Evaluation of markers of immunity in different metastatic immune microenvironments suggests more suppression within breast to liver metastases in breast cancer.

PURPOSE: Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. METHODS: 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. RESULTS: Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. CONCLUSIONS: LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.

Co-clinical FDG-PET radiomic signature in predicting response to neoadjuvant chemotherapy in triple-negative breast cancer.

PURPOSE: We sought to exploit the heterogeneity afforded by patient-derived tumor xenografts (PDX) to first, optimize and identify robust radiomic features to predict response to therapy in subtype-matched triple negative breast cancer (TNBC) PDX, and second, to implement PDX-optimized image features in a TNBC co-clinical study to predict response to therapy using machine learning (ML) algorithms. METHODS: TNBC patients and subtype-matched PDX were recruited into a co-clinical FDG-PET imaging trial to predict response to therapy. One hundred thirty-one imaging features were extracted from PDX and human-segmented tumors. Robust image features were identified based on reproducibility, cross-correlation, and volume independence. A rank importance of predictors using ReliefF was used to identify predictive radiomic features in the preclinical PDX trial in conjunction with ML algorithms: classification and regression tree (CART), Naïve Bayes (NB), and support vector machines (SVM). The top four PDX-optimized image features, defined as radiomic signatures (RadSig), from each task were then used to predict or assess response to therapy. Performance of RadSig in predicting/assessing response was compared to SUVmean, SUVmax, and lean body mass-normalized SULpeak measures. RESULTS: Sixty-four out of 131 preclinical imaging features were identified as robust. NB-RadSig performed highest in predicting and assessing response to therapy in the preclinical PDX trial. In the clinical study, the performance of SVM-RadSig and NB-RadSig to predict and assess response was practically identical and superior to SUVmean, SUVmax, and SULpeak measures. CONCLUSIONS: We optimized robust FDG-PET radiomic signatures (RadSig) to predict and assess response to therapy in the context of a co-clinical imaging trial.

Racial differences in no-show rates for screening mammography.

BACKGROUND: Differences in utilization of screening mammography partly explain the increased breast cancer mortality observed in African American (AA) women compared with non-Hispanic White women. However, the contribution of noncompliance from women who do not come for their scheduled screening mammography appointment (ie, no-shows) is unknown. The purpose of this study was to investigate racial differences in no-show rates for screening mammography. METHODS: Women scheduled for routine screening mammograms between January 2018 and March 2018 were identified from the Joanne Knight Breast Health Center at Siteman Cancer Center in St. Louis, Missouri. Using a case-control design, this study retrospectively identified patients who no-showed for their mammograms (cases) and randomly sampled an equal number of patients who completed their mammograms (controls). These participants were compared by race. The main outcome measure was whether AA race was associated with no-shows for screening mammography. RESULTS: During the study period, 5060 women were scheduled for screening mammography, and 316 (6.2%) did not keep their appointment (ie, they no-showed). Women who no-showed were more likely to be AA than women who kept their appointment (odds ratio, 2.64; 95% confidence interval, 1.90-3.67). Even after adjustments for marital status, insurance type, and place of residence, AA race was still significantly associated with no-shows for screening mammography. CONCLUSIONS: This study identified a no-show rate of 6.2% for screening mammography at the authors' institution. Women who no-showed were more likely to be AA than women who completed their mammogram even after adjustments for multiple factors. These data can be leveraged for future studies aimed at improving mammography attendance rates among AA women.

Early Assessment Window for Predicting Breast Cancer Neoadjuvant Therapy using Biomarkers, Ultrasound, and Diffuse Optical Tomography.

PURPOSE: The purpose of the study was to assess the utility of tumor biomarkers, ultrasound (US) and US-guided diffuse optical tomography (DOT) in early prediction of breast cancer response to neoadjuvant therapy (NAT). METHODS: This prospective HIPAA compliant study was approved by the institutional review board. Forty one patients were imaged with US and US-guided DOT prior to NAT, at completion of the first three treatment cycles, and prior to definitive surgery from February 2017 to January 2020. Miller-Payne grading was used to assess pathologic response. Receiver operating characteristic curves (ROCs) were derived from logistic regression using independent variables, including: tumor biomarkers, US maximum diameter, percentage reduction of the diameter (%US), pretreatment maximum total hemoglobin concentration (HbT) and percentage reduction in HbT (%HbT) at different treatment time points. Resulting ROCs were compared using area under the curve (AUC). Statistical significance was tested using two-sided two-sample student t-test with P < 0.05 considered statistically significant. Logistic regression was used for ROC analysis. RESULTS: Thirty-eight patients (mean age = 47, range 24-71 years) successfully completed the study, including 15 HER2 + of which 11 were ER + ; 12 ER + or PR + /HER2-, and 11 triple negative. The combination of HER2 and ER biomarkers, %HbT at the end of cycle 1 (EOC1) and %US (EOC1) provided the best early prediction, AUC = 0.941 (95% CI 0.869-1.0). Similarly an AUC of 0.910 (95% CI 0.810-1.0) with %US (EOC1) and %HbT (EOC1) can be achieved independent of HER2 and ER status. The most accurate prediction, AUC = 0.974 (95% CI 0.933-1.0), was achieved with %US at EOC1 and %HbT (EOC3) independent of biomarker status. CONCLUSION: The combined use of tumor HER2 and ER status, US, and US-guided DOT may provide accurate prediction of NAT response as early as the completion of the first treatment cycle. CLINICAL TRIAL REGISTRATION NUMBER: NCT02891681. https://clinicaltrials.gov/ct2/show/NCT02891681 , Registration time: September 7, 2016.

Immunogenomic profiling and pathological response results from a clinical trial of docetaxel and carboplatin in triple-negative breast cancer.

PURPOSE: Patients with triple-negative breast cancer (TNBC) who do not achieve pathological complete response (pCR) following neoadjuvant chemotherapy have a high risk of recurrence and death. Molecular characterization may identify patients unlikely to achieve pCR. This neoadjuvant trial was conducted to determine the pCR rate with docetaxel and carboplatin and to identify molecular alterations and/or immune gene signatures predicting pCR. EXPERIMENTAL DESIGN: Patients with clinical stages II/III TNBC received 6 cycles of docetaxel and carboplatin. The primary objective was to determine if neoadjuvant docetaxel and carboplatin would increase the pCR rate in TNBC compared to historical expectations. We performed whole-exome sequencing (WES) and immune profiling on pre-treatment tumor samples to identify alterations that may predict pCR. Thirteen matching on-treatment samples were also analyzed to assess changes in molecular profiles. RESULTS: Fifty-eight of 127 (45.7%) patients achieved pCR. There was a non-significant trend toward higher mutation burden for patients with residual cancer burden (RCB) 0/I versus RCB II/III (median 80 versus 68 variants, p 0.88). TP53 was the most frequently mutated gene, observed in 85.7% of tumors. EGFR, RB1, RAD51AP2, SDK2, L1CAM, KPRP, PCDHA1, CACNA1S, CFAP58, COL22A1, and COL4A5 mutations were observed almost exclusively in pre-treatment samples from patients who achieved pCR. Seven mutations in PCDHA1 were observed in pre-treatment samples from patients who did not achieve pCR. Several immune gene signatures including IDO1, PD-L1, interferon gamma signaling, CTLA4, cytotoxicity, tumor inflammation signature, inflammatory chemokines, cytotoxic cells, lymphoid, PD-L2, exhausted CD8, Tregs, and immunoproteasome were upregulated in pre-treatment samples from patients who achieved pCR. CONCLUSION: Neoadjuvant docetaxel and carboplatin resulted in a pCR of 45.7%. WES and immune profiling differentiated patients with and without pCR. TRIAL REGISTRATION: Clinical trial information: NCT02124902, Registered 24 April 2014 & NCT02547987, Registered 10 September 2015.

Predictors of Distant Metastases in Triple-Negative Breast Cancer Without Pathologic Complete Response After Neoadjuvant Chemotherapy.

BACKGROUND: Pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) for triple-negative breast cancer (TNBC) predicts decreased distant metastasis. However, most patients do not experience pCR, and other risk factors for distant metastasis after NAC are poorly characterized. This study investigated factors predictive of distant metastasis in TNBC without pCR after NAC. METHODS: Women with TNBC treated with NAC, surgery, and radiation therapy in 2000 through 2013 were reviewed. Freedom from distant metastasis (FFDM) was compared between patients with and without pCR using the Kaplan-Meier method. In patients without pCR, univariate and multivariable Cox analyses were used to determine factors predictive of distant metastasis. RESULTS: We identified 153 patients with median follow-up of 4.0 years (range, 0.5-14.0 years). After NAC, 108 had residual disease (pCR, 29%). Five-year FFDM was 98% and 55% in patients with and without pCR, respectively (P<.001). Factors independently predicting FFDM in patients without pCR were pathologic nodal positivity (hazard ratio, 3.08; 95% CI, 1.54-6.14; P=.001) and lymphovascular space invasion (hazard ratio, 1.91; 95% CI, 1.07-3.43; P=.030). Patients with a greater number of factors had worse FFDM; 5-year FFDM was 76.5% for patients with no factors (n=38) versus 54.9% and 27.5% for patients with 1 (n=44) and 2 factors (n=26), respectively (P<.001). CONCLUSIONS: Lack of pCR after NAC resulted in worse overall survival and FFDM, despite trimodality therapy. In patients with residual disease after NAC, pathologic lymph node positivity and lymphovascular space invasion predicted worse FFDM.

Assessing the effectiveness of the National Comprehensive Cancer Network genetic testing guidelines in identifying African American breast cancer patients with deleterious genetic mutations.

PURPOSE: Approximately, 10% of breast cancers are hereditary. Identifying women at high risk for hereditary breast and ovarian cancer allows for early detection, prevention, and individualized disease management for those diagnosed with breast cancer. There is limited data about breast cancer genetic risks among African Americans as the majority of the large studies have been conducted in European Americans. We examined the distribution of deleterious genetic mutations in African American breast cancer patients, and evaluated the effectiveness of the National Comprehensive Cancer Network (NCCN) guidelines for identifying African American women at high risk for deleterious genetic mutations. METHODS: African American participants with breast cancer underwent an interview regarding health and family history, and a 30-gene saliva test. Medical records were accessed to determine whether participants had received prior genetic testing as part of usual care, results of previous testing, and cancer characteristics. RESULTS: Two hundred and fifty participants were enrolled between February 2016 and May 2018. Twenty (8.0%) had a deleterious mutation in one of the 30 genes; BRCA2 had the highest frequency (40.0%). 187 (74.8%) met eligibility for testing based on NCCN guidelines. Only 110 (58.8%) of participants eligible for genetic testing, according to guidelines, had received prior testing as part of routine care. Using the 30-gene test, we identified deleterious mutations in 17 of 187 (9.1%) of those who met NCCN criteria for testing, and three of 63 (4.8%) of those who did not meet criteria for testing nonetheless had a deleterious mutation associated with breast cancer. CONCLUSIONS: Our results indicate that a large proportion of African American breast cancer patients who meet criteria for genetic testing do not receive it as part of routine care. Even in women who do not meet testing guidelines, nearly 5% have a known deleterious mutation associated with breast cancer.

Balixafortide plus eribulin in HER2-negative metastatic breast cancer: a phase 1, single-arm, dose-escalation trial.

BACKGROUND: The C-X-C chemokine receptor type 4 (CXCR4)-stromal cell-derived factor-1α (SDF-1α) axis regulates function and trafficking of immune cells and the tumour microenvironment. CXCR4 antagonists have been shown to enhance the activity of different anticancer treatments in preclinical models. We assessed the safety, tolerability, pharmacokinetics, and preliminary phase 1 activity of the CXCR4 antagonist, balixafortide, in combination with eribulin chemotherapy in patients with heavily pretreated, relapsed metastatic breast cancer. METHODS: This single-arm, dose-escalation, phase 1 trial enrolled patients at 11 sites in Spain and the USA. Eligible patients were women aged 18 years or older who had histologically confirmed HER2-negative metastatic breast cancer, evidence of tumour cell CXCR4 expression, an Eastern Cooperative Oncology Group performance status of 0 or 1, and who had previously received between one and three chemotherapy regimens for metastatic breast cancer, and at least one endocrine therapy if they had hormone receptor-positive disease, unless they were considered unsuitable for endocrine therapy. A standard 3+3 dose-escalation design was used, followed by an expanded cohort at the established maximum tolerated dose or highest dose if no dose-limiting toxicity was observed for the combination. After a treatment-related fatal adverse event in the first cohort who received 21-day cycles of treatment with eribulin and balixafortide, a protocol amendment modified the study design to be done in two parts. Patients enrolled to part 1 received an initial 28-day run-in cycle, with some cohorts receiving de-escalated doses of eribulin plus balixafortide to assess the safety and pharmacokinetics of the combination. The evaluation of part 1 did not confirm any dose-limiting toxicities or eribulin-balixafortide interactions, and therefore part 2 started enrolling patients to receive eribulin at the originally planned dose of 1·4 mg/m2 on days 2 and 9 of a 21-day cycle and balixafortide from a starting dose of 2 mg/kg with dose increments of 0·5 or 1 mg/kg on days 1-3 and 8-10 of the 21-day cycle. Both drugs were administered as intravenous infusions. All patients were to receive treatment until disease progression or unacceptable toxicity. The primary endpoints were dose-limiting toxicities and adverse events, and the establishment of a maximum tolerated dose or recommended phase 2 dose, and pharmacokinetic parameters. Safety analysis was done in all patients who received at least one dose of study treatment. Analysis of antitumour activity was done in all patients who received at least one full cycle of study treatment. The trial is registered at ClinicalTrials.gov, number NCT01837095, and is closed to accrual. FINDINGS: Between Jan 28, 2014, and Oct 4, 2016, 56 patients were enrolled into the trial. No dose-limiting toxicities were confirmed and the maximum tolerated dose was not reached. The highest dose was established as eribulin 1·4 mg/m2 on days 2 and 9, and balixafortide 5·5 mg/kg on days 1-3 and 8-10 of the 21-day cycle. Objective responses (all partial responses) were observed in 16 (30%; 95% CI 18-44) of 54 patients who were evaluable for antitumour activity. The most common treatment-emergent adverse events of any grade were fatigue (44 [79%] of 56 patients), neutropenia (32 [57%]), infusion-related reactions (27 [48%]), alopecia (26 [46%]), constipation (26 [46%]), and nausea (25 [45%]). Serious adverse events occurred in 21 (38%) of 56 patients, including febrile neutropenia in five (9%) of 56 patients, neutrophil count decrease in two (4%) patients, constipation in two (4%) patients, pneumonia in two (4%) patients, and urinary tract infection in three (5%) patients. Two (4%) of 56 patients died while receiving study treatment; one from septic shock and one from pneumonia. INTERPRETATION: The safety and tolerability of balixafortide plus eribulin seems to be similar to that of eribulin or balixafortide monotherapy, and the preliminary activity of the combination seems promising in patients with HER-negative metastatic breast cancer. The results suggest that balixafortide plus eribulin has potential to provide a new therapeutic option in heavily pretreated patients with metastatic breast cancer and warrants further investigation in randomised trials. FUNDING: Polyphor.

Differences in the mutational landscape of triple-negative breast cancer in African Americans and Caucasians.

BACKGROUND: Triple-negative breast cancer (TNBC) occurs at higher frequency in African Americans compared with Caucasians. It is unclear if the biology of TNBC is different in African American versus Caucasians. In this study, we sought to evaluate racial differences in the molecular pathology of TNBC. METHODS: Using data from The Cancer Genome Atlas, we identified TNBC patients with information on race. We analyzed differences in clinical characteristics, tumor somatic mutations, and gene expression patterns by race from whole exome and microarray data. RESULTS: 1104 patients were identified, of which 178 had TNBC. TNBC was more frequent in African Americans than Caucasians (33.3 vs 14.9%). Although more African Americans than Caucasians overall were classified as basal-like from PAM50 gene expression (34.8 vs 16.1%), no differences in the TNBC cohort were observed. Median tumor somatic mutation counts were higher in African Americans versus Caucasians (39.5 vs 34), but no racial differences in the mutation counts in TNBC were observed. Somatic mutation analysis revealed racial differences in specific high prevalence genes in all patients (TP53 46% in African Americans vs 27% in Caucasians; PIK3CA 23% in African Americans vs 34% in Caucasians; and MLL3 12% in African Americans vs 6% in Caucasians). TNBC patients did not have any specific high prevalence genes associated with racial differences. There were no racial differences in gene expression patterns in selected genes involved in breast cancer biology. Overall, African Americans had shorter TTP and worse DFS. Racial differences in clinical outcomes were not observed in TNBC. CONCLUSION: The mutational landscape of TNBC is similar between African Americans and Caucasians. The higher frequency of TNBC in African Americans is therefore not associated with a different genomic profile of commonly established tumor regulatory pathway genes. Other modifiable factors may exist that contribute to the racial disparity in TNBC.

Axillary Ultrasound Accurately Excludes Clinically Significant Lymph Node Disease in Patients With Early Stage Breast Cancer.

OBJECTIVE: Assess the performance characteristics of axillary ultrasound (AUS) for accurate exclusion of clinically significant axillary lymph node (ALN) disease. BACKGROUND: Sentinel lymph node biopsy (SLNB) is currently the standard of care for staging the axilla in patients with clinical T1-T2, N0 breast cancer. AUS is a noninvasive alternative to SLNB for staging the axilla. METHODS: Patients were identified using a prospectively maintained database. Sensitivity, specificity, and negative predictive value (NPV) were calculated by comparing AUS findings to pathology results. Multivariate analyses were performed to identify patient and/or tumor characteristics associated with false negative (FN) AUS. A blinded review of FN and matched true negative cases was performed by 2 independent medical oncologists to compare treatment recommendations and actual treatment received. Recurrence-free survival was described using Kaplan-Meier product limit methods. RESULTS: A total of 647 patients with clinical T1-T2, N0 breast cancer underwent AUS between January 2008 and March 2013. AUS had a sensitivity of 70%, NPV of 84%, and PPV of 56% for the detection of ALN disease. For detection of clinically significant disease (>2.0 mm), AUS had a sensitivity of 76% and NPV of 89%. FN AUS did not significantly impact adjuvant medical decision making. Patients with FN AUS had recurrence-free survival equivalent to patients with pathologic N0 disease. CONCLUSIONS: AUS accurately excludes clinically significant ALN disease in patients with clinical T1-T2, N0 breast cancer. AUS may be an alternative to SLNB in these patients, where axillary surgery is no longer considered therapeutic, and predictors of tumor biology are increasingly used to make adjuvant therapy decisions.

US breast cancer mortality trends in young women according to race.

BACKGROUND: Young age at diagnosis has a negative prognostic impact on outcome in patients with breast cancer (BC). In the current study, the authors sought to determine whether there is a differential effect of race and examined mortality trends according to race and age. METHODS: The Surveillance, Epidemiology, and End Results program was used to identify women aged <50 years with invasive BC diagnosed between 1990 and 2009. Multivariate regression analyses were performed to determine the risk-adjusted likelihood of survival for white and black patients. Annual hazards of BC death according to race and calendar period and adjusted relative hazards of death for white and black women stratified by age were computed. RESULTS: A total of 162,976 women were identified, 126,573 of whom were white, 20,405 of whom were black, and 15,998 of whom were of other races. At a median follow-up of 85 months, the 5-year disease specific survival rates were 90.1% for white patients and 79.3% for black patients. Annual hazards of death in white patients decreased by 26% at 5 years after diagnosis in contrast to the hazards in black patients, which decreased by only 19%. With 1990 as the referent year, the adjusted relative hazards of death in women aged <40 years in 2005 were 0.55 (95% confidence interval [95% CI], 0.46-0.66) and 0.68 (95% CI, 0.49-0.93), respectively, for white and black women. In women aged 40 to 49 years, adjusted hazards of death were 0.53 (95% CI, 0.47-0.60) and 0.78 (95% CI, 0.61-0.99), respectively, for white and black women. CONCLUSIONS: Among young women diagnosed with BC, black patients have a worse outcome compared with white patients. Mortality declines have been observed over time in both groups, although more rapid gains have been reported to occur in white women. Emphasis should be placed on improving outcomes for young patients with BC.

Time-trends in survival in young women with breast cancer in a SEER population-based study.

Mortality improvements in young women with breast cancer (BC) may be attributable to treatment advances; screening likely plays a less significant role as mammography is not recommended <40. We examined time-trends in outcome in a cohort of young women. Our goal was to determine the contributions of treatment and screening to mortality improvements and evaluate whether differential outcomes by ER status exist. Using SEER, patients (73,447) were divided into three categories by diagnosis year (1990-1994, 1995-1999, 2000-2004) and also categorized as <40 or 40-50 years. Multivariate analysis was done to investigate the association of survival with time period for both age groups by ER status. Hazard ratios (HR) for mortality in women 40-50 with ER positive BC declined over time. With 1990-1994 as referent, the HR in 1995-1999 was 0.77 (0.69-0.86) and 0.65 (0.59-0.71) in 2000-2004 (p < 0.001). Women <40 with ER positive BC also had improvements over time. In ER negative patients, the degree of improvements over time was less than that seen in ER positive women. We report a survival disparity over time in young women by ER status. Patients with ER negative disease have not had the degree of improvements over time as seen in ER positive disease. Therefore, mortality improvements in young women with ER positive BC may be attributed to treatment advances with endocrine agents.

Impact of the COVID-19 pandemic on breast, colorectal, lung, and prostate cancer stage at diagnosis according to race.

PURPOSE: To determine if the COVID-19 pandemic has further exacerbated racial disparities in late-stage presentation of breast, colorectal, lung, and prostate cancers. METHODS: We conducted a registry-based retrospective study of patients with newly reported diagnoses of breast, colorectal, lung, and prostate cancers between March 2019-June 2019 (pre-COVID-19) and March 2020-June 2020 (early-COVID-19). We compared the volume of new diagnoses and stage at presentation according to race between both periods. RESULTS: During the study period, a total of 3528 patients had newly diagnosed cancer; 3304 of which had known disease stages and were included in the formal analyses. 467 (14.1%) were Blacks, and 2743 were (83%) Whites. 1216 (36.8%) had breast, 415 (12.6%) had colorectal, 827 (25%) had lung, and 846 (25.6%) had prostate cancers, respectively. The pre-COVID-19 period included 2120 (64.2%), and the early-COVID-19 period included 1184 (35.8%), representing a proportional 44.2% decline in the volume of new cases of breast, colorectal, lung, and prostate cancers, p < 0.0001. Pre-COVID-19, 16.8% were diagnosed with metastatic disease, versus 20.4% early-COVID-19, representing a proportional increase of 21.4% in the numbers of new cases with metastatic disease, p = 0.01. There was a non-significant proportional decline of 1.9% in Black patients diagnosed with non-metastatic breast, colorectal, lung, and prostate cancers early-COVID-19 (p = 0.71) and a non-significant proportional increase of 7% in Black patients diagnosed with metastatic disease (p = 0.71). Difference-in-difference analyses showed no statistically significant differences in metastatic presentation comparing Black to White patients. CONCLUSION: While we identified substantial reductions in the volume of new cancer diagnoses and increases in metastatic presentations due to the COVID-19 pandemic, the impact was similar for White and Black patients.

Kinase Inhibitor Pulldown Assay Identifies a Chemotherapy Response Signature in Triple-negative Breast Cancer Based on Purine-binding Proteins.

Triple-negative breast cancer (TNBC) constitutes 10%-15% of all breast tumors. The current standard of care is multiagent chemotherapy, which is effective in only a subset of patients. The original objective of this study was to deploy a mass spectrometry (MS)-based kinase inhibitor pulldown assay (KIPA) to identify kinases elevated in non-pCR (pathologic complete response) cases for therapeutic targeting. Frozen optimal cutting temperature compound-embedded core needle biopsies were obtained from 43 patients with TNBC before docetaxel- and carboplatin-based neoadjuvant chemotherapy. KIPA was applied to the native tumor lysates that were extracted from samples with high tumor content. Seven percent of all identified proteins were kinases, and none were significantly associated with lack of pCR. However, among a large population of "off-target" purine-binding proteins (PBP) identified, seven were enriched in pCR-associated samples (P < 0.01). In orthogonal mRNA-based TNBC datasets, this seven-gene "PBP signature" was associated with chemotherapy sensitivity and favorable clinical outcomes. Functional annotation demonstrated IFN gamma response, nuclear import of DNA repair proteins, and cell death associations. Comparisons with standard tandem mass tagged-based discovery proteomics performed on the same samples demonstrated that KIPA-nominated pCR biomarkers were unique to the platform. KIPA is a novel biomarker discovery tool with unexpected utility for the identification of PBPs related to cytotoxic drug response. The PBP signature has the potential to contribute to clinical trials designed to either escalate or de-escalate therapy based on pCR probability. Significance: The identification of pretreatment predictive biomarkers for pCR in response to neoadjuvant chemotherapy would advance precision treatment for TNBC. To complement standard proteogenomic discovery profiling, a KIPA was deployed and unexpectedly identified a seven-member non-kinase PBP pCR-associated signature. Individual members served diverse pathways including IFN gamma response, nuclear import of DNA repair proteins, and cell death.

Society for Immunotherapy of Cancer (SITC) consensus definitions for resistance to combinations of immune checkpoint inhibitors with chemotherapy.

Although immunotherapy can offer profound clinical benefit for patients with a variety of difficult-to-treat cancers, many tumors either do not respond to upfront treatment with immune checkpoint inhibitors (ICIs) or progressive/recurrent disease occurs after an interval of initial control. Improved response rates have been demonstrated with the addition of ICIs to cytotoxic therapies, leading to approvals from the US Food and Drug Administration and regulatory agencies in other countries for ICI-chemotherapy combinations in a number of solid tumor indications, including breast, head and neck, gastric, and lung cancer. Designing trials for patients with tumors that do not respond or stop responding to treatment with immunotherapy combinations, however, is challenging without uniform definitions of resistance. Previously, the Society for Immunotherapy of Cancer (SITC) published consensus definitions for resistance to single-agent anti-programmed cell death protein 1 (PD-1). To provide guidance for clinical trial design and to support analyses of emerging molecular and cellular data surrounding mechanisms of resistance to ICI-based combinations, SITC convened a follow-up workshop in 2021 to develop consensus definitions for resistance to multiagent ICI combinations. This manuscript reports the consensus clinical definitions for combinations of ICIs and chemotherapies. Definitions for resistance to ICIs in combination with targeted therapies and with other ICIs will be published in companion volumes to this paper.

A phase II study of palbociclib plus letrozole plus trastuzumab as neoadjuvant treatment for clinical stages II and III ER+ HER2+ breast cancer (PALTAN).

Patients with ER+/HER2+ breast cancer (BC) are less likely to achieve pathological complete response (pCR) after chemotherapy with dual HER2 blockade than ER-/HER2+ BC. Endocrine therapy plus trastuzumab is effective in advanced ER+/HER2+ BC. Inhibition of CDK4/6 and HER2 results in synergistic cell proliferation reduction. We combined palbociclib, letrozole, and trastuzumab (PLT) as a chemotherapy-sparing regimen. We evaluated neoadjuvant PLT in early ER+/HER2+ BC. Primary endpoint was pCR after 16 weeks. Research biopsies were performed for whole exome and RNA sequencing, PAM50 subtyping, and Ki67 assessment for complete cell cycle arrest (CCCA: Ki67 ≤ 2.7%). After 26 patients, accrual stopped due to futility. pCR (residual cancer burden-RCB 0) was 7.7%, RCB 0/I was 38.5%. Grade (G) 3/4 treatment-emergent adverse events occurred in 19. Among these, G3/4 neutropenia was 50%, hypertension 26.9%, and leucopenia 7.7%. Analysis indicated CCCA in 85% at C1 day 15 (C1D15), compared to 27% at surgery after palbociclib was discontinued. Baseline PAM50 subtyping identified 31.2% HER2-E, 43.8% Luminal B, and 25% Luminal A. 161 genes were differentially expressed comparing C1D15 to baseline. MKI67, TK1, CCNB1, AURKB, and PLK1 were among the genes downregulated, consistent with CCCA at C1D15. Molecular Signatures Database gene-sets analyses demonstrated downregulated processes involved in proliferation, ER and mTORC1 signaling, and DNA damage repair at C1D15, consistent with the study drug's mechanisms of action. Neoadjuvant PLT showed a pCR of 7.7% and an RCB 0/I rate of 38.5%. RNA sequencing and Ki67 data indicated potent anti-proliferative effects of study treatments. ClinicalTrials.gov- NCT02907918.

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.

Aldehyde dehydrogenase 1A1 expression in breast cancer is associated with stage, triple negativity, and outcome to neoadjuvant chemotherapy.

Studies have shown that ALDH1A1 expression in the breast is associated with worse clinical outcome. ALDH1A1 inactivates cyclophosphamide, which is an integral agent in breast cancer chemotherapy regimens. The purposes of this study were to verify these results, to correlate ALDH1A1 expression with clinical outcome in patients treated with cyclophosphamide as part of the chemotherapy (adjuvant or neoadjuvant), and to evaluate ALDH1A1 as a useful marker to predict the clinical outcome of breast cancer subsets. A total of 513 primary breast cancers were studied. Tissue microarrays of the studied cases were stained with ALDH1A1. Key clinicopathological information was obtained. Disease-free survival and overall survival were calculated. Patients with neoadjuvant therapy who had substantial residual cancer burden (RCB) were included in the study. Fisher's exact test and Kaplan-Meier methods were used for statistical analysis. ALDH1A1 was expressed in 53 (10%) patients, with a higher frequency in triple negative, followed by HER2+, and finally hormonal receptor+/HER2- (P<0.0001). Tumors with advanced stage, node-positive, or larger tumor size were correlated with ALDH1A1 expression (P=0.006, P<0.0001, and P=0.05, respectively). ALDH1A1 expression was also correlated with worse disease-free survival (P<0.006) and overall survival (P<0.01) in patients who were treated with neoadjuvant chemotherapy. In all, 8 of 22 (36%) received neoadjuvant chemotherapy and died of disease-expressed ALDH1A1 (P=0.008). Similarly, 8 of 23 (35%) who received neoadjuvant chemotherapy and had tumor recurrence expressed this marker (P=0.002). The risk of recurrence was fivefold greater than negative ALDH1A1 tumors. The risk of recurrence became 11-fold greater when cyclophosphamide but not trastuzumab was part of the regimen. Our results are consistent with previous studies. Moreover, we found that ALDH1A1 could be a useful marker to predict worse clinical outcome after chemotherapy in the neoadjuvant setting with substantial RCB. However, a larger cohort is required to verify our results.