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Supplementary Table 1

Supplemental figures 1-11

Our understanding is limited concerning the tumor immune microenvironment of inflammatory breast cancer (IBC), an aggressive form of primary cancer with low rates of pathologic complete response to current neoadjuvant chemotherapy (NAC) regimens. We retrospectively identified pretreatment (N = 86) and matched posttreatment tissue (N = 27) from patients with stage III or de novo stage IV IBC who received NAC followed by a mastectomy. Immune profiling was performed including quantification of lymphoid and myeloid infiltrates by IHC and T-cell repertoire analysis. Thirty-four of 86 cases in this cohort (39.5%) achieved a pathologic complete response. Characterization of the tumor microenvironment revealed that having a lower pretreatment mast cell density was significantly associated with achieving a pathologic complete response to NAC (P = 0.004), with responders also having more stromal tumor-infiltrating lymphocytes (P = 0.035), CD8+ T cells (P = 0.047), and CD20+ B cells (P = 0.054). Spatial analysis showed close proximity of mast cells to CD8+ T cells, CD163+ monocytes/macrophages, and tumor cells when pathologic complete response was not achieved. PD-L1 positivity on tumor cells was found in fewer than 2% of cases and on immune cells in 27% of cases, but with no correlation to response. Our results highlight the strong association of mast cell infiltration with poor response to NAC, suggesting a mechanism of treatment resistance and a potential therapeutic target in IBC. Proximity of mast cells to immune and tumor cells may suggest immunosuppressive or tumor-promoting interactions of these mast cells.

Supplementary Figure 1. Immune profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 2. Myeloid cell profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 3. Increased contact between CD8 T cells and CD68 myeloid cells in non-responding patients to anti-CTLA-4 and anti-PD-1 therapy at pre-treatment CTLA-4 blockade, pre-treatment PD-1 blockade, and on-treatment PD-1 blockade time points. Supplementary Figure 4. Immune profiling of pre anti-PD-1, on-treatment anti-PD-1 and progression anti-PD-1 samples by immunohistochemistry. Supplementary Figure 5. Longitudinal increase in CD8, PD-1, and PD-L1 expression in responders to anti-PD-1 therapy. Supplementary Figure 6. Relative increase in CD8 T cell infiltrate at tumor center in responders to anti-PD-1 on treatment. Supplementary Figure 7. Significant increase in immune infiltrate between responders and non-responders to PD-1 blockade in absence of prior anti-CTLA-4 therapy. Supplementary Figure 8. Immune profiling of myeloid cells atpre-treatment and on-treatment PD-1 blockade time pointsby immunohistochemistry. Supplementary Figure 9. Heatmap of 54 NanoString samples. Supplementary Figure 10. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression CTLA-4 blockade samples. Supplementary Figure 11. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression PD-1 blockade samples. Supplementary Figure 12. Prior CTLA-4 blockade is not required for PD-1 early on-treatment profile. Supplementary Figure 13. Hierarchical clustering of gene expression across 54 samples confirms lack of batch effect.

Supplementary Figure Legends

Supplementary Table S1a. Patient Cohort Clinical Summary. Supplementary Table S1b. Patient clinical characteristics. Supplementary Table S1c. Immune profiling by IHC sample log. Supplementary Table S1d. Nanostring 54 sample log. Supplementary Table S2. Summary of immune profiling by immunohistochemistry. Supplementary Table S3. Summary of immune profiling by 4 additional myeloid markers. Supplementary Table S4. Summary of immune profiling by IHC of additional CTLA-4 blockade-naïve samples. Supplementary Table S5. Nanostring Gene List. Supplementary Table S6a. Nanostring summary 54 samples. Supplementary Table S6b. Differentially upregulated and downregulated genes in pre-treatment anti-CTLA-4 samples. Supplementary Table S6c. Differentially upregulated and downregulated genes in on-treatment anti-CTLA-4 samples. Supplementary Table S6d. Differentially upregulated and downregulated genes in pre-treatment anti-PD-1 samples. Supplementary Table S6e. Differentially upregulated and downregulated genes in on-treatment anti-PD-1 samples. Supplementary Table S7. Differentially upregulated and downregulated genes from pre- to on-treatment anti-CTLA-4. Supplementary Table S8. Differentially upregulated and downregulated genes from pre- to on-treatment anti-PD-1. Supplementary Table S9a. Nanostring gene list - chip used for comparison of CTLA-4 blockade-experienced vs -naive anti-PD1 samples (28 samples). Supplementary Table S9b. NanoString additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S9c. NanoString normalized data of additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S10. Commonly differentially regulated genes between pre- to on-treatment CTLA-4 blockade and PD-1 blockade. Supplementary Table S11a. Fold changes of significant change by anti-PD-1 therapy for paired samples. Supplementary Table S11b. Frequency of significant change by anti-PD1 therapy for paired samples.

SKBR3 breast cancer cells shed HER2 after treatment with chemotherapy or irradiation.

Early-phase clinical trials evaluating CD8+ T cell–eliciting, HER2-derived peptide vaccines administered to HER2+ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clinical trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy. Cancer Res; 77(19); 5374–83. ©2017 AACR.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

We present an integrated single-cell RNA-seq resource of the breast tumor microenvironment consisting of 236,363 cells from 119 biopsy samples across 8 publicly available datasets. In this computational study, we first leverage this novel resource to define cancer epithelial cell heterogeneity based on two clinically relevant markers and identify six new and distinct subsets of natural killer cells. We then illustrate how cancer epithelial cell heterogeneity impacts immune cell interactions. We develop T cell InteractPrint, which considers how cancer epithelial cell heterogeneity shifts the predicted strength of T cell interactions. We use InteractPrint to predict response to immune checkpoint inhibition (ICI) in two clinical trials testing immunotherapy in patients with breast cancer. T cell InteractPrint was predictive in both trials (AUC = 0.81 and 0.84), versus PD-L1 expression (AUC = 0.54 and 0.72). This result provides an alternative predictive biomarker to PD-L1 to select patients who should receive ICI.STATEMENT OF SIGNIFICANCEWe developed a novel integrated single-cell atlas of the breast tumor microenvironment to interrogate breast tumor cell heterogeneity and define how heterogenous cancer epithelial cell and immune cell interactions predict response to anti-PD-1 therapy.

The MYC oncogene is frequently amplified in triple-negative breast cancer (TNBC). Here, we show that MYC suppression induces immune-related hallmark gene set expression and tumor-infiltrating T cells in MYC-hyperactivated TNBCs. Mechanistically, MYC repressed stimulator of interferon genes (STING) expression via direct binding to the STING1 enhancer region, resulting in downregulation of the T-cell chemokines CCL5, CXCL10, and CXCL11. In primary and metastatic TNBC cohorts, tumors with high MYC expression or activity exhibited low STING expression. Using a CRISPR-mediated enhancer perturbation approach, we demonstrated that MYC-driven immune evasion is mediated by STING repression. STING repression induced resistance to PD-L1 blockade in mouse models of TNBC. Finally, a small-molecule inhibitor of MYC combined with PD-L1 blockade elicited a durable response in immune-cold TNBC with high MYC expression, suggesting a strategy to restore PD-L1 inhibitor sensitivity in MYC-overexpressing TNBC.

Background Inflammatory breast cancer (IBC) has a highly invasive and metastatic phenotype. However, little is known about its genetic drivers. To address this, we report the largest cohort of whole-genome sequencing (WGS) of IBC cases. Methods We performed WGS of 20 IBC samples and paired normal blood DNA to identify genomic alterations. For comparison, we used 23 matched non-IBC samples from the Cancer Genome Atlas Program (TCGA). We also validated our findings using WGS data from the International Cancer Genome Consortium (ICGC) and the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We examined a wide selection of genomic features to search for differences between IBC and conventional breast cancer. These include (i) somatic and germline single-nucleotide variants (SNVs), in both coding and non-coding regions; (ii) the mutational signature and the clonal architecture derived from these SNVs; (iii) copy number and structural variants (CNVs and SVs); and (iv) non-human sequence in the tumors (i.e., exogenous sequences of bacterial origin). Results Overall, IBC has similar genomic characteristics to non-IBC, including specific alterations, overall mutational load and signature, and tumor heterogeneity. In particular, we observed similar mutation frequencies between IBC and non-IBC, for each gene and most cancer-related pathways. Moreover, we found no exogenous sequences of infectious agents specific to IBC samples. Even though we could not find any strongly statistically distinguishing genomic features between the two groups, we did find some suggestive differences in IBC: (i) The MAST2 gene was more frequently mutated (20% IBC vs. 0% non-IBC). (ii) The TGF β pathway was more frequently disrupted by germline SNVs (50% vs. 13%). (iii) Different copy number profiles were observed in several genomic regions harboring cancer genes. (iv) Complex SVs were more frequent. (v) The clonal architecture was simpler, suggesting more homogenous tumor-evolutionary lineages. Conclusions Whole-genome sequencing of IBC manifests a similar genomic architecture to non-IBC. We found no unique genomic alterations shared in just IBCs; however, subtle genomic differences were observed including germline alterations in TGFβ pathway genes and somatic mutations in the MAST2 kinase that could represent potential therapeutic targets.

BackgroundCyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are a mainstay treatment for hormone receptor-positive breast cancer. While their principal mechanism is inhibition of cancer cell proliferation, preclinical and clinical evidence suggests that CDK4/6i can also promote antitumor T-cell responses. However, this pro-immunogenic property is yet to be successfully harnessed in the clinic, as combining CDK4/6i with immune checkpoint blockade (ICB) has not shown a definitive benefit in patients.MethodWe performed an in-depth analysis of the changes in the tumor immune microenvironment and systemic immune modulation associated with CDK4/6i treatment in muring breast cancer models and in patients with breast cancer using high dimensional flow cytometry and RNA sequencing. Gain and loss of function in vivo experiments employing cell transfer and depletion antibody were performed to uncover immune cell populations critical for CDK4/6i-mediated stimulation of antitumor immunity.ResultsWe found that loss of dendritic cells (DCs) within the tumor microenvironment resulting from CDK4/6 inhibition in bone marrow progenitors is a major factor limiting antitumor immunity after CDK4/6i and ICB. Consequently, restoration of DC compartment by adoptively transferring ex vivo differentiated DCs to mice treated with CDK4/6i and ICB therapy enabled robust tumor inhibition. Mechanistically, the addition of DCs promoted the induction of tumor-localized and systemic CD4 T-cell responses in mice receiving CDK4/6i-ICB-DC combination therapy, as characterized by enrichment of programmed cell death protein-1-negative T helper (Th)1 and Th2 cells with an activated phenotype. CD4 T-cell depletion abrogated the antitumor benefit of CDK4/6i-ICB-DC combination, with outgrowing tumors displaying an increased proportion of terminally exhausted CD8 T cells.ConclusionsOur findings suggest that CDK4/6i-mediated DC suppression limits CD4 T-cell responses essential for the sustained activity of CD8 T cells and tumor inhibition. Furthermore, they imply that restoring DC-CD4 T-cell crosstalk via DC transfer enables effective breast cancer immunity in response to CDK4/6i and ICB treatment.

Introduction: Triple negative breast cancers (TNBC) have shown limited responses to immune checkpoint blockade (ICB). Breast cancer is associated with defects in antigen presentation which may contribute to resistance to ICB. Flt3 ligand (Flt3L) is a growth factor that increases differentiation of DC1 dendritic cells, critical mediators of antigen presentation. CD40 agonist activates all 3 classes of antigen presenting cells - dendritic cells, B cells, and macrophages. Synergy has been demonstrated between Flt3L and CD40 agonist as well as between CD40 agonist and chemotherapy in other cancers, however the combination of all three has not been studied in breast cancer. Methods: 6-8 week old Balb/c mice were injected with 4T1 triple negative breast cancer cells. When tumors were 50 mm3, mice were first treated with intraperitoneal pegylated liposomal doxorubicin (PLD) once and subcutaneous Flt3L daily for 5 days in different sequencing schedules (PLD first followed by Flt3L immediately or 4 days later or Flt3L first followed by PLD 7 days or 10 days later). Based on optimal scheduling of PLD and Flt3L, mice were treated with PLD alone, Flt3L alone, CD40 agonist alone, PLD + Flt3L, PLD + CD40 agonist, PLD + PD-1 blockade, or PLD + CD40 agonist + Flt3L. In addition to serial tumor measurements, mice were sacrificed and tumors and lymphoid organs harvested for flow cytometry analyses. Results: Treatment with PLD at least 4 days before Flt3L led to an optimal increase in intra-tumoral DC1 cells (p=0.0002), increase in polyfunctional CD8 T cell response (p Conclusions: Novel triplet combination with PLD, CD40 agonist, and Flt3L leads to enhanced tumor control in the 4T1 TNBC mouse model. A clinical trial with this combination in metastatic TNBC patients is expected to begin recruitment soon (NCT05029999). Citation Format: Vijay Ramani, Shruthi Nooka, Yu-An Zhang, Serena Gibbs, Hai-Cheng Huang, Melanie Hullings, Suzanne Conzen, Carlos L. Arteaga, Isaac Chan, Sangeetha M. Reddy. Treatment with chemotherapy, CD40 agonist, and Flt3 ligand triplet combination enhances antigen presentation and leads to cures in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4197.

TPS617 Background: Local tumor destruction with cryoablation (cryo) induces inflammation and releases antigens that can activate tumor-specific immune responses. Pre-clinically, cryo with checkpoint inhibition augmented tumor-specific immune responses and prevented recurrence. Clinically, we established that peri-operative (peri-op) cryo with ipilimumab (ipi) +/- nivolumab (nivo) was not only safe in patients (pts) with operable, early stage breast cancer (ESBC) but also generated robust intra-tumoral and systemic immune responses. In this phase 2 study, we evaluate the disease specific impact of peri-op ipi/nivo/cryo in women with residual triple negative breast cancer (TNBC) after neoadjuvant chemotherapy (NAC), a subset at high risk of early relapse. Methods: Eligible pts are ≥18y, with ER < 10%, PR < 10%, HER2 negative (per ASCO/CAP definition), ≥ 1.0 cm, residual operable disease after taxane-based NAC. Approximately 80 pts will be enrolled and treated with ipi/nivo/cryo followed by breast surgery and adjuvant nivo. Pts undergo percutaneous, image-guided cryo with concurrent research core biopsy 7-10 days prior to surgery and will receive ipi (1mg/kg IV) with nivo (240mg IV) 1 to 5 days prior to cryo. After surgery, pts will receive 3 additional doses of nivo at 240mg IV Q2 weeks. Adjuvant capecitabine is recommended for all patients per local standard-of-care. Patients will be stratified by NAC platinum administration, NAC anthracycline administration, and clinical nodal status (positive versus negative). The primary endpoint is 3-year Event Free Survival (EFS). Secondary endpoints include Invasive Disease-Free Survival (IDFS), Distant Disease-Free Survival (DDFS), overall survival (OS) and safety. Exploratory correlative studies will be performed on tumor and serum to characterize the immunologic impact of the intervention and to explore predictors of efficacy and toxicity. Clinical trial information: NCT03546686.

TPS1126 Background: Only a subset of patients with metastatic triple-negative breast cancer demonstrate response to currently approved PD-1 immune checkpoint blockade, and few have durable responses. Antigen presentation defects may be a reason for this low response because deficiency of antigen-presenting DC1 dendritic cells is associated with poor anti-tumor immunity. CD40 agonists are a class of agents that activate antigen presenting cells including dendritic cells and B cells and also repolarize macrophages. Flt3 ligand is a growth factor that increases dendritic cells. In line with this, we recently demonstrated in pre-clinical models that the combination of liposomal-doxorubicin chemotherapy, a CD40 agonist, and a Flt3 ligand improves outcomes of breast cancer compared to alternate combinations. Methods: This is a single arm phase I pilot study of liposomal-doxorubicin, CDX-1140 (CD40 agonist), and CDX-301 (Flt3 ligand) combination therapy in patients with metastatic or unresectable locally advanced metastatic triple-negative breast cancer. Patients will be randomized to 3 lead-in arms (triplet therapy, doublet immunotherapy only, liposomal-doxorubicin only) prior to receiving full triplet therapy with fresh tissue biopsies before and after the lead-in treatment. CDX-301 will be discontinued after 2 cycles; liposomal-doxorubicin and CDX-1140 will be continued until disease progression or clinically limiting toxicities. Primary endpoint is determination of a recommended phase 2 dose based on treatment-related adverse events including dose-limiting toxicities. Secondary endpoints include anti-tumor immune response after triplet therapy, after immunotherapy alone, and after liposomal-doxorubicin alone; median progression-free survival, overall response rate, duration of response, and clinical benefit rate. Key eligibility criteria are unresectable stage III or stage IV triple-negative breast cancer (ER ≤10%, PR ≤10%, HER2/neu negative), 1st to 3rd line metastatic treatment setting (1st line patients need to be PD-L1 negative by 22C3 assay), measurable disease by RECIST 1.1 criteria, consent for pre-treatment and on-treatment biopsies of amenable soft tissue tumor lesions, no prior treatment with an anti-CD40 antibody or a Flt3 ligand, no anthracycline treatment in the metastatic setting, no prior progression while on anthracycline-based therapy or within 6 months of completing neoadjuvant chemotherapy, and no history of non-infectious pneumonitis or current pneumonitis. This trial will enroll up to 45 patients across multiple sites. Clinical trial information: NCT05029999.

Additional file 4: Fig. S1-S10. Supplementary Fig. S1-S10.

Axillary nodal status is one of the most important prognostic factors in breast cancer. While sentinel lymph node biopsy (SLNB) is a safe and validated procedure for clinically node-negative patients, axillary management of clinically node-positive patients has been more controversial. Patients with clinically detected axillary metastases often benefit from neoadjuvant chemotherapy (NAC). Those who convert to node-negative disease following NAC are important to identify, since they can often be spared significant morbidity from axillary dissection. SLNB has shown widely varying false-negative rates (FNR) but with the use of dual mapping and surgical biopsy of 3 or more nodes, it is considered an acceptable method to stage the axilla in clinically node-positive patients who receive NAC. Various methods including targeted axillary dissection (TAD) have been shown to decrease the FNR of SLNB. We will review appropriate methods to identify a metastatic node and subsequent ultrasound-guided biopsy with tissue marking techniques. We underscore key points in monitoring axillary response, techniques to accurately localize the biopsied and clipped known metastatic node for surgical excision and the effect of various methods in reducing the FNR of SLNB, including the emerging concept of TAD on patient care.

Complex tumor microenvironmental (TME) features influence the outcome of cancer immunotherapy (IO). Here we perform immunogenomic analyses on 67 intratumor sub-regions of a PD-1 inhibitor-resistant melanoma tumor and 2 additional metastases arising over 8 years, to characterize TME interactions. We identify spatially distinct evolution of copy number alterations influencing local immune composition. Sub-regions with chromosome 7 gain display a relative lack of leukocyte infiltrate but evidence of neutrophil activation, recapitulated in The Cancer Genome Atlas (TCGA) samples, and associated with lack of response to IO across three clinical cohorts. Whether neutrophil activation represents cause or consequence of local tumor necrosis requires further study. Analyses of T-cell clonotypes reveal the presence of recurrent priming events manifesting in a dominant T-cell clonotype over many years. Our findings highlight the links between marked levels of genomic and immune heterogeneity within the physical space of a tumor, with implications for biomarker evaluation and immunotherapy response., Immunotherapies now dominate the treatment landscape for melanoma, but why they only work in a subset of patients remains unclear. Here, the authors perform an immunogenomic analysis on 67 intratumor sub-regions of a PD-1 inhibitor resistant melanoma, and 2 additional metastases from a single patient, mapping the spatial relationships between genomic and immune heterogeneity at high resolution.

Introduction: In March 2019, atezolizumab became the first immune checkpoint inhibitor to receive a breast cancer-specific approval. Based on a significant improvement in progression-free survival as well as a 10-month improvement in overall survival (on interim analysis) seen in the IMpassion 130 trial, the combination of atezolizumab and nab-paclitaxel was approved for patients with unresectable locally advanced or metastatic triple-negative breast cancer (TNBC).Areas covered: This article reviews current data and ongoing research on atezolizumab for the treatment of breast cancer. Results of atezolizumab monotherapy trials in the context of other early immune checkpoint blockade trials in breast cancer are discussed as well as data from combination clinical trials with chemotherapy in both early-stage and metastatic breast cancer. We focus on the safety and efficacy analyses from the phase III IMpassion trial that led to FDA and EMA approval of atezolizumab and nab-paclitaxel in patients whose tumor tested positive for PD-L1 by the Ventana SP142 companion diagnostic immunohistochemical assay.Expert opinion: The FDA and EMA approvals of atezolizumab mark an important advance for treatment of metastatic TNBC. However, ongoing investigations need to define better biomarkers of response, determine resistance mechanisms, and identify strategies to increase response rates.

Multiomic profiling of several cohorts of patients treated with immune checkpoint blockade highlights the presence and potential role of B cells and tertiary lymphoid structures in promoting therapy response.Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers(1-10) and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity(11-15), although these have been less well-studied in ICB treatment(16). A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling(17) that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter(18)) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Preclinical studies suggest that treatment with neoadjuvant immune checkpoint blockade is associated with enhanced survival and antigen-specific T cell responses over adjuvant treatment1; however, optimal regimens have not been defined. Herein, we report results from a randomized phase II study of neoadjuvant nivolumab versus combined ipilimumab with nivolumab in 23 patients with high-risk resectable melanoma (NCT02519322). RECIST overall response rates (ORR), pathologic complete response rates (pCR), treatment-related adverse events (trAEs), and immune correlates of response were assessed. Treatment with combined ipilimumab and nivolumab yielded high response rates (RECIST ORR 73%, pCR 45%) but substantial toxicity (73% grade 3 trAEs), whereas treatment with nivolumab monotherapy yielded modest responses (ORR 25%, pCR 25%) and low toxicity (8% grade 3 trAEs). Immune correlates of response were identified, demonstrating higher lymphoid infiltrates in responders to both therapies and a more clonal and diverse T cell infiltrate in responders to nivolumab monotherapy. These results are the first to describe the feasibility of neoadjuvant immune checkpoint blockade in melanoma and emphasize the need for additional studies to optimize treatment regimens and to validate putative biomarkers.

Summary Background Dual BRAF and MEK inhibition produces a response in a large number of patients with stage IV BRAF-mutant melanoma. The existing standard of care for patients with clinical stage III melanoma is upfront surgery and consideration for adjuvant therapy, which is insufficient to cure most patients. Neoadjuvant targeted therapy with BRAF and MEK inhibitors (such as dabrafenib and trametinib) might provide clinical benefit in this high-risk p opulation. Methods We undertook this single-centre, open-label, randomised phase 2 trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Eligible participants were adult patients (aged ≥18 years) with histologically or cytologically confirmed surgically resectable clinical stage III or oligometastatic stage IV BRAFV600E or BRAFV600K (ie, Val600Glu or Val600Lys)-mutated melanoma. Eligible patients had to have an Eastern Cooperative Oncology Group performance status of 0 or 1, a life expectancy of more than 3 years, and no previous exposure to BRAF or MEK inhibitors. Exclusion criteria included metastases to bone, brain, or other sites where complete surgical excision was in doubt. We randomly assigned patients (1:2) to either upfront surgery and consideration for adjuvant therapy (standard of care group) or neoadjuvant plus adjuvant dabrafenib and trametinib (8 weeks of neoadjuvant oral dabrafenib 150 mg twice per day and oral trametinib 2 mg per day followed by surgery, then up to 44 weeks of adjuvant dabrafenib plus trametinib starting 1 week after surgery for a total of 52 weeks of treatment). Randomisation was not masked and was implemented by the clinical trial conduct website maintained by the trial centre. Patients were stratified by disease stage. The primary endpoint was investigator-assessed event-free survival (ie, patients who were alive without disease progression) at 12 months in the intent-to-treat population. This trial is registered at ClinicalTrials.gov, number NCT02231775. Findings Between Oct 23, 2014, and April 13, 2016, we randomly assigned seven patients to standard of care, and 14 to neoadjuvant plus adjuvant dabrafenib and trametinib. The trial was stopped early after a prespecified interim safety analysis that occurred after a quarter of the participants had been accrued revealed significantly longer event-free survival with neoadjuvant plus adjuvant dabrafenib and trametinib than with standard of care. After a median follow-up of 18·6 months (IQR 14·6–23·1), significantly more patients receiving neoadjuvant plus adjuvant dabrafenib and trametinib were alive without disease progression than those receiving standard of care (ten [71%] of 14 patients vs none of seven in the standard of care group; median event-free survival was 19·7 months [16·2–not estimable] vs 2·9 months [95% CI 1·7–not estimable]; hazard ratio 0·016, 95% CI 0·00012–0·14, p Interpretation Neoadjuvant plus adjuvant dabrafenib and trametinib significantly improved event-free survival versus standard of care in patients with high-risk, surgically resectable, clinical stage III–IV melanoma. Although the trial finished early, limiting generalisability of the results, the findings provide proof-of-concept and support the rationale for further investigation of neoadjuvant approaches in this disease. This trial is currently continuing accrual as a single-arm study of neoadjuvant plus adjuvant dabrafenib and trametinib. Funding Novartis Pharmaceuticals Corporation.

Background: IBCs that do not completely respond to chemotherapy often have dysregulated immune pathways, and novel therapies are needed to improve outcomes in recurrent/metastatic disease. One-third of IBCs express the atezolizumab target PD-L1, and cobimetinib increases PD-L1 expression; thus, we hypothesize that atezolizumab and cobimetinib may act synergistically in IBC. The FDA-approved agent eribulin is active in IBC and has anti-stem cell activity and can reverse the IBC phenotype of epithelial-to-mesenchymal transition. Hence the use of eribulin as a chemotherapy backbone in combination with other novel agents is well justified. Trial Design: This single-arm, open-label trial is enrolling patients with recurrent IBC or de novo metastatic IBC that has progressed on at least 1 line of standard chemotherapy. During a 4-week pharmacodynamic window, patients have an upfront biopsy, receive atezolizumab and cobimetinib treatment for 4 weeks, and have a second biopsy. Triple-combination treatment then commences, with standard eribulin dosing. After 4 cycles of eribulin, patients receive maintenance targeted therapy until disease progression or intolerable toxicity. Eligibility Criteria: Patients with metastatic IBC of any molecular subtype must have measurable disease (per RECIST 1.1) amenable to biopsy. Patients with HER2+ disease must have received both pertuzumab and T-DM1. Patients with treated stable brain metastases are allowed. Patients must have recovered from the acute effects of any prior therapies and have adequate hematologic, organ, and cardiac function. Patients with autoimmune diseases or a history of pneumonitis are ineligible. Specific Aims: The primary objective is to determine the overall response rate (ORR) of the combination therapy. Secondary objectives include determining the safety and tolerability, clinical benefit rate, response duration, progression-free survival, 2-year overall survival rate and predictive biomarker analyses. Statistical Methods: The trial will enroll up to 9 patients in its phase I/safety lead-in portion and up to 33 patients total. A Bayesian optimal interval design is used to efficiently determine the maximum tolerated cobimetinib dose in phase I. Patients start cobimetinib at the FDA-approved dose of 60 mg/day with a target toxicity rate is 0.3. Phase II will enroll 24 patients to determine the efficacy of the triple-combination therapy. The historical ORR in metastatic IBC is 10%; our sample size provides 80% power to detect an ORR improvement to 25%. Accrual: The trial has enrolled 7 patients since its start in August 2017. Citation Format: Alexander A, Marx AN, Reddy SM, Reuben JM, Le-Petross HC, Lane D, Huang ML, Krishnamurthy S, Gong Y, Gombos DS, Patel N, Tung CI, Allen RC, Kandl TJ, Wu J, Liu S, Patel AB, Futreal A, Wistuba I, Layman RM, Valero V, Tripathy D, Ueno NT, Lim B. Phase II study of atezolizumab, cobimetinib, and eribulin in patients with recurrent or metastatic inflammatory breast cancer (IBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-05-04.

Background: We counsel our triple-negative breast cancer (TNBC) patients that the risk of recurrence is highest in the first 5 years after diagnosis. However, there are limited data with extended follow-up on the frequency, characteristics, and predictors of late events. Methods: We queried the MD Anderson Breast Cancer Management System database to identify patients with stage I–III TNBC who were disease free at 5 years from diagnosis. The Kaplan–Meier method was used to estimate yearly recurrence-free interval (RFI), recurrence-free survival (RFS), and distant relapse-free survival (DRFS), as defined by the STEEP criteria. Cox proportional hazards model was used to compute hazard ratios (HRs) and 95% confidence intervals (CIs). Results: We identified 873 patients who were disease free at least 5 years from diagnosis with median follow-up of 8.3 years. The 10-year RFI was 97%, RFS 91%, and DRFS 92% the 15-year RFI was 95%, RFS 83%, and DRFS 84%. On a subset of patients with oestrogen receptor and progesterone receptor percentage recorded, low hormone receptor positivity conferred higher risk of late events on multivariable analysis for RFS only (RFI: HR=1.98, 95% CI=0.70–5.62, P-value=0.200; RFS: HR=1.94, 95% CI=1.05–3.56, P-value=0.034; DRFS: HR=1.72, 95% CI=0.92–3.24, P-value=0.091). Conclusions: The TNBC survivors who have been disease free for 5 years have a low probability of experiencing recurrence over the subsequent 10 years. Patients with low hormone receptor-positive cancers may have a higher risk of late events as measured by RFS but not by RFI or DRFS.

Major advances have been made in melanoma treatment with the use of molecularly targeted therapies and immunotherapies, and numerous regimens are now approved by the US Food and Drug Administration for patients with stage IV disease. However, therapeutic resistance remains an issue to both classes of agents, and reliable biomarkers of therapeutic response and resistance are lacking. Mechanistic insights are being gained through preclinical studies and translational research, offering potential strategies to enhance responses and survival in treated patients. A comprehensive understanding of the immune effects of common mutations at play in melanoma is critical, as is an appreciation of the molecular mechanisms contributing to therapeutic resistance to immunotherapy. These mechanisms and the interplay between them are discussed herein. Cancer 2017;123:2130-42. © 2017 American Cancer Society.

Goal: Inflammatory breast cancer (IBC) is a rare, aggressive form of breast cancer that is characterized by a highly metastatic phenotype. Numerous previous attempts failed to identify, recurrent, IBC-specific gene expression or DNA copy number alterations. We performed whole genome sequencing (WGS) of IBC biopsies obtained before any therapy to define a comprehensive genomic landscape of this disease. Methods: Illumina paired-end whole genome sequencing (WGS) of 20 IBC (n=9 ER+, n=11 ER-) and matched normal samples were performed with median coverage of 60X and 40X for cancer and normal, the percentages of mapped reads were 99.3% and 99.2%, respectively. We identified germ-line and somatic variants, indels as well as large scale structural variants, using GATK Haplotype Caller, MuTect and CREST, respectively. We performed the same analysis on WGS data from 23, age, race and ER and HER2 matched, non-IBC (n=12 ER+, n=11 ER-) from the TCGA for comparison. Variants in both coding and noncoding sequences were categorized by FunSeq to identify potential drivers. Mutation clustering in each gene, as well as significantly mutated non-coding regulatory modules, were identified using LARVA. DeconstructSigs were used to decompose the mutational spectrum of each cancer into 30 validated, mutational signatures provided by COSMIC. Contributions of each validated signature to mutations in IBC vs. non-IBC were compared using Welch's t-test. Results: We identified 118,818 somatic variants in the IBC samples (median: 3,856; minimum: 1,109; maximum: 24,815) including 1,060 variants (~0.9%) in coding regions. 5,287 somatic indels and 5,959 large scale structural variants were detected including 1,028 insertions and 1,857 deletions. Recurrent, non-synonymous mutations were detected in the coding region of GRIN2A gene in 3/20 IBC samples (15%), (previously reported as a potential driver mutation in 1.7% of breast cancers). Other significant mutations in coding regions included GRHL1, PIK3R2, ESR1, FLG2 and etc. Three DNase I hypersensitive sites (DHSs) in non-coding regions were altered in 20% (4/20) IBC samples vs. fewer than 8.7% (2/23) in non-IBC. Mutational frequency of GATA3 is 80.0% vs. 47.8% (p=0.03), and PTEN is 45.0% vs. 73.9% (p=0.05), in IBC vs. non-IBC samples when including both coding and non-coding variants. Contributions of mutational signature 9, that is associated with polymerase η , were significantly higher in IBC cohort than non-IBC cohort (p-value=0.056). Conclusion: This is the first whole genome sequencing analysis of IBC and comparison with the results from non-IBC. We identified promising candidate drivers in the coding sequence and in non-coding regulatory modules of expressed genes. We also identified mutational signature 9, and mutations in several DHS as significantly more frequent alterations in IBC compared to non-IBC. Citation Format: Li X, Krishnamurthy S, Kumar S, Reddy S, Woodward W, Reuben J, Hatzis C, Ueno NT, Gerstein M, Pusztai L. Landscape of somatic mutations in inflammatory breast cancer whole-genome sequences [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-01.

Background: Treatment of inflammatory breast cancer (IBC) includes neoadjuvant chemotherapy (NAC) followed by mastectomy and radiation. Responses are limited however with low pathological complete response (pCR) rates and poor survival. Recent RNA expression studies suggest that activated T cell signaling pathways and immunomodulatory markers such as PD-L1 are associated with a higher pCR rate in IBC; however comprehensive studies of tumor infiltrating lymphocytes (TIL) and protein expression of immunomodulatory molecules are lacking. There is a critical need therefore to study molecular and immune determinants of therapeutic response in IBC, with the goal of identifying biomarkers and actionable strategies to improve treatment outcomes. Methods: Baseline core biopsies from 36 IBC patients, 22 with stage III and 14 with stage IV disease were evaluated. Of these, 21 stage III and 10 stage IV patients underwent mastectomy following NAC, the latter for palliative purposes. Tumor subtype distribution was 14 patients with HER2-/HR-, 6 with HER2+/HR-, 11 with HER2-/HR+, and 5 with HER2+/HR+ disease. TIL infiltration in the tumor stroma was quantified on H&E slides based on consensus guidelines as well as by immunohistochemistry (IHC) staining for CD8. PD-L1 expression in the TIL and invasive tumor was evaluated by IHC in tumors with >1%TIL. Results: Stromal TIL were found in the invasive tumor on pretreatment biopsies in 26 (72%) patients, with TIL percentages ranging from 1% to 60% (mean=11.6; sd=13.8); of note, 1 patient biopsy sample only had tumor emboli on the tissue block and therefore was not evaluable. Higher TIL infiltrate was noted in stage III versus stage IV disease (mean TIL 11.6% versus 3.5%, p=0.028). Mean TIL infiltrate was 11.5% in HER2-/HR-, 10.0% in HER2+/HR-, 10.4% in HER2+/HR+, and 3.6% in HER2-/HR+ tumors (p=NS). At mastectomy, 7/21 stage III patients and 1/10 stage IV patients achieved a pCR. Mean TIL was 13.4% in the pCR group versus 8.2% in the non-pCR group (p=0.37) CD8 and PD-L1 staining was performed on samples with >1%TIL (n=15, of which 14 samples were available for additional staining). An average of 42% of TIL stained positive for CD8 (range 10-80%). There was no significant relationship between %CD8 and pCR, stage, or receptor status. None of these 14 patients demonstrated membranous PD-L1 positivity but all had focal weak cytoplasmic staining in the lymphocytes. Conclusions: Differences exist in the presence of stromal TIL in distinct groups within IBC (stage III versus stage IV disease and across histologic subtypes) and may contribute to differential responses to therapy. When comparing these results to published non-IBC literature (FinHER trial), our IBC patient cohort had lower TIL infiltrate in several histologic subtypes (HER2-/HR- 11.5% vs 25%, p=0.015), HER2+/HR-(10% vs 20%, p=0.10), and HER-/HR+ disease (3.6 vs 7.5%, p=0.01); TIL was comparable for HER2+/HR+ disease. Additional studies are underway (including multiplex analysis of myeloid and lymphoid markers, T cell receptor sequencing, and molecular profiling) in pre-treatment and surgical samples to better understand mechanisms of treatment response and resistance. Citation Format: Reddy SM, Wargo JA, Reuben A, Reuben J, Woodward W, Ueno N, Mittendorf EA, Krishnamurthy S. Immune characterization of inflammatory breast cancer and correlation to pathological complete response [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-16-01.

Background: Patients who have TNBC or triple negative-IBC (TN-IBC) and do not achieve pathological complete response after neoadjuvant chemotherapy are at significant risk for distant relapse and death from recurrent disease. Apart from capecitabine, there are no proven adjuvant therapies that may improve these poor outcomes of patients with chemo-resistant tumors. Therefore, there is an unmet need for effective systemic therapy for this subset of patients with TNBC. Epidemiological evidence reveals that statin use after diagnosis is associated with improved breast cancer relapse-free survival and decreased mortality. However, direct evidence of in vivo mechanisms explaining this association are lacking. Preclinical studies using statins in breast cancer reveal pathways that statins can inhibit proliferation, stem cell self-renewal and metastatic potential. Trial Design: This is a pilot study designed in 2 phases to assess feasibility of completion while providing a signal of efficacy in biomarker changes. In the first phase, we will follow the initial 30 patients who meet eligibility for atorvastatin treatment for the 2-year treatment window, or until disease recurrence. We will collect blood samples prior to, and during atorvastatin treatment for circulating tumor cells (CTCs), cytokine and inflammatory biomarker analyses. We defined a positive outcome as CTCs remaining non-detected at 6 months when baseline CTC is undetected, or a reduction in the number of CTCs at 6 months compared to baseline. If we observe a positive outcome among the initial 30 patients, then we will open the second phase of this study for an additional 50 patients. Here we will follow both patient cohorts who receive and not receive atorvastatin treatment to collect longitudinal data on biomarkers as a function of the natural history of TNBC to better understand the activity of atorvastatin. Trial Eligibility: Patients with stage II-III TNBC who have residual cancer burden (RCB)-II or RCB-III or stage 3 TN-IBC with any amount of residual disease, and are not taking a statin or any other anti-lipidemic agent are candidates for the study. Patients must have adequate hematologic, organ, and cardiac function and must have recovered from the acute effects of any prior treatments. Baseline lipid profile will be assessed by a cardiologist to determine the patients’ eligibility to take atorvastatin based on current ACC/AHA guideline, and to select between moderate (20mg) or high intensity treatment (40mg). Specific Aims: The primary objective is to determine the proportion of patients with undetectable CTCs at 6 months with and without atorvastatin therapy. Secondary objectives include correlation of baseline lipid profiles/lipid profile changes with 2 year-relapse free survival (RFS), CTC counts and inflammatory biomarkers. Statistical Methods: The total estimated enrollment is 80 patients, including at least 5 treated with adjuvant capecitabine and at least 5 without adjuvant capecitabine. The study overall is powered with the assumption that 48 patients will receive atorvastatin and 32 will not, and this will allow us to estimate the percent of patients with negative CTCs at 6 months with a standard error not larger than 7% and 9%, respectively. All other analyses including inflammatory biomarkers and RFS differences between groups are exploratory and considered hypothesis-generating rather than conclusive. Citation Format: Angela Alexander, Takeo Fujii, Michael C Stauder, Wendy A Woodward, James M Reuben, Yu Shen, Diane Liu, Sangeetha M Reddy, Vicente Valero, Susan C Gilchrist, Bora Lim, Anthony Lucci, Naoto T Ueno, Carlos H Barcenas. A pilot study to examine the feasibility of measuring CTC and inflammatory biomarker changes resulting from atorvastatin as adjuvant therapy in TNBC and TN-IBC patients with residual disease after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT1-02-02.

Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with poor response rates to current chemotherapy regimens. With recent successes of immune targeted therapies in other solid tumors and a growing understanding of how the immune tumor microenvironment (TME) affects non-IBC outcomes, we sought to characterize the immune TME in IBC to identify biomarkers of treatment response and potential targets for drug development. Methods: Pre-treatment core biopsy samples were identified from the MD Anderson Cancer Center IBC tissue bank from patients with stage III and de novo stage IV (with T4d) disease who received neoadjuvant chemotherapy (NAC) with intent to take to mastectomy. Lymphocytes were characterized by stromal tumor infiltrating lymphocyte (sTIL) quantification, CD8 T cell quantification, and T cell receptor sequencing. PD-L1 expression was assessed using DAKO 22C3 clone on tumor and immune cells. Myeloid cells were characterized using a multiplex immunohistochemistry approach, using CD68 and CD163 for macrophage markers, tryptase for mast cell marker, HLA-DR for class II antigen presentation marker, and cytokeratin as tumor marker. Spatial analyses were performed by determining probabilities of finding cell 1 of interest within 20 uM of cell 2 of interest and computing area under the curve for statistical comparison. Results: 91 patients with stage III (N=62) or de novo stage IV (n=29) disease were identified. Breast cancer subtype included 25 triple negative, 34 HER2+ and 32 HER2-HR+. 86 patients received a mastectomy, of whom 33 (38.4%) patients experienced a pathologic complete response (pCR). sTIL was higher in stage III tumors (11.9 vs 4.8%, p Conclusions: Higher TIL and CD8 T cell density are correlated with improved responses to NAC in IBC. Mast cell infiltration and HLA-DR expression on tumor cells are inversely correlated to response and suggest possible mechanisms of resistance. Mast cells could present potential therapeutic target in IBC. Citation Format: Reddy SM, Reuben A, Jiang H, Roszik J, Tetzlaff MT, Reuben J, Wang L, Tsujikawa T, Barua S, Rao A, Villareal L, Wood A, Woodward W, Ueno NT, Krishnamurthy S, Wargo JA, Mittendorf EA. Lymphoid and myeloid cell characterization of inflammatory breast cancer tumor microenvironment and correlation to pathological complete response [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-08.

Immune checkpoint inhibitors (ICIs) have improved overall survival for cancer patients, however, optimal duration of ICI therapy has yet to be defined. Given ICIs were first used to treat patients with metastatic melanoma, a condition that at the time was incurable, little attention was initially paid to how much therapy would be needed for a durable response. As the early immunotherapy trials have matured past 10 years, a significant per cent of patients have demonstrated durable responses; it is now time to determine whether patients have been overtreated, and if durable remissions can still be achieved with less therapy, limiting the physical and financial toxicity associated with years of treatment. Well-designed trials are needed to identify optimal duration of therapy, and to define biomarkers to predict who would benefit from shorter courses of immunotherapy. Here, we outline key questions related to health, financial and societal toxicities of over treating with ICI and present four unique clinical trials aimed at exposing criteria for early cessation of ICI. Taken together, there is a serious liability to overtreating patients with ICI and future work is warranted to determine when it is safe to stop ICI.

Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte–associated antigen-4 (CTLA4) blockade (n = 53) followed by programmed death-1 (PD-1) blockade at progression (n = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified. Significance: These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. Cancer Discov; 6(8); 827–37. ©2016 AACR. See related commentary by Teng et al., p. 818. This article is highlighted in the In This Issue feature, p. 803

Immune monitoring in the tumor microenvironment allows for important insights into immune mechanisms of response and resistance to various cancer treatments; however clinical challenges exist using current strategies. Significant questions remain regarding monitoring of archival versus fresh tissue, assessment of static versus dynamic markers, evaluation of limited tissue samples, and the translation of insights gained from immunologically “hot” tumors such as melanoma to other “cold” tumor microenvironments prevalent in other cancer types. Current and emerging immune monitoring strategies will be examined herein, and genomic-based assays complementing these techniques will also be discussed. Finally, host genomic and external environmental factors influencing anti-tumor immune responses will be considered, including the role of the gut microbiome. Though optimal immune monitoring techniques are in evolution, great promise exists in recent advances that will help guide patient selection as far as type, sequence, and combination of therapeutic regimens to enhance anti-tumor immunity and clinical responses.

Purpose Oxaliplatin neurotoxicity has a spectrum of manifestations from an often reversible acute neurotoxicity to a more irreversible “stocking and glove” chronic neuropathy that is associated with high morbidity. Quantitative sensory testing (QST) is a noninvasive psychometric testing method that can potentially be used in the clinic setting to measure subclinical neurologic changes early on to identify patients that will experience chronic oxaliplatin-induced peripheral neuropathy at 1 year. Patients and Methods Thirty patients with gastrointestinal malignancies who were receiving oxaliplatin were recruited. QST and patient-reported outcomes were assessed at baseline; during infusion cycles 1, 2, 4, and 6; and at 1 year. National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), version 4.0, chronic neuropathy scores were assessed at the 1-year time point. The variables at each time point were evaluated for prediction of 1-year chronic neuropathy scores. Results We found that patients with preexisting subclinical neuropathy were more likely to experience grades 2 and 3 chronic neuropathy than were those who did not have this condition (heat detection threshold, Spearman correlation coefficient (r s ) = 0.39; P = .037; pellet retrieval time, r s = 0.47; P = .024). Patients in whom thermal and cutaneous sensory deficits developed with cycle 1 infusion were also more likely to experience grades 2 and 3 neuropathy at 1 year (cold detection threshold, r s = 0.50; P = .007; heat detection threshold, r s = 0.39; P = .042; cutaneous detection threshold, r s = 0.42; P = .043). Conclusion QST provides a noninvasive, commercially available, and feasible clinical test to select patients, even before oxaliplatin treatment, who are likely to experience moderate to severe chronic peripheral neuropathy.

Background: Stage I-III TNBC patients have a high risk of disease relapse during the first 5 years after diagnosis. However, there is limited data on the risk of late relapse in TNBC survivors who are disease free at 5 years or more from diagnosis. We sought to characterize this risk in a cohort of TNBC long-term survivors from a large institutional database. Methods: The MD Anderson Breast Cancer Management System database was queried for TNBC survivors who were disease free 5 years or more from diagnosis. Demographic, tumor, and treatment data was extracted. Electronic medical records were searched to confirm pathology reports for invasive breast cancer diagnosis, triple negative receptor status, and hormone receptor percentage (%). The primary and secondary outcomes of interest were relapse free survival (RFS) and distant relapse free survival (DRFS). Patients were censored at time of developing a second primary breast cancer or at last follow-up time for those who were alive during the study. We used ACP-ASCO definition of ER and PR Results: We identified 1038 patients who had a median follow-up of 8.0 years. Receptor % information was available on 69% of patients, with 78% of them meeting current TNBC definition. From the total cohort of 130, 12.5% suffered event(s) that occurred after 5 years from diagnosis, with 86.2% of them occurring within 5-10 years of diagnosis. The event rate was 16.4% among patients with ER/PR 1-9% versus 11.3% among patients with ER/PR Conclusions: TNBC long term survivors are still at risk for relapse events after 5 years from diagnosis, and it is important to quantity this risk when counseling our patients. Frequency of late events was higher among patients with low hormone receptor positivity. Multivariate modeling of predictors of late recurrence is ongoing. Table 1: RFS and DRFS by Year from Diagnosis All PatientsER/PR Table 2: Site of Initial Distant RecurrenceSiteN (%)Lung/Pleura28 (54.9)Bone19 (37.3)Distant Lymph Nodes19 (37.3)Liver11 (21.6)Brain/Spinal Cord8 (15.7)Colorectal/Pancreas/Kidney/Adrenal6 (11.7)Other2 (3.9)*Patients presenting with multiple sites of distant recurrence are counted in each category. Citation Format: Reddy SM, Sinha A, Syed M, Barcenas C, Valero V. Relapse-free survival of triple negative breast cancer long term survivors and characterization of late events in MD Anderson experience. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-10-09.

Our understanding is limited concerning the tumor immune microenvironment of inflammatory breast cancer (IBC), an aggressive form of primary cancer with low rates of pathologic complete response to current neoadjuvant chemotherapy (NAC) regimens. We retrospectively identified pretreatment (N = 86) and matched posttreatment tissue (N = 27) from patients with stage III or de novo stage IV IBC who received NAC followed by a mastectomy. Immune profiling was performed including quantification of lymphoid and myeloid infiltrates by IHC and T-cell repertoire analysis. Thirty-four of 86 cases in this cohort (39.5%) achieved a pathologic complete response. Characterization of the tumor microenvironment revealed that having a lower pretreatment mast cell density was significantly associated with achieving a pathologic complete response to NAC (P = 0.004), with responders also having more stromal tumor-infiltrating lymphocytes (P = 0.035), CD8+ T cells (P = 0.047), and CD20+ B cells (P = 0.054). Spatial analysis showed close proximity of mast cells to CD8+ T cells, CD163+ monocytes/macrophages, and tumor cells when pathologic complete response was not achieved. PD-L1 positivity on tumor cells was found in fewer than 2% of cases and on immune cells in 27% of cases, but with no correlation to response. Our results highlight the strong association of mast cell infiltration with poor response to NAC, suggesting a mechanism of treatment resistance and a potential therapeutic target in IBC. Proximity of mast cells to immune and tumor cells may suggest immunosuppressive or tumor-promoting interactions of these mast cells.

Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8(+) T cells were defined by clustering, and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8(+) T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states, and demonstrated enhanced anti-tumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms and targets for enhancing checkpoint immunotherapy.

Good bacteria help fight cancer Resident gut bacteria can affect patient responses to cancer immunotherapy (see the Perspective by Jobin). Routy et al. show that antibiotic consumption is associated with poor response to immunotherapeutic PD-1 blockade. They profiled samples from patients with lung and kidney cancers and found that nonresponding patients had low levels of the bacterium Akkermansia muciniphila . Oral supplementation of the bacteria to antibiotic-treated mice restored the response to immunotherapy. Matson et al. and Gopalakrishnan et al. studied melanoma patients receiving PD-1 blockade and found a greater abundance of “good” bacteria in the guts of responding patients. Nonresponders had an imbalance in gut flora composition, which correlated with impaired immune cell activity. Thus, maintaining healthy gut flora could help patients combat cancer. Science , this issue p. 91 , p. 104 , p. 97 ; see also p. 32

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (, Melanoma: Tumor differences within a patient may explain heterogeneous responses Patients with metastatic melanoma display molecular and immune differences across tumor sites associated with differential drug responses. A team led by Jennifer Wargo from the University of Texas MD Anderson Cancer Center, Houston, USA, studied the radiological responses of 60 patients with metastatic melanoma, half of whom received targeted drug therapy and half of whom received an immune checkpoint inhibitor. The majority (83%) showed differences in responses across metastases. The group then profiled tumors in a subset, and found molecular and immune heterogeneity in different tumors within the same patient. Heterogeneity in mutational and immune profiles within tumors from individual patients could explain differences in treatment response. Knowing this, the authors emphasize the importance of acquiring biopsies from more than one tumor site in order to best tailor therapies to the features of metastatic cancer.

Immune checkpoint blockade produces clinical benefit in many patients. However better biomarkers of response are still needed, and mechanisms of resistance remain incompletely understood. To address this, we recently studied a cohort of melanoma patients treated with sequential checkpoint blockade against cytotoxic T lymphocyte antigen-4 (CTLA-4) followed by programmed death receptor-1 (PD-1), and identified immune markers of response and resistance. Building on these studies, we performed deep molecular profiling including T-cell receptor sequencing (TCR-seq) and whole exome sequencing (WES) within the same cohort, and demonstrated that a more clonal T cell repertoire was predictive of response to PD-1 but not CTLA-4 blockade. Analysis of copy number alterations identified a higher burden of copy number loss in non-responders to CTLA-4 and PD-1 blockade and found that it was associated with decreased expression of genes in immune-related pathways. The effect of mutational load and burden of copy number loss on response was non-redundant, suggesting the potential utility of a combinatorial biomarker to optimize patient care with checkpoint blockade therapy.

Purpose: We sought to investigate if the use of HMG Co-A reductase inhibitors (statins) has an impact on outcomes among patients with triple negative breast cancer (TNBC). Methods: We reviewed the cases of women with invasive, non-metastatic TNBC, diagnosed 1997-2012. Clinical outcomes were compared based on statin use (defined as ever use during treatment vs. never use). We identified a subset of women for whom a 5-value lipid panel (5VLP) was available, including total cholesterol, low density lipoprotein, high density lipoprotein, very low density lipoprotein, and triglycerides. The Kaplan-Meier method was used to estimate median overall survival (OS), distant metastases-free survival (DMFS), and local-regional recurrence-free survival (LRRFS). A Cox proportional hazards regression model was used to test the statistical significance of prognostic factors. Results: 869 women were identified who met inclusion criteria, with a median follow-up time of 75.1 months (range 2.4-228.9 months). 293 (33.7%) patients used statins and 368 (42.3%) had a 5VLP. OS, DMFS, and LRRFS were not significant based on statin use or type. Controlling for the 5VLP values, on multivariable analysis, statin use was significantly associated with OS (HR 0.10, 95% CI 0.01-0.76), but not with DMFS (HR 0.14, 95% CI 0.01-1.40) nor LRRFS (HR 0.10 95% CI 0.00-3.51). Conclusions: Statin use among patients with TNBC is not associated with improved OS, although it may have a benefit for a subset of patients. Prospective assessment would be valuable to better assess the potential complex correlation between clinical outcome, lipid levels, and statin use.

Background: The pCR rate to conventional chemotherapy in hormone receptor positive IBC has historically been low (7.4% for HR+ HER2-, and 30% for HR+ HER2+), and despite the use of adjuvant endocrine therapy, the recurrence rate is still as high as 40%. To date, no targeted agent is proven to improve the efficacy of adjuvant endocrine therapy within the IBC population to improve this poor disease free survival (DFS). One plausible reason for the poor efficacy of endocrine therapy is a suppressed immune system, which allows tumor cells to avoid detection despite expression of potential immunogenic surface antigens. Trial Design: This is a single arm trial that will enroll stage III HR+ IBC patients who have completed neoadjuvant therapy but had residual disease at mastectomy. Enrollment should be before or within 2 months of beginning endocrine therapy. Monitoring of DFS will be done with radiological imaging every 3 cycles (starting at cycle 4) as clinically indicated, per standard of care. Pembrolizumab is given on day 1 of each 21 day cycle for up to 2 years if the disease is controlled, and hormonal therapy will be administered per standard of care. Eligibility Criteria: Clinical stage 3 IBC ER+/PR+ and HER2 negative patients who completed neoadjuvant chemotherapy and surgery with evidence of residual cancer in the breast or lymph nodes, but be clinically disease-free with good performance status at the start of study. Patients also must have adequate hematologic and organ function, and have recovered from the acute effects from prior treatments. Specific Aims: The primary objective is to determine the disease free survival (DFS) at 2 years of patients with adjuvant therapy using Pembrolizumab in combination with standard adjuvant hormonal therapy. The secondary objective is to determine the safety and toxicity profile of this combination. Statistical Methods: With a sample size of 37 patients, assuming that 80% are alive (20% increase from historical data) and disease-free at 2 years, and all patients are followed for >2 years after enrollment with no dropout, a 95% confidence interval around the 2-year estimate of DFS will be generated. DFS will then be compared with the historical control rate of 60% by year 2 using a one-sided exponential MLE test. Accrual: To date we have enrolled 3 patients since activation in January 2017, and the target enrollment is 37 patients. Contact information: For more information or to refer a patient, please contact study coordinator, Angela Alexander - aalexand@mdanderson.org Citation Format: Alexander A, Willey J, Sun H, Parker CA, Marx AN, Wood AL, Reddy SM, Reuben JM, Bassett RL, Le-Petross HT, Krishnamurthy S, Gong Y, Woodward WA, Valero V, Ueno NT, Lim B. A single arm phase II study of adjuvant anti-PD1 (pembrolizumab) in combination with hormonal therapy in patients with hormone receptor (HR)-positive localized inflammatory breast cancer (IBC) who did not achieve a pathological complete response (pCR) to neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT1-02-05.

Sustained periods of apparent clinical benefit despite lack of objective response are well known in a subpopulation of advanced melanoma patients. Inter-individual heterogeneity in response of separate tumors is common, characterizing complex overall response patterns. The molecular and cellular dynamics facilitating such long-term survival and heterogeneous response is poorly understood, particularly in the era of exposure to multiple potentially active therapies. We studied an exceptional case of long-term survival in a patient with non-responding metastatic melanoma in order to characterize the clonal and microenvironmental factors active across 3 time points. We performed immunogenomic analyses of 3 metachronous tumors, including a systemic therapy-naïve mass, 67 intratumor sub-regions of a non-responding mass during PD-1 inhibitor therapy, and a post-PD-1 inhibitor mass. We profiled samples using whole exome sequencing, RNA-sequencing (RNA-seq), immunohistochemistry (IHC), and T cell receptor sequencing. Longitudinal, spatial, and cross-modal analyses were performed. Longitudinal analyses identified mutations in several genes known to be associated with targeted or immune therapy resistance affecting distinct metastases. Genomic intratumoral heterogeneity (ITH) was primarily driven by subclonal copy number alterations that showed evidence of spatially-distinct evolution which may be in response to selective pressures at the tumor margin. RNA-seq revealed an unexpectedly high degree of ITH characterized by limited group-level gene or pathway associations with physical or immune characteristics of each site. Spatially-distinct pockets of immune activation and suppression were observed throughout the PD-1 inhibitor resistant metastasis despite a largely immune-excluded phenotype seen on IHC. A specific T cell Vβ CDR3 rearrangement was identified as dominant and recurrent not only across multiple spatial points within a single tumor mass, but also across metachronous tumors spanning the patient’s disease course. Immunophenotyping of the T cell population with single-cell RNA-seq suggested repeated T-cell priming events leading to the persistence of both activated and exhausted T cells bearing the same TCR-β at any given time. Our findings highlight an unexpected level of genomic and immune heterogeneity in metastatic melanoma tumors of a long-term surviving patient. The observed degree of ITH across local tumor microenvironments reiterates the inherent limitations to identifying robust and reproducible predictive biomarkers of therapy response based on limited physical sampling of tumors. Further spatiotemporal analysis of metastatic lesions in the context of immune checkpoint blockade will be required to determine how the mechanisms driving convergent microenvironmental phenotypes may be harnessed for therapeutic gain. Citation Format: Akash Mitra, Miles C. Andrews, Whijae Roh, Mariana P. de Macedo, Alexandre Reuben, Fernando Carapeto, Feng Wang, Sangeetha M. Reddy, Khalida Wani, Christine Spencer, John Miller, Aislyn Schalck, Latasha D. Little, Donald A. Sakellariou-Thompson, Curtis Gumbs, Wen-Jen Hwu, Chantale Bernatchez, Jianhua Zhang, Patrick Hwu, Nicholas Navin, Padmanee Sharma, James P. Allison, Jennifer Wargo, Alexander J. Lazar, Philip A. Futreal. Spatially resolved immunogenomic analyses reveal diverse sub tumoral microenvironments in the context of melanoma immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3776.

Background: While treatment with immune checkpoint blockade (ICB) has markedly improved outcomes in advanced melanoma patients and other malignancies, predicting response remains a challenge. Biomarkers including tumor mutational burden (TMB), T-cell infiltration, and PD-L1 expression, have been identifiedbut remain inadequate. Other components of innate and adaptive immunity, including B-cells and tertiary lymphoid structures (TLS), have been implicated in the response to other cancer therapies, andpreclinical data suggests B cells may contribute in the response to immunotherapy. Here, we use targeted protein expression profiling-via NanoString digital spatial profiling (DSP) technology (research use only)-to demonstrate a role for B cells in the response to ICB in patients with high-risk resectable melanoma; furthermore, we characterize the B cell subsets that enable this function. Methods: We conducted a phase 2 clinical trial of neoadjuvant ICB therapy in patients with high-risk resectable melanoma (PD-1 blockade monotherapy or combined CTLA-4/PD-1 blockade) (NCT02519322). Longitudinal tumor samples were taken during therapy. Formalin-fixed paraffin embedded tissue sections from tumor samples (n=10 responders [R], n=10 non-responders [NR]) were analyzed by NanoString DSP technology and stained with a cocktail of S100B, CD45, CD19 and a 40-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. Regions of interest (ROI) were delineated using immunofluorescence followed by UV excitation of the defined ROIs, releasing the DNA barcodes for downstream quantitation on the NanoString nCounter®platform. Utilizing masking strategies, we define the unique expression pattern within discrete subsets of immune cells. These same tumors have concurrently been analyzed by RNAseq, immunohistochemistry, CYTOF, and single-cell RNAseq. Results: Biomarker counts are highly concordant across samples from the same patient’s tumor. High concordance between DSP and quantitative fluorescence is seen as a validation for the DSP method. We identify B cells as components of TLS; the B cells are closely integrated with CD4 and CD8 T cells and follicular dendritic cells. There are significantly more TLS identified in R compared to NR patients. Utilizing CYTOF, we concurrently identify specific subsets of B cells present within the TLS associated with response. We characterize the spatial relationship of these same B cell subsets with other components of the TLS and define the protein expression patterns of these cells. Conclusion: NanoString DSP data complement our deep molecular and immune profiling of tumors from melanoma patients treated with ICB; together, they provide a novel predictive role for B-cells and TLS in the response to ICB and, importantly, provide mechanistic insight into their potential contribution in the response to cancer therapy. Citation Format: Beth A. Helmink, Sangeetha Reddy, Jorge Blando, Yan Liang, Sarah Warren, Vancheswaran Gopalakrishnan, Hussein A. Tawbi, Rodabe N. Amaria, Michael Davies, Jeffrey E. Gershenwald, Elizabeth Burton, Rafet Basar, Alexander J. Lazar, Courtney W. Hudgens, Katy Rezvani, James P. Allison, Padmanee Sharma, Joseph M. Beechem, Jennifer A. Wargo, Michael T. Tetzlaff. NanoString®GeoMx®digital spatial profiling further defines the role of B cells in the response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 499.