients, we observed differences by ancestry in the frequency of somatic alterations, recapitulating known associations and revealing novel associations. Despite the comparable overall prevalence of driver alterations by ancestry group, the proportion of patients with clinically actionable alterations was lower for African (30%) compared with European (33%) ancestry. Although this result is largely explained by population-specific cancer subtype differences, it reveals an inequity in the degree to which different populations are served by existing precision oncology interventions.Significance:We performed a comprehensive analysis of ancestral associations with somatic mutations in a real-world pan-cancer cohort, including >5,000 non-European individuals. Using an FDA-authorized tumor sequencing panel and an FDA-recognized oncology knowledge base, we detected differences in the prevalence of clinically actionable alterations, potentially contributing to health care disparities affecting underrepresented populations.This article is highlighted in the In This Issue feature, p. 2483

Supplementary Figure from Patients Recently Treated for B-lymphoid Malignancies Show Increased Risk of Severe COVID-19

Supplementary Data from Genetic Ancestry Correlates with Somatic Differences in a Real-World Clinical Cancer Sequencing Cohort

Supplementary Figures 1-7

Supplementary Figure from Optimal Strategy and Benefit of Pulsed Therapy Depend On Tumor Heterogeneity and Aggressiveness at Time of Treatment Initiation

S1. Generation of MCF-10A isogenic cells via TP53 knockout and AKT1 E17K knock-in. S2. Frequent co-occurrence of AKT1, TP53 and CDH1 mutations in breast cancers. S3. TP53 CRISPR knockout in MCF-10A cells. S4. Phosphorylation of the AKT1 effector PRAS40 was AKT-dependent in MCF-10A p53ko/E17K cells. S5. Immunofluorescence using a pT308 specific AKT1 antibody of MCF-10A cells following lentiviral infection with V5 tagged (V5 tag) wildtype (WT) or E17K mutant AKT1. S6. Knock-in of the AKT1 E17K mutation did not enhance sensitivity to the pan-AKT kinase inhibitor AZD5363 or the allosteric pan-AKT inhibitor MK2206 in 2Dculture conditions. S7. Survey of previously postulated mechanisms for AKT1-mediated inhibition of cell migration in breast cancer cells. S8. CDH1 promoter methylation status in the MCF-10A isogenic cells. S9. β-catenin interacts with the kinase domain of AKT1. S10. Expression of AKT1 E17K decreased phosphorylation of AKT2 in MCF-10A cells. S11. AKT2 knockdown induces E-cadherin expression in MCF-10A p53ko cells. S12. Model of the mechanism of paradoxical inhibition of cell migration by activated AKT1. Table S1. List of oligonucleotide sequences Table S2. List of antibodies Table S3. List of plasmids

Supplementary Fig 3

Supplementary Fig 2

Supplementary Figure from INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors

Supplementary Data from Optimal Strategy and Benefit of Pulsed Therapy Depend On Tumor Heterogeneity and Aggressiveness at Time of Treatment Initiation

Supplementary Methods, Tables 1 - 9, Figures 1 - 8. Supplementary Table 1. Functional classification for validated genes. Supplementary Table 2. Validated 43 candidate genes' genetic alteration status in the invasive breast cancer (TCGA) database. Supplementary Table 3. Receptor status, mutational status and intrinsic subtypes of breast cancer cell lines used in this study. Supplementary Table 4. GI50 of BYL719 in different breast cancer cell lines with overexpression of PIM isoforms. Supplementary Table 5. PIM1 and AKT phosphorylation sites in PRAS40, BAD, p21 and p27. Supplementary Table 6. PIM gene expression levels in PIK3CA-mutant breast cancer cell lines. Supplementary Table 7. GI50 of BYL719 in the presence of LGH447 (1μM) or DMSO in PIK3CA-mutant breast cancer cell lines. Supplementary Table 8. Signature genes that changed concordantly in patient sample pairs and PIM signature. Supplementary Table 9. Hormonal receptor status by immunohistochemistry (IHC), HER2 status by FISH and IHC of PIM1 amplified or overexpressed breast cancer tumor samples from the invasive breast cancer (TCGA) database. Supplementary Figure 1. Lentiviral infection efficacy quality in the large-scale ORF screen. Supplementary Figure 2. PIM1 confers resistance to breast cancer cell lines with different PIK3CA mutations and various intrinsic subtypes. Supplementary Figure 3. PIM1 confers resistance to inhibitors targeting key components of PI3K pathway. Supplementary Figure 4. PIM1 inhibitors alone have minimal inhibitory effects on T47D cells with or without PIM1 overexpression. Supplementary Figure 5. PIM1 expression abrogates cell-cycle arrest in cells treated with BYL719. Supplementary Figure 6. BYL719 and AZD1208 or LGH447 demonstrated synergy in PIM1 expressing T47D cells. Supplementary Figure 7. PIM activation signature. Supplementary Figure 8. PIM and PI3K pathway genetic mutations showed mutual exclusivity in treatment-naive breast cancers.

Supplementary Tables 1-4

Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C–cyclin D–CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance.Significance:CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6–INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.This article is highlighted in the In This Issue feature, p. 275

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.Significance:T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627

Supplementary Fig 5

Supplementary Figures 1 - 8, Tables 1 - 3

Supplementary Methods

Mutational activation of the PI3K/AKT pathway is among the most common pro-oncogenic events in human cancers. The clinical utility of PI3K and AKT inhibitors has, however, been modest to date. Here, we used CRISPR-mediated gene editing to study the biological consequences of AKT1 E17K mutation by developing an AKT1 E17K–mutant isogenic system in a TP53-null background. AKT1 E17K expression under the control of its endogenous promoter enhanced cell growth and colony formation, but had a paradoxical inhibitory effect on cell migration and invasion. The mechanistic basis by which activated AKT1 inhibited cell migration and invasion was increased E-cadherin expression mediated by suppression of ZEB1 transcription via altered β-catenin subcellular localization. This phenotypic effect was AKT1-specific, as AKT2 activation had the opposite effect, a reduction in E-cadherin expression. Consistent with the opposing effects of AKT1 and AKT2 activation on E-cadherin expression, a pro-migratory effect of AKT1 activation was not observed in breast cancer cells with PTEN loss or expression of an activating PIK3CA mutation, alterations which induce the activation of both AKT isoforms. The results suggest that the use of AKT inhibitors in patients with breast cancer could paradoxically accelerate metastatic progression in some genetic contexts and may explain the frequent coselection for CDH1 mutations in AKT1-mutated breast tumors.Implications:AKT1 E17K mutation in breast cancer impairs migration/invasiveness via sequestration of β-catenin to the cell membrane leading to decreased ZEB1 transcription, resulting in increased E-cadherin expression and a reversal of epithelial–mesenchymal transition.

Supplementary Table 1

Supplementary Fig 6

Supplementary Fig 4

RPPA data analysis

Supplementary Figure 1. Comparison of tumor mutational burden, dominant mutational signatures and MSISensor scores of Lynch syndrome BC cohort to control-matched BC cohort using MSK-IMPACT targeted sequencing.