Virtual patient analysis identifies strategies to improve the performance of predictive biomarkers for PD-1 blockade.

Patients with metastatic triple-negative breast cancer (TNBC) show variable responses to PD-1 inhibition. Efficient patient selection by predictive biomarkers would be desirable, but is hindered by the limited performance of existing biomarkers. Here, we leveraged in-silico patient cohorts generated using a quantitative systems pharmacology model of metastatic TNBC, informed by transcriptomic and clinical data, to explore potential ways to improve patient selection. We tested 90 biomarker candidates, including various cellular and molecular species, by a cutoff-based biomarker testing algorithm combined with machine learning-based feature selection. Combinations of pre-treatment biomarkers improved the specificity compared to single biomarkers at the cost of reduced sensitivity. On the other hand, early on-treatment biomarkers, such as the relative change in tumor diameter from baseline measured at two weeks after treatment initiation, achieved remarkably higher sensitivity and specificity. Further, blood-based biomarkers had a comparable ability to tumor- or lymph node-based biomarkers in identifying a subset of responders, potentially suggesting a less invasive way for patient selection.

Bi-level Graph Learning Unveils Prognosis-Relevant Tumor Microenvironment Patterns from Breast Multiplexed Digital Pathology.

The tumor microenvironment is widely recognized for its central role in driving cancer progression and influencing prognostic outcomes. Despite extensive research efforts dedicated to characterizing this complex and heterogeneous environment, considerable challenges persist. In this study, we introduce a data-driven approach for identifying patterns of cell organizations in the tumor microenvironment that are associated with patient prognoses. Our methodology relies on the construction of a bi-level graph model: (i) a cellular graph, which models the intricate tumor microenvironment, and (ii) a population graph that captures inter-patient similarities, given their respective cellular graphs, by means of a soft Weisfeiler-Lehman subtree kernel. This systematic integration of information across different scales enables us to identify patient subgroups exhibiting unique prognoses while unveiling tumor microenvironment patterns that characterize them. We demonstrate our approach in a cohort of breast cancer patients, where the identified tumor microenvironment patterns result in a risk stratification system that provides complementary, new information with respect to alternative standards. Our results, which are validated in a completely independent cohort, allow for new insights into the prognostic implications of the breast tumor microenvironment, and this methodology could be applied to other cancer types more generally.

Epithelial Expressed B7-H4 Drives Differential Immunotherapy Response in Murine and Human Breast Cancer.

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.

NKG2A Is a Therapeutic Vulnerability in Immunotherapy Resistant MHC-I Heterogeneous Triple-Negative Breast Cancer.

Despite the success of immune checkpoint inhibition (ICI) in treating cancer, patients with triple-negative breast cancer (TNBC) often develop resistance to therapy, and the underlying mechanisms are unclear. MHC-I expression is essential for antigen presentation and T-cell-directed immunotherapy responses. This study demonstrates that TNBC patients display intratumor heterogeneity in regional MHC-I expression. In murine models, loss of MHC-I negates antitumor immunity and ICI response, whereas intratumor MHC-I heterogeneity leads to increased infiltration of natural killer (NK) cells in an IFNγ-dependent manner. Using spatial technologies, MHC-I heterogeneity is associated with clinical resistance to anti-programmed death (PD) L1 therapy and increased NK:T-cell ratios in human breast tumors. MHC-I heterogeneous tumors require NKG2A to suppress NK-cell function. Combining anti-NKG2A and anti-PD-L1 therapies restores complete response in heterogeneous MHC-I murine models, dependent on the presence of activated, tumor-infiltrating NK and CD8+ T cells. These results suggest that similar strategies may enhance patient benefit in clinical trials. SIGNIFICANCE: Clinical resistance to immunotherapy is common in breast cancer, and many patients will likely require combination therapy to maximize immunotherapeutic benefit. This study demonstrates that heterogeneous MHC-I expression drives resistance to anti-PD-L1 therapy and exposes NKG2A on NK cells as a target to overcome resistance. This article is featured in Selected Articles from This Issue, p. 201.

Atezolizumab in Combination With Carboplatin and Survival Outcomes in Patients With Metastatic Triple-Negative Breast Cancer: The TBCRC 043 Phase 2 Randomized Clinical Trial.

Importance: Agents targeting programmed death ligand 1 (PD-L1) have demonstrated efficacy in triple-negative breast cancer (TNBC) when combined with chemotherapy and are now the standard of care in patients with PD-L1-positive metastatic disease. In contrast to microtubule-targeting agents, the effect of combining platinum compounds with programmed cell death 1 (PD-1)/PD-L1 immunotherapy has not been extensively determined. Objective: To evaluate the efficacy of atezolizumab with carboplatin in patients with metastatic TNBC. Design, Setting, and Participants: This phase 2 randomized clinical trial was conducted in 6 centers from August 2017 to June 2021. Interventions: Patients with metastatic TNBC were randomized to receive carboplatin area under the curve (AUC) 6 alone or with atezolizumab, 1200 mg, every 3 weeks until disease progression or unacceptable toxic effects with a 3-year duration of follow-up. Main Outcome and Measures: The primary end point was investigator-assessed progression-free survival (PFS). Secondary end points included overall response rate (ORR), clinical benefit rate (CBR), and overall survival (OS). Other objectives included correlation of response with tumor PD-L1 levels, tumor-infiltrating lymphocytes (TILs), tumor DNA- and RNA-sequenced biomarkers, TNBC subtyping, and multiplex analyses of immune markers. Results: All 106 patients with metastatic TNBC who were enrolled were female with a mean (range) age of 55 (27-79) years, of which 12 (19%) identified as African American/Black, 1 (1%) as Asian, 73 (69%) as White, and 11 (10%) as unknown. Patients were randomized and received either carboplatin (n = 50) or carboplatin and atezolizumab (n = 56). The combination improved PFS (hazard ratio [HR], 0.66; 95% CI, 0.44-1.01; P = .05) from a median of 2.2 to 4.1 months, increased ORR from 8.0% (95% CI, 3.2%-18.8%) to 30.4% (95% CI, 19.9%-43.3%), increased CBR at 6 months from 18.0% (95% CI, 9.8%-30.1%) to 37.5% (95% CI, 26.0%-50.6%), and improved OS (HR, 0.60; 95% CI, 0.37-0.96; P = .03) from a median of 8.6 to 12.6 months. Subgroup analysis showed PD-L1-positive tumors did not benefit more from adding atezolizumab (HR, 0.62; 95% CI, 0.23-1.65; P = .35). Patients with high TILs (HR, 0.12; 95% CI, 0.30-0.50), high mutation burden (HR, 0.50; 95% CI, 0.23-1.06), and prior chemotherapy (HR, 0.59; 95% CI, 0.36-0.95) received greater benefit on the combination. Patients with obesity and patients with more than 125 mg/dL on-treatment blood glucose levels were associated with better PFS (HR, 0.35; 95% CI, 0.10-1.80) on the combination. TNBC subtypes benefited from adding atezolizumab, except the luminal androgen receptor subtype. Conclusions and Relevance: In this randomized clinical trial, the addition of atezolizumab to carboplatin significantly improved survival of patients with metastatic TNBC regardless of PD-L1 status. Further, lower risk of disease progression was associated with increased TILs, higher mutation burden, obesity, and uncontrolled blood glucose levels. Trial Registration: ClinicalTrials.gov Identifier: NCT03206203.

The PD-1- and LAG-3-targeting bispecific molecule tebotelimab in solid tumors and hematologic cancers: a phase 1 trial.

Tebotelimab, a bispecific PD-1×LAG-3 DART molecule that blocks both PD-1 and LAG-3, was investigated for clinical safety and activity in a phase 1 dose-escalation and cohort-expansion clinical trial in patients with solid tumors or hematologic malignancies and disease progression on previous treatment. Primary endpoints were safety and maximum tolerated dose of tebotelimab when administered as a single agent (n = 269) or in combination with the anti-HER2 antibody margetuximab (n = 84). Secondary endpoints included anti-tumor activity. In patients with advanced cancer treated with tebotelimab monotherapy, 68% (184/269) experienced treatment-related adverse events (TRAEs; 22% were grade ≥3). No maximum tolerated dose was defined; the recommended phase 2 dose (RP2D) was 600 mg once every 2 weeks. There were tumor decreases in 34% (59/172) of response-evaluable patients in the dose-escalation cohorts, with objective responses in multiple solid tumor types, including PD-1-refractory disease, and in LAG-3+ non-Hodgkin lymphomas, including CAR-T refractory disease. To enhance potential anti-tumor responses, we tested margetuximab plus tebotelimab. In patients with HER2+ tumors treated with tebotelimab plus margetuximab, 74% (62/84) had TRAEs (17% were grade ≥3). The RP2D was 600 mg once every 3 weeks. The confirmed objective response rate in these patients was 19% (14/72), including responses in patients typically not responsive to anti-HER2/anti-PD-1 combination therapy. ClinicalTrials.gov identifier: NCT03219268 .

Feasibility of Symptom Monitoring During the First Year of Endocrine Therapy for Early Breast Cancer Using Patient-Reported Outcomes Collected via Smartphone App.

PURPOSE: Treatment-associated symptoms drive early discontinuation of adjuvant endocrine therapy (ET) for breast cancer. We hypothesized that symptom monitoring with electronic patient-reported outcomes (ePROs) during adjuvant ET will enhance symptom detection, symptom management, and persistence. METHODS: Eligible patients were initiating ET for stage 0-III breast cancer. Participants completed ePRO surveys via smartphone at baseline and 1, 3, 6, and 12 months. Measures included Patient-Reported Outcomes Measurement Information System Anxiety, Depression, Fatigue, and Vaginal Discomfort; plus Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events items assessing joint pain, hot flashes, vaginal dryness, concentration problems, and memory problems. Scores surpassing prespecified thresholds triggered alerts, and recommended symptom management pathways were provided to clinicians. The primary objective was to evaluate feasibility, assessed by survey completion rates, with targets of >65% for the baseline survey and ≥1 follow-up survey during the first 6 months. Secondary objectives included 12-month ET discontinuation rate (target: ≤15%), describing symptoms and evaluating pathway implementation. RESULTS: Among 250 participants, 73.2% completed the baseline survey and 69.6% completed ≥1 follow-up survey during the first 6 months. Thirty-one percent of participants had ≥1 symptom alert at baseline and 74% had ≥1 symptom alert during follow-up. The proportions of participants for whom pathway-concordant symptom management was documented at each time point ranged from 12.8% to 36.6%. Twenty-eight participants (11.2%) discontinued ET by 12 months. CONCLUSION: Symptom monitoring with ePROs during adjuvant ET is feasible. Despite infrequent documentation of pathway-concordant symptom management after symptom alerts, ePROs were associated with favorable short-term ET persistence.

A transcriptome-informed QSP model of metastatic triple-negative breast cancer identifies predictive biomarkers for PD-1 inhibition.

Triple-negative breast cancer (TNBC), a highly metastatic breast cancer subtype, has limited treatment options. While a small number of patients attain clinical benefit with single-agent checkpoint inhibitors, identifying these patients before the therapy remains challenging. Here, we developed a transcriptome-informed quantitative systems pharmacology model of metastatic TNBC by integrating heterogenous metastatic tumors. In silico clinical trial with an anti-PD-1 drug, pembrolizumab, predicted that several features, such as the density of antigen-presenting cells, the fraction of cytotoxic T cells in lymph nodes, and the richness of cancer clones in tumors, could serve individually as biomarkers but had a higher predictive power as combinations of two biomarkers. We showed that PD-1 inhibition neither consistently enhanced all antitumorigenic factors nor suppressed all protumorigenic factors but ultimately reduced the tumor carrying capacity. Collectively, our predictions suggest several candidate biomarkers that might effectively predict the response to pembrolizumab monotherapy and potential therapeutic targets to develop treatment strategies for metastatic TNBC.

Exceptional responses to PARP inhibitors in patients with metastatic breast cancer in oncologic crisis.

PURPOSE: Cancers deficient in homologous recombination DNA repair, such as those with BRCA1 or BRCA2 (BRCA1/2) mutations rely on a pathway mediated by the enzyme poly(adenosine diphosphate-ribose) polymerase (PARP). PARP inhibitors (PARPi's) have demonstrated efficacy in treating patients with germline (g)BRCA1/2, somatic (s)BRCA1/2, and gPALB2 mutations in clinical trials. However, patients with a poor performance status (PS) and those with severe organ impairment are often excluded from clinical trials and cancer-directed treatment. METHODS: We report the cases of two patients with metastatic breast cancer who had poor PS, significant visceral disease, and gPALB2 and sBRCA mutations, who derived significant clinical benefit from treatment with PARP inhibition. RESULTS: Patient A had germline testing demonstrating a heterozygous PALB2 pathogenic mutation (c.3323delA) and a BRCA2 variant of unknown significance (c.9353T>C), and tumor sequencing revealed PALB2 (c.228_229del and c.3323del) and ESR1 (c.1610A>C) mutations. Patient B was negative for pathologic BRCA mutations upon germline testing, but tumor sequencing demonstrated somatic BRCA2 copy number loss and a PIK3CA mutation (c.1633G>A). Treatment with PARPi's in these two patients with an initial PS of 3-4 and significant visceral disease resulted in prolonged clinical benefit. CONCLUSION: Patients with a poor PS, such as those described here, may still have meaningful clinical responses to cancer treatments targeting oncogenic drivers. More studies evaluating PARPi's beyond gBRCA1/2 mutations and in sub-optimal PS would help identify patients who may benefit from these therapies.

A Phase II Single-arm Study of Palbociclib in Patients With HER2-positive Breast Cancer With Brain Metastases and Analysis of ctDNA in Patients With Active Brain Metastases.

INTRODUCTION: Palbociclib is highly efficacious and well tolerated in hormone-receptor positive (HR+) metastatic breast cancer (BC) but its activity for HER2+ BC with brain metastases (BM) is unknown. METHODS: In a single-arm phase II study we evaluated palbociclib with trastuzumab for patients with HER2+ MBC and BM. The primary endpoint was BM response rate. Circulating tumor DNA (ctDNA) was evaluated at baseline, and in a subset of patients at cycle 3 and progression. We also retrospectively identified additional patients with metastatic BC, active BM, and a ctDNA assessment prior to therapy for BM. RESULTS: Twelve patients with HER2+ MBC were enrolled, 4 with HR+ and 8 with HR- disease. No responses were seen. Best response was stable disease for 6 patients and progressive disease for 6 patients. The median PFS was 2.2 months, interquartile range (IQR) was 1.56 to 3.63 months. The median OS was 13.1 months and IQR was 9.4 to 23.8 months The CNS was the primary site of progression for all patients. The median variant allele fraction (VAF) of the dominant variant in each patient was 0.18% (interquartile range [IQR] 0.12%-0.47%) with a median number of somatic alterations of 1. We additionally evaluated ctDNA results from 26 patients with BC and active BM, among whom the median VAF was 11.8% (IQR 3.9%-27.3%) with a median number of alterations was 6 (IQR 4-9). Notably, progressive systemic disease was significantly less frequent in the trial cohort compared with additional retrospectively identified patients (8% vs. 81%). CONCLUSION: Palbociclib did not demonstrate activity in HER2+ MBC with BM. Patients with progressive BM but stable, responding, or absent systemic disease have low VAF and number of alterations detected by ctDNA analysis from blood.

Spatial and Compositional Biomarkers in Tumor Microenvironment Predicts Clinical Outcomes in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited treatment options, which warrants identification of novel therapeutic targets. Deciphering nuances in the tumor microenvironment (TME) may unveil insightful links between anti-tumor immunity and clinical outcomes, yet such connections remain underexplored. Here we employed a dataset derived from imaging mass cytometry of 58 TNBC patient specimens at single-cell resolution and performed in-depth quantifications with a suite of multi-scale computational algorithms. We detected distinct cell distribution patterns among clinical subgroups, potentially stemming from different infiltration related to tumor vasculature and fibroblast heterogeneity. Spatial analysis also identified ten recurrent cellular neighborhoods (CNs) - a collection of local TME characteristics with unique cell components. Coupling of the prevalence of pan-immune and perivasculature immune hotspot CNs, enrichment of inter-CN interactions was associated with improved survival. Using a deep learning model trained on engineered spatial data, we can with high accuracy (mean AUC of 5-fold cross-validation = 0.71) how a separate cohort of patients in the NeoTRIP clinical trial will respond to treatment based on baseline TME features. These data reinforce that the TME architecture is structured in cellular compositions, spatial organizations, vasculature biology, and molecular profiles, and suggest novel imaging-based biomarkers for treatment development in the context of TNBC.

A randomized phase 2 study of neoadjuvant carboplatin and paclitaxel with or without atezolizumab in triple negative breast cancer (TNBC) - NCI 10013.

Atezolizumab with chemotherapy has shown improved progression-free and overall survival in patients with metastatic PD-L1 positive triple negative breast cancer (TNBC). Atezolizumab with anthracycline- and taxane-based neoadjuvant chemotherapy has also shown increased pathological complete response (pCR) rates in early TNBC. This trial evaluated neoadjuvant carboplatin and paclitaxel with or without atezolizumab in patients with clinical stages II-III TNBC. The co-primary objectives were to evaluate if chemotherapy and atezolizumab increase pCR rate and tumor infiltrating lymphocyte (TIL) percentage compared to chemotherapy alone in the mITT population. Sixty-seven patients (ages 25-78 years; median, 52 years) were randomly assigned - 22 patients to Arm A, and 45 to Arm B. Median follow up was 6.6 months. In the modified intent to treat population (all patients evaluable for the primary endpoints who received at least one dose of combination therapy), the pCR rate was 18.8% (95% CI 4.0-45.6%) in Arm A, and 55.6% (95% CI 40.0-70.4%) in Arm B (estimated treatment difference: 36.8%, 95% CI 8.5-56.6%; p = 0.018). Grade 3 or higher treatment-related adverse events occurred in 62.5% of patients in Arm A, and 57.8% of patients in Arm B. One patient in Arm B died from recurrent disease during the follow-up period. TIL percentage increased slightly from baseline to cycle 1 in both Arm A (mean ± SD: 0.6% ± 21.0%) and Arm B (5.7% ± 15.8%) (p = 0.36). Patients with pCR had higher median TIL percentages (24.8%) than those with non-pCR (14.2%) (p = 0.02). Although subgroup analyses were limited by the small sample size, PD-L1-positive patients treated with chemotherapy and atezolizumab had a pCR rate of 75% (12/16). The addition of atezolizumab to neoadjuvant carboplatin and paclitaxel resulted in a statistically significant and clinically relevant increased pCR rate in patients with clinical stages II and III TNBC. (Funded by National Cancer Institute).

Integrating Immunotherapy in Early-Stage Triple-Negative Breast Cancer: Practical Evidence-Based Considerations.

The KEYNOTE-522 study is a practice-changing phase III randomized study that demonstrated that the addition of pembrolizumab to polychemotherapy improves outcomes in patients with high-risk early-stage triple-negative breast cancer (TNBC). This regimen is highly efficacious with unprecedented pathologic complete response (pCR) rates, and clinically meaningful improvements in event-free survival (EFS). However, the combination is also associated with significant high-grade treatment-related toxicity. The backbone regimen deviated from common practice, including the addition of carboplatin, lack of dose dense anthracyclines, and adjuvant capecitabine for residual disease, thus brining important questions regarding real-world translation of these results. This brief report practically addresses some of the most relevant questions physicians and patients face in optimizing care using the best available evidence.

Dynamics of tumor-associated macrophages in a quantitative systems pharmacology model of immunotherapy in triple-negative breast cancer.

Quantitative systems pharmacology (QSP) modeling is an emerging mechanistic computational approach that couples drug pharmacokinetics/pharmacodynamics and the course of disease progression. It has begun to play important roles in drug development for complex diseases such as cancer, including triple-negative breast cancer (TNBC). The combination of the anti-PD-L1 antibody atezolizumab and nab-paclitaxel has shown clinical activity in advanced TNBC with PD-L1-positive tumor-infiltrating immune cells. As tumor-associated macrophages (TAMs) serve as major contributors to the immuno-suppressive tumor microenvironment, we incorporated the dynamics of TAMs into our previously published QSP model to investigate their impact on cancer treatment. We show that through proper calibration, the model captures the macrophage heterogeneity in the tumor microenvironment while maintaining its predictive power of the trial results at the population level. Despite its high mechanistic complexity, the modularized QSP platform can be readily reproduced, expanded for new species of interest, and applied in clinical trial simulation.

Neoadjuvant study of niraparib in patients with HER2-negative, BRCA-mutated, resectable breast cancer.

This single-arm pilot study (NCT03329937) evaluated neoadjuvant niraparib antitumor activity and safety in patients with localized HER2-negative, BRCA-mutated breast cancer. Twenty-one patients received niraparib 200 mg once daily in 28-day cycles. After 2 cycles, tumor response (≥30% reduction from baseline) by MRI was 90.5% and 40.0% (6 of 15) of patients who received only niraparib (2-6 cycles) had pathological complete response; no new safety signals were identified. High niraparib intratumoral concentration was observed.

Immunotherapy Combined with Radiation Therapy in Breast Cancer: A Rapidly Evolving Landscape.

Immune checkpoint inhibitors (ICI) can have significant anticancer activity, and are approved for many different cancer types, including breast cancer. In breast cancer, programmed cell death 1 (PD-1) inhibitors in combination with chemotherapy have demonstrated significant clinical benefit in early-stage and metastatic settings; however, these combinations can have significant side effects, and there are still many breast cancer patients who do not respond to these approaches. Novel combinations with immunotherapy are needed to improve responses. Given the effects of radiation therapy (RT) on the tumor micro-environment, combinations of RT with immune checkpoint blockade are active areas of investigation. In this review, we discuss experience ICI in breast cancer, including current clinical indications, emerging data in combination with RT, and ongoing studies exploring optimal dosing of RT, and novel combinations with other therapeutics.