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BackgroundPathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence.Patients and methodsCell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years.ResultsAt T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P = 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5).ConclusionsLack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Cancer cell lines are a cornerstone of cancer research but previous studies have shown that not all cell lines are equal in their ability to model primary tumors. Here we present a comprehensive pan-cancer analysis utilizing transcriptomic profiles from The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia to evaluate cell lines as models of primary tumors across 22 tumor types. We perform correlation analysis and gene set enrichment analysis to understand the differences between cell lines and primary tumors. Additionally, we classify cell lines into tumor subtypes in 9 tumor types. We present our pancreatic cancer results as a case study and find that the commonly used cell line MIA PaCa-2 is transcriptionally unrepresentative of primary pancreatic adenocarcinomas. Lastly, we propose a new cell line panel, the TCGA-110-CL, for pan-cancer studies. This study provides a resource to help researchers select more representative cell line models.

Abstract: Background: WISDOM is a 100,000 healthy women preference-tolerant, pragmatic study comparing annual to personalized risk-based breast screening. The novelty of WISDOM personalized screening is the integration of previously validated genetic and clinical risk factors (age, family history, breast biopsy results, ethnicity, mammographic density) into a single risk assessment model that directs the starting age, timing, and frequency of screening. The goal of WISDOM is to determine if personalized screening, compared to annual screening, is as safe, less morbid, enables prevention, and is preferred by women. The study is registered on ClinicalTrials.gov, NCT02620852. Methods: Women aged 40-74 years with no history of breast cancer or DCIS, and no previous double mastectomy can join the study online at wisdomstudy.org. Participants can elect randomization or self-select a study arm, and provide electronic consent and Release for Medical Information using DocuSign. For all participants, 5-year risk of developing breast cancer is calculated according to the Breast Cancer Screening Consortium (BCSC) model. Participants in the personalized arm undergo panel-based mutation testing, and their 5-year risk is calculated using the BCSC score combined with a Polygenic Risk Score (BCSC-PRS) that includes 75 single nucleotide polymorphisms (SNPs, increase to 229) known to increase breast cancer risk. SNPs and mutations (BRCA1, BRCA2, TP53, PTEN, STK11, CDH1, ATM, PALB2, and CHEK2) are assessed by saliva-based testing through Color Genomics. 5-year risk level thresholds are used to stratify for low-, moderate- and high risk. Risk stratification determines age to start, stop, and frequency of screening. Enrollment: As of July 2018, the WISDOM study is open to all eligible women in California, North Dakota, South Dakota, Minnesota and Iowa. To date, 23,329 eligible women have registered and 14,393 women have consented to participate in the trial. We analyzed 3,255 participants who have completed risk assessment in the personalized arm. The median age was 56 years. 82% were Caucasian, 1% African-American, and 6% Asian. 9% self-reported as Hispanic. We are partnering with health insurers and self-insured companies using coverage with evidence progression. To strengthen generalizability, we are expanding to other states. WISDOM enrollment will continue past 2019. Feasibility: To evaluate the addition of PRS, we used paired statistical tests (McNemar) to compare the distributions of BCSC, and BCSC-PRS risk estimates around low-risk (6%) thresholds, the latter corresponding to 5-year risk of a BRCA mutation carrier. The median 5-year risk was 1.5% (IQR 1.0-2.1%) using the BCSC model, and 1.4% (IQR 0.8-2.5%) using the BCSC-PRS model. The BCSC-PRS model classified more women into the low (

BackgroundSomatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer.MethodsA single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC).ResultsRs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95-0.99, P=4.6 × 10(-3)), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96-1.01, P=0.139).ConclusionCommon germline variation in PIK3CA does not have a strong influence on the risk of breast cancer.

BackgroundUnderstanding the molecular biology of colorectal cancer (CRC) provides opportunities for effective personalised patient management. We evaluated whether chromosomal aberrations, mutations in the PI(3)K signalling pathway and the CpG-island methylator phenotype (CIMP) in primary colorectal tumours can predict liver metastases.MethodsFormalin-fixed paraffin-embedded material from primary colorectal tumours of three different groups were investigated: patients with CRC without metastases (M0, n=39), patients who were treated with hyperthermal intraperitoneal chemotherapy for CRC metastases confined to the peritoneum (PM, n=46) and those who had isolated hepatic perfusion for CRC metastases confined to the liver (LM, n=48).ResultsAll samples were analysed for DNA copy number changes, PIK3CA, KRAS, BRAF mutations, CIMP and microsatellite instability. The primary CRCs of the LM group had significantly higher frequency of amplified chromosome 20q (P=0.003), significantly fewer mutations in the PI(3)K signalling pathway (P=0.003) and fewer CIMP high tumours (P=0.05). There was a strong inverse correlation between 20q and the PI(3)K pathway mutations.ConclusionThe development of CRC liver metastases is associated with amplification of chromosome 20q and not driven by mutations in the PI(3)K signalling pathway.

Backgroundoverexpression of HER-2 is observed in 15-25% of breast cancers, and is associated with increased risk of recurrence. Current guidelines recommend trastuzumab and chemotherapy for most HER-2-positive patients. However, the majority of patients does not recur and might thus be overtreated with adjuvant systemic therapy. We investigated whether the 70-gene MammaPrint signature identifies HER-2-positive patients with favourable outcome.Methodsin all, 168 T1-3, N0-1, HER-2-positive patients were identified from a pooled database, classified by the 70-gene signature as good or poor prognosis, and correlated with long-term outcome. A total of 89 of these patients did not receive adjuvant chemotherapy.Resultsin the group of 89 chemotherapy-naive patients, after a median follow-up of 7.4 years, 35 (39%) distant recurrences and 29 (33%) breast cancer-specific deaths occurred. The 70-gene signature classified 20 (22%) patients as good prognosis, with 10-year distant disease-free survival (DDFS) of 84%, compared with 69 (78%) poor prognosis patients with 10-year DDFS of 55%. The estimated hazard ratios (HRs) were 4.5 (95% confidence interval (CI) 1.1-18.7, P=0.04) and 3.8 (95% CI 0.9-15.8, P=0.07) for DDFS and breast cancer-specific survival (BCSS), respectively. In multivariate analysis adjusted for known prognostic factors and hormonal therapy, HRs were 5.8 (95% CI 1.3-26.7, P=0.03) and 4.7 (95% CI 1.0-21.7, P=0.05) for DDFS and BCSS, respectively.Interpretationthe 70-gene prognosis signature is an independent prognostic indicator that identifies a subgroup of HER-2-positive early breast cancer with a favourable long-term outcome.

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 μM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7±2.9 adducts/108 nucleotides, n=8), followed by lymphocytes (5.9±1.7, n=8), and granulocytes (0.5±0.2, n=8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7±1.0, n=5) > monocytes (2.0±0.5, n=8)  lymphocytes (1.6±0.4, n=8) >granulocytes (0.8±0.2, n=8) by NP1-enrichment and monocytes (9.0±3.2, n=5)  lymphocytes (8.0±2.1, n=6) > granulocytes (2.1±0.3, n=7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n=78). Adduct levels in MNC were related to daily exposure to cigarette tar (r=0.31, P<0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6 fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r=0.86, P<0.001, n=12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts. [ABSTRACT FROM PUBLISHER]

Among the goals of patient-centric care are the advancement of effective personalized treatment, while minimizing toxicity. The phase 2 I-SPY2.2 trial uses a neoadjuvant sequential therapy approach in breast cancer to further these goals, testing promising new agents while optimizing individual outcomes. Here we tested datopotamab-deruxtecan (Dato-DXd) in the I-SPY2.2 trial for patients with high-risk stage 2/3 breast cancer. I-SPY2.2 uses a sequential multiple assignment randomization trial design that includes three sequential blocks of biologically targeted neoadjuvant treatment: the experimental agent(s) (block A), a taxane-based regimen tailored to the tumor subtype (block B) and doxorubicin-cyclophosphamide (block C). Patients are randomized into arms consisting of different investigational block A treatments. Algorithms based on magnetic resonance imaging and core biopsy guide treatment redirection after each block, including the option of early surgical resection in patients predicted to have a high likelihood of pathological complete response, the primary endpoint. There are two primary efficacy analyses: after block A and across all blocks for the six prespecified breast cancer subtypes (defined by clinical hormone receptor/human epidermal growth factor receptor 2 (HER2) status and/or the response-predictive subtypes). We report results of 103 patients treated with Dato-DXd. While Dato-DXd did not meet the prespecified threshold for success (graduation) after block A in any subtype, the treatment strategy across all blocks graduated in the hormone receptor-negative HER2 - Immune - DNA repair deficiency - subtype with an estimated pathological complete response rate of 41%. No new toxicities were observed, with stomatitis and ocular events occurring at low grades. Dato-DXd was particularly active in the hormone receptor-negative/HER2 - Immune - DNA repair deficiency - signature, warranting further investigation, and was safe in other subtypes in patients who followed the treatment strategy. ClinicalTrials.gov registration: NCT01042379 ., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Sequential adaptive trial designs can help accomplish the goals of personalized medicine, optimizing outcomes and avoiding unnecessary toxicity. Here we describe the results of incorporating a promising antibody-drug conjugate, datopotamab-deruxtecan (Dato-DXd) in combination with programmed cell death-ligand 1 inhibitor, durvalumab, as the first sequence of therapy in the I-SPY2.2 phase 2 neoadjuvant sequential multiple assignment randomization trial for high-risk stage 2/3 breast cancer. The trial includes three blocks of treatment, with initial randomization to different experimental agent(s) (block A), followed by a taxane-based regimen tailored to tumor subtype (block B), followed by doxorubicin-cyclophosphamide (block C). Subtype-specific algorithms based on magnetic resonance imaging volume change and core biopsy guide treatment redirection after each block, including the option of early surgical resection in patients predicted to have a high likelihood of pathologic complete response, which is the primary endpoint assessed when resection occurs. There are two primary efficacy analyses: after block A and across all blocks for six prespecified HER2-negative subtypes (defined by hormone receptor status and/or response-predictive subtypes). In total, 106 patients were treated with Dato-DXd/durvalumab in block A. In the immune-positive subtype, Dato-DXd/durvalumab exceeded the prespecified threshold for success (graduated) after block A; and across all blocks, pathologic complete response rates were equivalent to the rate expected for the standard of care (79%), but 54% achieved that result after Dato-DXd/durvalumab alone (block A) and 92% without doxorubicin-cyclophosphamide (after blocks A + B). The treatment strategy across all blocks graduated in the hormone-negative/immune-negative subtype. No new toxicities were observed. Stomatitis was the most common side effect in block A. No patients receiving block A treatment alone had adrenal insufficiency. Dato-DXd/durvalumab is a promising therapy combination that can eliminate standard chemotherapy in many patients, particularly the immune-positive subtype.ClinicalTrials.gov registration: NCT01042379 ., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: MammaPrint (MP) determines distant metastatic risk and may improve patient selection for extended endocrine therapy (EET). This study examined MP in predicting extended letrozole therapy (ELT) benefit in patients with early-stage breast cancer (BC) from the NSABP B-42 trial., Patients and Methods: MP was tested in 1,866 patients randomly assigned to receive ELT or placebo. The primary end point was distant recurrence (DR). Secondary end points were disease-free survival (DFS) and BC-free interval (BCFI). Tumors were classified as MP high risk (MP-HR) or low risk (MP-LR). MP-LR tumors were further classified as ultralow risk (MP-UL) or low non-ultralow risk (MP-LNUL)., Results: There was no statistically significant difference in ELT benefit on DR between MP-HR and MP-LR (interaction P = .38). MP-LR tumors (n = 1,160) exhibited a statistically significant 10-year benefit of 3.7% for DR (hazard ratio [HR], 0.43 [95% CI, 0.25 to 0.74]; P = .002), whereas MP-HR tumors (n = 706) exhibited a nonsignificant 2.4% benefit (HR, 0.65 [95% CI, 0.34 to 1.24]; P = .19). The 10-year ELT benefit was significant for DFS (7.8%) and BCFI (7.0%) for MP-LR tumors, whereas MP-HR tumors did not significantly benefit (interaction DFS: P = .015, BCFI: P = .006). In exploratory analysis, the 10-year ELT benefit was significant and more pronounced in MP-LNUL (n = 908) tumors: 4.0% for DR, 9.5% for DFS, and 7.9% for BCFI; the benefit in MP-UL (n = 252) tumors was not significant: 3% for DR, 1.8% for DFS, and 4.1% for BCFI., Conclusion: The primary hypothesis of predictive ability of MP on DR was not confirmed. However, the secondary outcomes demonstrated MP was predictive of ELT response and identified a subset of patients with early-stage hormone receptor-positive BC (MP-LR) with improved outcomes from ELT. These data could have important clinical implications in patient selection beyond clinical risk assessment for EET.

Purpose: We previously demonstrated the clinical significance of circulating tumor DNA (ctDNA) in patients with HER2-negative breast cancer receiving neoadjuvant chemotherapy (NAC). Here, we compared its predictive and prognostic value with cell-free DNA (cfDNA) concentration measured in the same samples from the same patients., Experimental Design: 145 patients with hormone receptor (HR)-positive/HER2-negative and 138 triple-negative breast cancer (TNBC) with ctDNA data from a previous study were included in the analysis. Associations of serial cfDNA concentration with residual cancer burden (RCB) and distant recurrence-free survival (DRFS) were examined., Results: In TNBC, we observed a modest negative correlation between cfDNA concentration 3 weeks after treatment initiation and RCB, but none of the other timepoints showed significant correlation. In contrast, ctDNA was significantly positively correlated with RCB at all timepoints (all R > 0.3 and P < 0.05). In the HR-positive/HER2-negative group, cfDNA concentration did not associate with response to NAC, but survival analysis showed that high cfDNA shedders at pretreatment had a significantly worse DRFS than low shedders (hazard ratio, 2.12; P = 0.037). In TNBC, the difference in survival between high versus low cfDNA shedders at all timepoints was not statistically significant. In contrast, as previously reported, ctDNA at all timepoints was significantly correlated with DRFS in both subtypes., Conclusions: In TNBC, cfDNA concentrations during therapy were not strongly correlated with response or prognosis. In the HR-positive/HER2-negative group, pretreatment cfDNA concentration was prognostic for DRFS. Overall, the predictive and prognostic value of cfDNA concentration was more limited than that of ctDNA., (©2024 American Association for Cancer Research.)

Biomarkers for evaluating tumor response to therapy and estimating the risk of disease relapse represent tremendous areas of clinical need. To evaluate treatment efficacy, tumor response is routinely assessed using different imaging modalities like positron emission tomography/computed tomography or magnetic resonance imaging. More recently, the development of circulating tumor DNA detection assays has provided a minimally invasive approach to evaluate tumor response and prognosis through a blood test (liquid biopsy). Integrating imaging- and circulating tumor DNA-based biomarkers may lead to improvements in the prediction of patient outcomes. For this mini-review, we searched the scientific literature to find original articles that combined quantitative imaging and circulating tumor DNA biomarkers to build prediction models. Seven studies reported building prognostic models to predict distant recurrence-free, progression-free, or overall survival. Three discussed building models to predict treatment response using tumor volume, pathologic complete response, or objective response as endpoints. The limited number of articles and the modest cohort sizes reported in these studies attest to the infancy of this field of study. Nonetheless, these studies demonstrate the feasibility of developing multivariable response-predictive and prognostic models using regression and machine learning approaches. Larger studies are warranted to facilitate the building of highly accurate response-predictive and prognostic models that are generalizable to other datasets and clinical settings.

Combinations of immune checkpoint inhibitors (ICI, including anti-PD-1/PD-L1) and chemotherapy have been FDA approved for metastatic and early-stage triple-negative breast cancer (TNBC), but most patients do not benefit. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with anti-PD-L1 resistance. However, little is known about its regulation and effect on immune cell function in breast cancers. We assessed murine and human breast cancer cells to identify regulation mechanisms of B7-H4 in vitro. We used an immunocompetent anti-PD-L1-sensitive orthotopic mammary cancer model and induced ectopic expression of B7-H4. We assessed therapy response and transcriptional changes at baseline and under treatment with anti-PD-L1. We observed B7-H4 was highly associated with epithelial cell status and transcription factors and found to be regulated by PI3K activity. EMT6 tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. In addition, tumor-infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. Paradoxically, in human breast cancer, B7-H4 expression was associated with survival benefit for patients with metastatic TNBC treated with carboplatin plus anti-PD-L1 and was associated with no change in response or survival for patients with early breast cancer receiving chemotherapy plus anti-PD-1. While B7-H4 induces tumor resistance to anti-PD-L1 in murine models, there are alternative mechanisms of signaling and function in human cancers. In addition, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 independent of PD-L1., Significance: This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: A number of studies are currently investigating de-escalation of radiation therapy in patients with a low risk of in-breast relapses on the basis of clinicopathologic factors and molecular tests. We evaluated whether 70-gene risk score is associated with risk of locoregional recurrence (LRR) and estimated 8-year cumulative incidences for LRR in patients with early-stage breast cancer treated with breast conservation., Methods: In this exploratory substudy of European Organisation for Research and Treatment of Cancer 10041/BIG 03-04 MINDACT trial, we evaluated women with a known clinical and genomic 70-gene risk score test result and who had breast-conserving surgery (BCS). The primary end point was LRR at 8 years, estimated by cumulative incidences. Distant metastasis and death were considered competing risks., Results: Among 6,693 enrolled patients, 5,470 (81.7%) underwent BCS, of whom 98% received radiotherapy. At 8-year follow-up, 189 patients experienced a LRR, resulting in an 8-year cumulative incidence of 3.2% (95% CI, 2.7 to 3.7). In patients with a low-risk 70-gene signature, the 8-year LRR incidence was 2.7% (95% CI, 2.1 to 3.3). In univariable analysis, adjusted for chemotherapy, five of 12 variables were associated with LRR, including the 70-gene signature. In multivariable modeling, adjuvant endocrine therapy and to a lesser extent tumor size and grade remained significantly associated with LRR., Conclusion: This exploratory analysis of the MINDACT trial estimated an 8-year low LRR rate of 3.2% after BCS. The 70-gene signature was not independently predictive of LRR perhaps because of the low number of events observed and currently cannot be used in clinical decision making regarding LRR. The overall low number of events does provide an opportunity to design trials toward de-escalation of local therapy.

Purpose: At the primary analysis, the APHINITY trial reported a statistically significant but modest benefit of adding pertuzumab to standard adjuvant chemotherapy plus trastuzumab in patients with histologically confirmed human epidermal growth factor receptor 2 (HER2)-positive early-stage breast cancer. This study evaluated whether the 80-gene molecular subtyping signature (80-GS) could identify patients within the APHINITY population who derive the most benefit from dual anti-HER2 therapy., Methods: In a nested case-control study design of 1,023 patients (matched event to control ratio of 3:1), the 80-GS classified breast tumors into functional luminal type, HER2 type, or basal type. Additionally, 80-GS distinguished tumor subtypes that exhibited a single-dominant functional pathway versus tumors with multiple activated pathways. The primary end point was invasive disease-free survival (IDFS). Hazard ratios (HRs) were evaluated by Cox regression. After excluding patients without appropriate consent and those with missing data, 964 patients were included., Results: The 80-GS classified 50% (n = 479) of tumors as luminal type, 28% (n = 275) as HER2 type, and 22% (n = 209) as basal type. Most luminal-type tumors (86%) displayed a single-activated pathway, whereas 49% of HER2-type and 42% of basal-type tumors were dual activated. There was no significant difference in IDFS among different conventional 80-GS subtypes (single- and dual-activated subtypes combined). However, basal single-subtype tumors were significantly more likely to have an IDFS event (hazard ratio, 1.69 [95% CI, 1.12 to 2.54]) compared with other subtypes. HER2 single-subtype tumors displayed a trend toward greater beneficial effect on the addition of pertuzumab (hazard ratio, 0.56 [95% CI, 0.27 to 1.16]) compared with all other subtypes., Conclusion: The 80-GS identified subgroups of histologically confirmed HER2-positive tumors with distinct biological characteristics. Basal single-subtype tumors exhibit an inferior prognosis compared with other subgroups and may be candidates for additional therapeutic strategies. Preliminary results suggest patients with HER2-positive, genomically HER2 single-subtype tumors may particularly benefit from added pertuzumab, which warrants further investigation.

Molecular subtyping of breast cancer is based mostly on HR/HER2 and gene expression-based immune, DNA repair deficiency, and luminal signatures. We extend this description via functional protein pathway activation mapping using pre-treatment, quantitative expression data from 139 proteins/phosphoproteins from 736 patients across 8 treatment arms of the I-SPY 2 Trial (ClinicalTrials.gov: NCT01042379). We identify predictive fit-for-purpose, mechanism-of-action-based signatures and individual predictive protein biomarker candidates by evaluating associations with pathologic complete response. Elevated levels of cyclin D1, estrogen receptor alpha, and androgen receptor S650 associate with non-response and are biomarkers for global resistance. We uncover protein/phosphoprotein-based signatures that can be utilized both for molecularly rationalized therapeutic selection and for response prediction. We introduce a dichotomous HER2 activation response predictive signature for stratifying triple-negative breast cancer patients to either HER2 or immune checkpoint therapy response as a model for how protein activation signatures provide a different lens to view the molecular landscape of breast cancer and synergize with transcriptomic-defined signatures., Competing Interests: Declaration of interests J.W. reports honoraria from DAVA Oncology, consults for Baylor College of Medicine, has ownership in Theralink, and is co-inventor of RPPA technology and p-HER2 and -EGFR response predictors with filed patents. C.Y. consults for NantOmics, LLC. M.C.L. reports support from Eisai, Genentech, GRAIL, Menarini Silicon Biosystems, Merck, Novartis, Seattle Genetics, and Tesaro. W.F.S. is a co-founder of Delphi Diagnostics and co-inventor/patent holder for a free residual cancer burden calculator, holds shares in IONIS Pharmaceuticals and Eiger Biopharmaceuticals, and is an unpaid advisor/steering committee member for Roche trials. H.S.R. reports support from Pfizer, Merck, Novartis, Lilly, Roche, Daiichi, Seattle Genetics, Macrogenics, Sermonix, Boehringer Ingelheim, Polyphor, AstraZeneca, Astellas, and Gilead; honoraria from Puma Biotechnology, Samsung, Chugai, Blueprint, and NAPO; and travel support from GE Healthcare. C.I. consults for Seattle Genetics, Genentech, AstraZeneca, Novartis, PUMA, Pfizer, and Esai. A.M.D. reports support from Novartis, Pfizer, Genentech, Calithera, and Menarini. D.Y. receives unrelated support from Boehringer Ingleheim and consults with Martell Diagnostics unrelated to this topic. P.R.P. reports leadership and stock in Immunonet BioSciences and honoraria from ASCO, Dava Oncology, OncLive (Courses), and Frontiers (Editorship); consults for Personalized Cancer Therapy, Immunonet BioSciences, Sirtex, CARIS Lifesciences, OncoPlex Diagnostics, Pfizer, Heron, Puma, AbbVie, BOLT, and SEAGEN; and is an occasional speaker for Genentech and Roche. G.L.H. is a partner and holds stock (<1%) in NanoString, Moderna, Gilead Sciences, and Exact Sciences. L.J.E. is an unpaid member of the board of directors of Quantum Leap Healthcare Collaborative, received grant support for the I-SPY 2 trial, is on the Blue Cross/Blue Shield Medical Advisory Panel and receives support for time and travel, and receives unrelated research support from Merck. L.J.v.V. is a part-time employee and stockholder of Agendia, NV. E.F.P. reports leadership, stock/ownership, and consulting/advisory and travel funds from Theralink Technologies, Inc., Perthera, Inc., and Ceres Nanosciences, Inc.; support from Ceres Nanosciences, GlaxoSmithKline, Abbvie, Symphogen, Deciphera Pharmaceuticals, Inc, Springworks Therapeutics, Inc, Mirati, Inc. and Genentech; patents/royalties from NIH; and patents/roylaties for anti-HER2/EGFR and anti-mTOR response predictors., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Importance: There has been little consideration of genomic risk of recurrence by breast cancer subtype despite evidence of racial disparities in breast cancer outcomes., Objective: To evaluate associations between clinical trial end points, namely pathologic complete response (pCR) and distant recurrence-free survival (DRFS), and race and examine whether gene expression signatures are associated with outcomes by race., Design, Setting, and Participants: This retrospective cohort study used data from the Investigation of Serial Studies to Predict Your Therapeutic Response With Imaging and Molecular Analysis 2 (I-SPY 2) multicenter clinical trial of neoadjuvant chemotherapy with novel agents and combinations for patients with previously untreated stage II/III breast cancer. Analyses were conducted of associations between race and short- and long-term outcomes, overall and by receptor subtypes, and their association with 28 expression biomarkers. The trial enrolled 990 female patients between March 30, 2010, and November 5, 2016, with a primary tumor size of 2.5 cm or greater and clinical or molecular high risk based on MammaPrint or hormone receptor (HR)-negative/ERBB2 (formerly HER2 or HER2/neu)-positive subtyping across 9 arms. This data analysis was performed between June 10, 2021, and October 20, 2022., Exposure: Race, tumor receptor subtypes, and genomic biomarker expression of early breast cancer., Main Outcomes and Measures: The primary outcomes were pCR and DRFS assessed by race, overall, and by tumor subtype using logistic regression and Cox proportional hazards regression models. The interaction between 28 expression biomarkers and race, considering pCR and DRFS overall and within subtypes, was also evaluated., Results: The analytic sample included 974 participants (excluding 16 self-reporting as American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, or multiple races due to small sample sizes), including 68 Asian (7%), 120 Black (12%), and 786 White (81%) patients. Median (range) age at diagnosis was 47 (25-71) years for Asian, 49 (25-77) for Black, and 49 (23-73) years for White patients. The pCR rates were 32% (n = 22) for Asian, 30% for Black (n = 36), and 32% for White (n = 255) patients (P = .87). Black patients with HR-positive/ERBB2-negative tumors not achieving pCR had significantly worse DRFS than their White counterparts (hazard ratio, 2.28; 95% CI, 1.24-4.21; P = .01), with 5-year DRFS rates of 55% (n = 32) and 77% (n = 247), respectively. Black patients with HR-positive/ERBB2-negative tumors, compared with White patients, had higher expression of an interferon signature (mean [SD], 0.39 [0.87] and -0.10 [0.99]; P = .007) and, compared with Asian patients, had a higher mitotic score (mean [SD], 0.07 [1.08] and -0.69 [1.06]; P = .01) and lower estrogen receptor/progesterone receptor signature (mean [SD], 0.31 [0.90] and 1.08 [0.95]; P = .008). A transforming growth factor β signature had a significant association with race relative to pCR and DRFS, with a higher signature associated with lower pCR and worse DRFS outcomes among Black patients only., Conclusions and Relevance: The findings show that women with early high-risk breast cancer who achieve pCR have similarly good outcomes regardless of race, but Black women with HR-positive/ERBB2-negative tumors without pCR may have worse DRFS than White women, highlighting the need to develop and test novel biomarker-informed therapies in diverse populations.

Background: Despite major improvements in treatment of HER2-positive metastatic breast cancer (MBC), only few patients achieve complete remission and remain progression free for a prolonged time. The tumor immune microenvironment plays an important role in the response to treatment in HER2-positive breast cancer and could contain valuable prognostic information. Detailed information on the cancer-immune cell interactions in HER2-positive MBC is however still lacking. By characterizing the tumor immune microenvironment in patients with HER2-positive MBC, we aimed to get a better understanding why overall survival (OS) differs so widely and which alternative treatment approaches may improve outcome., Methods: We included all patients with HER2-positive MBC who were treated with trastuzumab-based palliative therapy in the Netherlands Cancer Institute between 2000 and 2014 and for whom pre-treatment tissue from the primary tumor or from metastases was available. Infiltrating immune cells and their spatial relationships to one another and to tumor cells were characterized by immunohistochemistry and multiplex immunofluorescence. We also evaluated immune signatures and other key pathways using next-generation RNA-sequencing data. With nine years median follow-up from initial diagnosis of MBC, we investigated the association between tumor and immune characteristics and outcome., Results: A total of 124 patients with 147 samples were included and evaluated. The different technologies showed high correlations between each other. T-cells were less prevalent in metastases compared to primary tumors, whereas B-cells and regulatory T-cells (Tregs) were comparable between primary tumors and metastases. Stromal tumor-infiltrating lymphocytes in general were not associated with OS. The infiltration of B-cells and Tregs in the primary tumor was associated with unfavorable OS. Four signatures classifying the extracellular matrix of primary tumors showed differential survival in the population as a whole., Conclusions: In a real-world cohort of 124 patients with HER2-positive MBC, B-cells, and Tregs in primary tumors are associated with unfavorable survival. With this paper, we provide a comprehensive insight in the tumor immune microenvironment that could guide further research into development of novel immunomodulatory strategies., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Purpose: Oligometastatic breast cancer (OMBC) has a more favorable outcome than widespread metastatic breast cancer. Some patients with OMBC achieve long-term remission if treated with multimodality therapy, including systemic and locally ablative therapies. However, not all patients with OMBC benefit from such treatment, while all experience toxicity. To explore biomarkers identifying patients with OMBC and potential long-term survival, we compared tumor-immune characteristics of patients with OMBC and long-term versus shorter-term survival., Materials and Methods: We collected tumor tissue of 97 patients with de novo OMBC (≤5 metastases) via the Dutch nationwide cancer and pathology registries using a case-control design. Long-term survivors (LTS) were defined as patients alive ≥10 years since OMBC diagnosis. Fifty-five LTS and 42 shorter-term survivors (STS) were included. Median follow-up was 15 years (IQR, 14-16). Tumor characteristics and infiltrating immune cells were assessed by immunohistochemistry and next-generation RNA-sequencing. Association of the resulting 52 biomarkers with long-term survival was assessed using logistic regression. Associations with survival within LTS were assessed using Cox-proportional hazards modeling. P values were adjusted for multiple hypothesis testing., Results: Most patients had estrogen receptor (ER)-positive OMBC (n = 86; 89%) and 23 (24%) had human epidermal growth factor receptor 2-positive disease. ER positivity in primary tumors distinguished LTS from STS. In addition, extracellular matrix (ECM)2-low and ECM4-high distinguished between long-term and shorter-term survival. Immune levels in the primary tumor did not associate with LTS. However, within the LTS subset, higher immune levels associated with improved progression-free survival., Conclusion: We identified tumor and ECM features in the primary tumor of patients with de novo OMBC that were associated with long-term survival. Our data should be validated in other patients with OMBC before they can be used in clinical practice.

Circulating tumor DNA (ctDNA) analysis may improve early-stage breast cancer treatment via non-invasive tumor burden assessment. To investigate subtype-specific differences in the clinical significance and biology of ctDNA shedding, we perform serial personalized ctDNA analysis in hormone receptor (HR)-positive/HER2-negative breast cancer and triple-negative breast cancer (TNBC) patients receiving neoadjuvant chemotherapy (NAC) in the I-SPY2 trial. ctDNA positivity rates before, during, and after NAC are higher in TNBC than in HR-positive/HER2-negative breast cancer patients. Early clearance of ctDNA 3 weeks after treatment initiation predicts a favorable response to NAC in TNBC only. Whereas ctDNA positivity associates with reduced distant recurrence-free survival in both subtypes. Conversely, ctDNA negativity after NAC correlates with improved outcomes, even in patients with extensive residual cancer. Pretreatment tumor mRNA profiling reveals associations between ctDNA shedding and cell cycle and immune-associated signaling. On the basis of these findings, the I-SPY2 trial will prospectively test ctDNA for utility in redirecting therapy to improve response and prognosis., Competing Interests: Declaration of interests R.W.S. owns stock in Pfizer Inc., AstraZeneca, and Moderna Inc. D.R., E.K., A.R., H.S., A.A., M.C.L., and M.R. are employees of and/or hold stock or stock options in Natera Inc. A.L.D. reports honoraria from the Department of Defense and the California Breast Cancer Research Program (CBCRP). M.C.L. reports funding from Eisai, Exact Sciences, Genentech, Genomic Health, GRAIL, Menarini Silicon Biosystems, Merck, Novartis, and Seattle Genetics; participation on advisory boards (no personal compensation) of Adela, Astra Zeneca, Celgene, Roche/Genentech, Genomic Health, GRAIL, Ionis, Merck, Pfizer, Seattle Genetics, Syndax; meeting support from Agena, Astra Zeneca, Celgene, Cynvenio, Genomic Health, GRAIL, Ionis, Menarini Silicon Biosystems, Merck, Pfizer. KA reports support from Merck, Seattle Genetics, Amgen, Genentech-Roche; Daiichi Sankyo, and AstraZeneca; participation on an advisory board for Genomic Health/Exact Sciences, Genentech-Roche, and a data and safety monitoring board for Seattle Genetics/Axio. A.J.C. reports funding from Novartis. A.F.-T. is an employee of Seagen. C.I. reports funding from Tesaro/GlaxoSmithKline, Seattle Genetics, Pfizer, AstraZeneca, Bristol Myers Squibb, Genentech, Novartis, PUMA, Eisai, Sanofi, ION, and Gilead. R.N. reports funding from Arvinas, AstraZeneca, Celgene, Corcept Therapeutics, Genentech/Roche, Gilead/Immunomedics, Merck, OBI Pharma Inc., OncoSec Medical, Pfizer, Relay Therapeutics, Seattle Genetics, Sun Pharmaceutical Industries Ltd., Taiho Pharmaceutica, BeyondSpring Inc., FUJIFILM Pharmaceuticals, Infintiy Pharmaceuticals Inc., ITeos Therapeutics, and Seagen. J.P. reports honoraria from Methods in Clinical Research. W.F.S. reports funding from AstraZeneca and Pfizer; owns stock in IONIS Pharmaceuticals and Eiger Biopharmaceuticals; and receives royalties for patents licensed by the MD Anderson Cancer Center to Delphi Diagnostics, Inc. D.Y. reports funding from Fusion Pharmaceutical, Boehringer Ingelheim, Martell Diagnostics, and Akston Biosciences. L.J.E. reports funding from Merck & Co.; participation on an advisory board for Blue Cross Blue Shield; and personal fees from UpToDate. A.M.D. reports funding from Pfizer, Genentech, Novartis, Inivata Ltd., and Calithera Biosciences. H.S.R. reports funding from Pfizer, Merck, Novartis, Lilly, Roche, Daiichi, Seattle Genetics, Macrogenics, Sermonix, Boehringer Ingelheim, Polyphor, AstraZeneca, Ayala, Astellas, Gilead, Puma, Samsung, Chugai, Blueprint, NAPO, and GE Healthcare. L.J.v.V. is a part-time employee and owns stock in Agendia. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: ROR1 and ROR2 are Type 1 tyrosine kinase-like orphan receptors for Wnt5a that are associated with breast cancer progression. Experimental agents targeting ROR1 and ROR2 are in clinical trials. This study evaluated whether expression levels of ROR1 or ROR2 correlated with one another or with clinical outcomes., Methods: We interrogated the clinical significance of high-level gene expression of ROR1 and/or ROR2 in the annotated transcriptome dataset from 989 patients with high-risk early breast cancer enrolled in one of nine completed/graduated/experimental and control arms in the neoadjuvant I-SPY2 clinical trial (NCT01042379)., Results: High ROR1 or high ROR2 was associated with breast cancer subtypes. High ROR1 was more prevalent among hormone receptor-negative and human epidermal growth factor receptor 2-negative (HR-HER2-) tumors and high ROR2 was less prevalent in this subtype. Although not associated with pathologic complete response, high ROR1 or high ROR2 each was associated with event-free survival (EFS) in distinct subtypes. High ROR1 associated with a worse EFS in HR + HER2- patients with high post-treatment residual cancer burden (RCB-II/III) (HR 1.41, 95% CI = 1.11-1.80) but not in patients with minimal post-treatment disease (RCB-0/I) (HR 1.85, 95% CI = 0.74-4.61). High ROR2 associated with an increased risk of relapse in patients with HER2 + disease and RCB-0/I (HR 3.46, 95% CI = 1.33-9.020) but not RCB-II/III (HR 1.07, 95% CI = 0.69-1.64)., Conclusion: High ROR1 or high ROR2 distinctly identified subsets of breast cancer patients with adverse outcomes. Further studies are warranted to determine if high ROR1 or high ROR2 may identify high-risk populations for studies of targeted therapies., (© 2023. The Author(s).)

Purpose: A polygenic risk score (PRS) consisting of 313 common genetic variants (PRS 313 ) is associated with risk of breast cancer and contralateral breast cancer. This study aimed to evaluate the association of the PRS 313 with clinicopathologic characteristics of, and survival following, breast cancer., Methods: Women with invasive breast cancer were included, 98,397 of European ancestry and 12,920 of Asian ancestry, from the Breast Cancer Association Consortium (BCAC), and 683 women from the European MINDACT trial. Associations between PRS 313 and clinicopathologic characteristics, including the 70-gene signature for MINDACT, were evaluated using logistic regression analyses. Associations of PRS 313 (continuous, per standard deviation) with overall survival (OS) and breast cancer-specific survival (BCSS) were evaluated with Cox regression, adjusted for clinicopathologic characteristics and treatment., Results: The PRS 313 was associated with more favorable tumor characteristics. In BCAC, increasing PRS 313 was associated with lower grade, hormone receptor-positive status, and smaller tumor size. In MINDACT, PRS 313 was associated with a low risk 70-gene signature. In European women from BCAC, higher PRS 313 was associated with better OS and BCSS: hazard ratio (HR) 0.96 (95% CI, 0.94 to 0.97) and 0.96 (95% CI, 0.94 to 0.98), but the association disappeared after adjustment for clinicopathologic characteristics (and treatment): OS HR, 1.01 (95% CI, 0.98 to 1.05) and BCSS HR, 1.02 (95% CI, 0.98 to 1.07). The results in MINDACT and Asian women from BCAC were consistent., Conclusion: An increased PRS 313 is associated with favorable tumor characteristics, but is not independently associated with prognosis. Thus, PRS 313 has no role in the clinical management of primary breast cancer at the time of diagnosis. Nevertheless, breast cancer mortality rates will be higher for women with higher PRS 313 as increasing PRS 313 is associated with an increased risk of disease. This information is crucial for modeling effective stratified screening programs.

BRAF V600E mutation confers a poor prognosis in metastatic colorectal cancer (CRC) despite combinatorial targeted therapies based on the latest understanding of signaling circuitry. To identify parallel resistance mechanisms induced by BRAF-MEK-EGFR co-targeting, we used a high-throughput kinase activity mapping platform. Here we show that SRC kinases are systematically activated in BRAF V600E CRC following targeted inhibition of BRAF ± EGFR and that coordinated targeting of SRC with BRAF ± EGFR increases treatment efficacy in vitro and in vivo. SRC drives resistance to BRAF ± EGFR targeted therapy independently of ERK signaling by inducing transcriptional reprogramming through β-catenin (CTNNB1). The EGFR-independent compensatory activation of SRC kinases is mediated by an autocrine prostaglandin E 2 loop that can be blocked with cyclooxygenase-2 (COX2) inhibitors. Co-targeting of COX2 with BRAF + EGFR promotes durable suppression of tumor growth in patient-derived tumor xenograft models. COX2 inhibition represents a drug-repurposing strategy to overcome therapeutic resistance in BRAF V600E CRC., (© 2023. The Author(s).)

Background: Despite advances in early detection and therapies, cancer is still one of the most common causes of death worldwide. Since each tumor is unique, there is a need to implement personalized care and develop robust tools for monitoring treatment response to assess drug efficacy and prevent disease relapse., Main Body: Recent developments in liquid biopsies have enabled real-time noninvasive monitoring of tumor burden through the detection of molecules shed by tumors in the blood. These molecules include circulating tumor nucleic acids (ctNAs), comprising cell-free DNA or RNA molecules passively and/or actively released from tumor cells. Often highlighted for their diagnostic, predictive, and prognostic potential, these biomarkers possess valuable information about tumor characteristics and evolution. While circulating tumor DNA (ctDNA) has been in the spotlight for the last decade, less is known about circulating tumor RNA (ctRNA). There are unanswered questions about why some tumors shed high amounts of ctNAs while others have undetectable levels. Also, there are gaps in our understanding of associations between tumor evolution and ctNA characteristics and shedding kinetics. In this review, we summarize current knowledge about ctNA biology and release mechanisms and put this information into the context of tumor evolution and clinical utility., Conclusions: A deeper understanding of the biology of ctDNA and ctRNA may inform the use of liquid biopsies in personalized medicine to improve cancer patient outcomes., (© 2023. The Author(s).)

Purpose: To assess the long-term (20-year) endocrine therapy benefit in premenopausal patients with breast cancer., Methods: Secondary analysis of the Stockholm trial (STO-5, 1990-1997) randomly assigning 924 premenopausal patients to 2 years of goserelin (3.6 mg subcutaneously once every 28 days), tamoxifen (40 mg orally once daily), combined goserelin and tamoxifen, or no adjuvant endocrine therapy (control) is performed. Random assignment was stratified by lymph node status; lymph node-positive patients (n = 459) were allocated to standard chemotherapy (cyclophosphamide, methotrexate, and fluorouracil). Primary tumor immunohistochemistry (n = 731) and gene expression profiling (n = 586) were conducted in 2020. The 70-gene signature identified genomic low-risk and high-risk patients. Kaplan-Meier analysis, multivariable Cox proportional hazard regression, and multivariable time-varying flexible parametric modeling assessed the long-term distant recurrence-free interval (DRFI). Swedish high-quality registries allowed a complete follow-up of 20 years., Results: In estrogen receptor-positive patients (n = 584, median age 47 years), goserelin, tamoxifen, and the combination significantly improved long-term distant recurrence-free interval compared with control (multivariable hazard ratio [HR], 0.49; 95% CI, 0.32 to 0.75, HR, 0.57; 95% CI, 0.38 to 0.87, and HR, 0.63; 95% CI, 0.42 to 0.94, respectively). Significant goserelin-tamoxifen interaction was observed ( P = .016). Genomic low-risk patients (n = 305) significantly benefitted from tamoxifen (HR, 0.24; 95% CI, 0.10 to 0.60), and genomic high-risk patients (n = 158) from goserelin (HR, 0.24; 95% CI, 0.10 to 0.54). Increased risk from the addition of tamoxifen to goserelin was seen in genomic high-risk patients (HR, 3.36; 95% CI, 1.39 to 8.07). Moreover, long-lasting 20-year tamoxifen benefit was seen in genomic low-risk patients, whereas genomic high-risk patients had early goserelin benefit., Conclusion: This study shows 20-year benefit from 2 years of adjuvant endocrine therapy in estrogen receptor-positive premenopausal patients and suggests differential treatment benefit on the basis of tumor genomic characteristics. Combined goserelin and tamoxifen therapy showed no benefit over single treatment. Long-term follow-up to assess treatment benefit is critical.

HSP90 inhibitors destabilize oncoproteins associated with cell cycle, angiogenesis, RAS-MAPK activity, histone modification, kinases and growth factors. We evaluated the HSP90-inhibitor ganetespib in combination with standard chemotherapy in patients with high-risk early-stage breast cancer. I-SPY2 is a multicenter, phase II adaptively randomized neoadjuvant (NAC) clinical trial enrolling patients with stage II-III breast cancer with tumors 2.5 cm or larger on the basis of hormone receptors (HR), HER2 and Mammaprint status. Multiple novel investigational agents plus standard chemotherapy are evaluated in parallel for the primary endpoint of pathologic complete response (pCR). Patients with HER2-negative breast cancer were eligible for randomization to ganetespib from October 2014 to October 2015. Of 233 women included in the final analysis, 140 were randomized to the standard NAC control; 93 were randomized to receive 150 mg/m 2 ganetespib every 3 weeks with weekly paclitaxel over 12 weeks, followed by AC. Arms were balanced for hormone receptor status (51-52% HR-positive). Ganetespib did not graduate in any of the biomarker signatures studied before reaching maximum enrollment. Final estimated pCR rates were 26% vs. 18% HER2-negative, 38% vs. 22% HR-negative/HER2-negative, and 15% vs. 14% HR-positive/HER2-negative for ganetespib vs control, respectively. The predicted probability of success in phase 3 testing was 47% HER2-negative, 72% HR-negative/HER2-negative, and 19% HR-positive/HER2-negative. Ganetespib added to standard therapy is unlikely to yield substantially higher pCR rates in HER2-negative breast cancer compared to standard NAC, and neither HSP90 pathway nor replicative stress expression markers predicted response. HSP90 inhibitors remain of limited clinical interest in breast cancer, potentially in other clinical settings such as HER2-positive disease or in combination with anti-PD1 neoadjuvant chemotherapy in triple negative breast cancer.Trial registration: www.clinicaltrials.gov/ct2/show/NCT01042379., (© 2022. The Author(s).)