Faculty Bibliography

BACKGROUND AND PURPOSE: Resistance to endocrine therapies in hormone receptor (HR)-positive breast cancer is a significant challenge. Prior studies have shown that low-dose oral cyclophosphamide can transiently deplete regulatory T cells (Tregs) and improve anti-tumor immunity. We investigated the combination of exemestane with cyclophosphamide in patients with advanced HR-positive breast cancer and assessed changes in circulating immune cell subsets. METHODS: This was a single-arm phase II trial of exemestane with cyclophosphamide in patients with metastatic HR-positive/HER2-negative breast cancer who had progressed on prior endocrine therapy (ClinicalTrials.gov: NCT01963481). Primary endpoint was progression-free survival (PFS) at 3 months (RECIST 1.1). Secondary objectives included median PFS, objective response rate, duration of response, and safety. Circulating Tregs (FOXP3+Helios+) and other immune cell subsets were monitored during treatment and compared with healthy controls. RESULTS: Twenty-three patients were enrolled. Treatment was well tolerated, without grade 4/5 toxicities. Objective responses were seen in 6/23 patients (26.1%; 95% CI 10.2-48.4%) and were durable (median 11.6 months). Three-month PFS rate was 50.1% (95% CI 33.0-76.0%); median PFS was 4.23 months (95% CI 2.8-11.7). No treatment-related decrease in Tregs was observed. However, elevated baseline levels of Naive Tregs [greater than 2.5 (the median of the naive Tregs)] were associated with relative risk of disease progression or death [hazard ratio 11.46 (95% CI 2.32-56.5)]. In addition, the baseline levels of Naive Tregs (adj-p = 0.04), Memory Tregs (adj-p = 0.003), CD4 + Central Memory T cells (adj-p = 0.0004), PD-1 + CD4 + Central Memory T cells (adj-p = 0.008), and PD-1 + CD4 + Effector Memory T cells (adj-p = 0.009) were significantly greater in the patients than in the healthy controls; the baseline levels of %CD4 + Naive T cells (adj-p = 0.0004) were significantly lower in patients compared with healthy controls (n = 40). CONCLUSION: Treg depletion was not observed with low-dose cyclophosphamide when assessed by the specific marker FOXP3 + Helios +; however, baseline naive Tregs were associated with 3-month PFS. Exemestane/cyclophosphamide combination had favorable safety profile with evidence of clinical activity in heavily pretreated patients.

Environmental nutrient levels impact cancer cell metabolism, resulting in context-dependent gene essentiality. Here, using loss-of-function screening based on RNA interference, we show that environmental oxygen levels are a major driver of differential essentiality between in vitro model systems and in vivo tumours. Above the 3-8% oxygen concentration typical of most tissues, we find that cancer cells depend on high levels of the iron-sulfur cluster biosynthetic enzyme NFS1. Mammary or subcutaneous tumours grow despite suppression of NFS1, whereas metastatic or primary lung tumours do not. Consistent with a role in surviving the high oxygen environment of incipient lung tumours, NFS1 lies in a region of genomic amplification present in lung adenocarcinoma and is most highly expressed in well-differentiated adenocarcinomas. NFS1 activity is particularly important for maintaining the iron-sulfur co-factors present in multiple cell-essential proteins upon exposure to oxygen compared to other forms of oxidative damage. Furthermore, insufficient iron-sulfur cluster maintenance robustly activates the iron-starvation response and, in combination with inhibition of glutathione biosynthesis, triggers ferroptosis, a non-apoptotic form of cell death. Suppression of NFS1 cooperates with inhibition of cysteine transport to trigger ferroptosis in vitro and slow tumour growth. Therefore, lung adenocarcinomas select for expression of a pathway that confers resistance to high oxygen tension and protects cells from undergoing ferroptosis in response to oxidative damage.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

PURPOSE: Diffusion-weighted imaging (DWI) and 18 F-fluorodeoxyglucose-positron emission tomography (18 F-FDG-PET) independently correlate with malignancy in breast cancer, but the relationship between their structural and metabolic metrics is not completely understood. This study spatially correlates diffusion, perfusion, and glucose avidity in breast cancer with simultaneous PET/MR imaging and compares correlations with clinical prognostics. METHODS: In this Health Insurance Portability and Accountability Act-compliant prospective study, with written informed consent and approval of the institutional review board and using simultaneously acquired FDG-PET and DWI, tissue diffusion (Dt ), and perfusion fraction (fp ) from intravoxel incoherent motion (IVIM) analysis were registered to FDG-PET within 14 locally advanced breast cancers. Lesions were analyzed using 2D histograms and correlation coefficients between Dt , fp , and standardized uptake value (SUV). Correlations were compared with prognostics from biopsy, metastatic burden from whole-body PET, and treatment history. RESULTS: SUV||Dt correlation coefficient significantly distinguished treated (0.11 +/- 0.24) from nontreated (-0.33 +/- 0.26) patients (P = 0.005). SUV||fp correlations were on average negative for the whole cohort (-0.17 +/- 0.13). CONCLUSION: Simultaneously acquired and registered FDG-PET/DWI allowed quantifiable descriptions of breast cancer microenvironments that may provide a framework for monitoring and predicting response to treatment. Magn Reson Med, 2016. (c) 2016 International Society for Magnetic Resonance in Medicine.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

BACKGROUND: Targeted therapies in HER2-positive metastatic breast cancer significantly improve outcomes but efficacy is limited by therapeutic resistance. HER2 is an acutely sensitive Heat Shock Protein 90 (HSP90) client and HSP90 inhibition can overcome trastuzumab resistance. Preclinical data suggest that HSP90 inhibition is synergistic with taxanes with the potential for significant clinical activity. We therefore tested ganetespib, a HSP90 inhibitor, in combination with paclitaxel and trastuzumab in patients with trastuzumab-refractory HER2-positive metastatic breast cancer. METHODS: In this phase I dose-escalation study, patients with trastuzumab-resistant HER2-positive metastatic breast cancer received weekly trastuzumab (2 mg/kg) and paclitaxel (80 mg/m2) on days 1, 8, 15, and 22 of a 28-day cycle with escalating doses of ganetespib (100 mg/m2, 150 mg/m2, and a third cohort of 125 mg/m2 if needed) on days 1, 8, and 15. Therapy was continued until disease progression or toxicity. The primary objective was to establish the safety and maximum tolerated dose and/or recommended phase II dose (RP2D) of this therapy. The secondary objectives included evaluation of the effects of ganetespib on the pharmacokinetics of paclitaxel, and to make a preliminary assessment of the efficacy of the combination therapy. RESULTS: Dose escalation was completed for the two main cohorts without any observed dose-limiting toxicities. Nine patients received treatment. The median prior lines of anti-HER2 therapy numbered three (range 2-4), including prior pertuzumab in 9/9 patients and ado-trastuzumab emtansine (T-DM1) in 8/9 patients. The most common grade 1/2 adverse events (AEs) were diarrhea, fatigue, anemia, and rash. There were no grade 4 AEs related to ganetespib. The overall response rate was 22% (2/9 patients had partial response) and stable disease was seen in 56% (5/9 patients). The clinical benefit rate was 44% (4/9 patients). The median progression-free survival was 20 weeks (range 8-55). CONCLUSION: The RP2D of ganetespib is 150 mg/m2 in combination with weekly paclitaxel plus trastuzumab. The combination was safe and well tolerated. Despite prior taxanes, pertuzumab, and T-DM1, clinical activity of this triplet regimen in this heavily pretreated cohort is promising and warrants further study in HER2-positive metastatic breast cancer. TRIAL REGISTRATION: ClinicalTrials.gov NCT02060253 . Registered 30 January 2014.

Frequent overexpression of programmed death-ligand 1 has recently been demonstrated in metaplastic breast cancer, which is a rare breast cancer subtype with limited treatment options. This report describes the clinical course of a patient with metastatic metaplastic breast cancer who had a remarkable response to anti-programmed death-1 therapy with pembrolizumab in combination with nab-paclitaxel. Tissue correlates are presented including tumor-infiltrating lymphocytes and high-programmed death-ligand 1 expression in the tumor.

Importance: The presence of tumor-infiltrating lymphocytes (TILs) is a favorable prognostic factor in breast cancer, and TILs may synergize with chemotherapy and immune checkpoint inhibitor therapy for improved clinical response. A more detailed understanding of the variation in lymphocytic infiltration in breast cancer may aid in identifying subtypes more amenable to immunomodulation. Objective: To determine the median percentage of patients with breast cancer with no, intermediate, or high levels of TIL and assess variations in lymphocytic cell subsets across breast cancer subtypes. Evidence Review: Eligible studies (PubMed, 1990-2015) analyzed tumor lymphocytic, CD8+, and FOXP3+ cellular infiltrates, and used multivariable analyses and quantitative methods for enumerating cell populations. Selection of of studies was performed in accordance with PRISMA guidelines and evaluated by 2 independent appraisers. Findings: Fifteen studies (n = 13914) met prespecified criteria and were reviewed in December 2015. A median of 11% (range, 5%-26%) of breast cancers demonstrate lymphocyte-predominant breast cancer (LPBC), with approximately 16% of cancers showing no evidence of TILs. Triple-negative (TN) breast cancers demonstrated the highest incidence of LPBC (20%; range, 4%-37%). This incidence is similar to that of breast cancers that are human epidermal growth factor 2 positive and either hormone receptor positive or negative (HER2+) at 16% (range 11%-24%). Hormone receptor positive/HER2- (HR+) breast cancer showed the lowest incidence of LPBC at 6% (range, 3%-12%). CD8+ T-cell infiltrates, indicative of type I immunity, were found in 48% of all breast cancers (range, 32%-80%) with similar levels observed in TN (60%; range, 40%-91%) and HER2+ disease (61%; range, 40%-83%). Fewer HR+ tumors demonstrated CD8+ TIL (43%; range, 30%-73%). The highest levels of FOXP3+ cells were observed in TN (70%; range, 65%-76%) and HER2+ disease (67%; range, 61%-74%). A minority of HR+ breast cancers demonstrated high levels of tumor-infiltrating FOXP3+ cells (38%; range, 35%-41%). Conclusions and Relevance: The magnitude of TIL is variable within and between breast cancer subtypes. Levels of lymphocytic subpopulations may identify breast cancers more amenable to immunomodulation and indicate additional strategies to enhance immunity in patients with low to moderate levels of TILs.

Breast cancer has the second highest death toll in women worldwide, despite significant progress in early diagnosis and treatments. The main cause of death is metastatic disease. Matrix metalloproteinases (MMPs) are required for the initial steps of metastasis, and have therefore been considered as ideal pharmacological targets for anti-metastatic therapy. However, clinical trials of MMP inhibitors were unsuccessful. These trials were conducted in patients with advanced disease, beyond the stage when these compounds could have been effective. We hypothesized that early treatment with a selective MMP inhibitor between the time of diagnosis and definitive surgery, the so-called "window-of-opportunity," can inhibit metastasis and thereby improve survival. To investigate our hypothesis we used the 4T1 mouse model of aggressive mammary carcinoma. We treated the animals with SD-7300, an oral inhibitor of MMP-2, -9 and -13, starting after the initial detection of the primary tumor. Seven days later the primary tumors were excised and analyzed for MMP activity, and the SD-7300 treatment was discontinued. After four weeks the animals were sacrificed and their lungs analyzed histologically for number of metastases and metastatic burden (metastases' area / lung section area). SD-7300 treatment inhibited 70-80% of tumor-associated MMP activity (P = 0.0003), reduced metastasis number and metastatic burden by 50-60% (P = 0.002; P = 0.0082, respectively), and increased survival (92% vs. 66.7%; P = 0.0409), relative to control vehicle. These results show that treatment of early invasive breast cancer with selective MMP inhibitors can lower the risk of recurrence and increase long-term disease-free survival.

The huge communities of residential microbes, including bacteria, viruses, Archaea, and Eukaryotes, that colonize humans are increasingly recognized as playing important roles in health and disease. A complex populous ecosystem, the human gastrointestinal (GI) tract harbors up to 1011 bacterial cells per gram of luminal content, whose collective genome, the gut metagenome, contains a vastly greater number of individual genes than the human genome. In health, the function of the microbiome might be considered to be in dynamic equilibrium with the host, exerting both local and distant effects. However, 'disequilibrium' may contribute to the emergence of disease, including malignancy. In this review, we discuss how the intestinal bacterial microbiome and in particular how an 'estrobolome,' the aggregate of enteric bacterial genes capable of metabolizing estrogens, might affect women's risk of developing postmenopausal estrogen receptor-positive breast cancer. Estrobolome composition is impacted by factors that modulate its functional activity. Exploring variations in the composition and activities of the estrobolome in healthy individuals and in women with estrogen-driven breast cancer may lead to development of microbiome-based biomarkers and future targeted interventions to attenuate cancer risk.