Connecting blood and intratumoral Treg cell activity in predicting future relapse in breast cancer.

Regulatory T (Treg) cells play a major role in the development of an immunosuppressive tumor microenvironment. The origin of intratumoral Treg cells and their relationship with peripheral blood Treg cells remain unclear. Treg cells consist of at least three functionally distinct subpopulations. Here we show that peripheral blood CD45RA-FOXP3hi Treg cells (Treg II cells) are phenotypically closest to intratumoral Treg cells, including in their expression of CCR8. Analyses of T cell antigen receptor repertoires further support the hypothesis that intratumoral Treg cells may originate primarily from peripheral blood Treg II cells. Moreover, the signaling responsiveness of peripheral blood Treg II cells to immunosuppressive, T helper type 1 (TH1) and T helper type 2 (TH2) cytokines reflects intratumoral immunosuppressive potential, and predicts future relapse in two independent cohorts of patients with breast cancer. Together, our findings give important insights into the relationship between peripheral blood Treg cells and intratumoral Treg cells, and highlight cytokine signaling responsiveness as a key determinant of intratumoral immunosuppressive potential and clinical outcome.

Clinical features associated with immune checkpoint inhibitor nephritis: a single-center clinical case series.

BACKGROUND: Acute kidney injury (AKI) has been well described as a complication of immune checkpoint inhibitor therapy. We present a series of patients, the majority with lung adenocarcinoma, who developed AKI while actively receiving immune checkpoint inhibitors. METHODS: This is a retrospectively analyzed clinical case series of six patients treated at City of Hope Comprehensive Cancer Center. Data were collected on gender, age, ethnicity, comorbidities, concomitant medications, type of malignancy, treatments, and renal function. All patients underwent renal biopsy for classification of the mechanism of AKI. Comprehensive genomic profiling (CGP) was performed on tumor tissue for all patients. RESULTS: Patterns of AKI included acute interstitial nephritis and acute tubular necrosis. Contributing factors included the use of concomitant medications known to contribute to AKI. All but two patients had full resolution of the AKI with the use of steroids. There were several mutations found on CGP that was notable including an Exon 20 insertion as well as multiple NF1 and TP53 mutations. There was high PD-L1 expression on tumor tissue noted in two out of six patients. In addition to AKI, a subset of patients had proteinuria with biopsies revealing corresponding glomerular lesions of minimal change disease and focal and segmental glomerulosclerosis. CONCLUSIONS: Our case series demonstrates that AKI from immune checkpoint inhibitors has a variable presentation that may require an individualized treatment approach. Further studies are needed to identify biomarkers that may help identify those at risk and guide the management of this condition.

Phase 2 prospective open label study of neoadjuvant nab-paclitaxel, trastuzumab, and pertuzumab in patients with HER2-positive primary breast cancer.

BACKGROUND: The objective of this study was to evaluate the safety and efficacy of nab-paclitaxel, trastuzumab, and pertuzumab as neoadjuvant therapy (NAT) in patients with human epidermal growth factor receptor 2 HER2+ breast cancer (HER2+ BC) to determine pathologic complete response (pCR), invasive disease-free survival (iDFS), and overall survival. METHODS: Forty-five patients with HER2+ BC Stages II-III were to be enrolled from 2013 to 2017. Patients were treated with weekly nab-paclitaxel (100 mg/m2 intravenously), weekly trastuzumab (4 mg/kg loading dose, then 2 mg/kg), and six cycles of pertuzumab (840 mg loading dose, then 420 mg intravenously day 1 every 21 days). RESULTS: Median follow-up was 60 months (95% CI, 32.3-55.6) and pCR was 29/45 (64%). The 5-year iDFS for patients who achieved pCR (N = 29) was 96.3% (95% CI, 76.5-99.5) and non-pCR patients (N = 16) was 74.3% (95% CI, 39.1-91.0). The 5-year overall survival (N = 45) was 94.1% (95% CI, 77.6-98.5). Based on hormonal status, the 5-year iDFS for HR+ pCR patients (N = 14) was 92.3% (95% CI, 56.6-98.9) and for HR- (N = 15) was 100% (p = .3). CONCLUSIONS: This anthracycline/carboplatin-free regimen with nab-paclitaxel achieved a pCR rate of 64% in patients with HER2+ BC. The 5-year iDFS in patients with and without pCR was 96.3% and 74.3%, respectively. The pCR rate is comparable with docetaxel, carboplatin, trastuzumab, and pertuzumab therapy in the NAT setting, but with fewer treatment-associated toxicities. This finding suggests the possibility of safe avoidance of anthracyclines and carboplatin as components of NAT in patients with HER2+ BC.

Phase I Trial of Ipatasertib Plus Carboplatin, Carboplatin/Paclitaxel, or Capecitabine and Atezolizumab in Metastatic Triple-Negative Breast Cancer.

BACKGROUND: This trial evaluated the safety and efficacy of ipatasertib in combination with carboplatin, carboplatin/paclitaxel, or capecitabine/atezolizumab in patients with metastatic triple-negative breast cancer (mTNBC). METHODS: Eligibility criteria were mTNBC, RECIST 1.1 measurable disease, no prior use of platinum for metastatic disease (Arms A and B), and no prior exposure to immune checkpoint inhibitor (Arm C). Primary endpoints were safety and RP2D. Secondary endpoints were progression-free survival (PFS), response rate, and overall survival. RESULTS: RP2D for Arm A (n = 10) was ipatasertib 300 mg daily, carboplatin AUC2, and paclitaxel 80 mg m-2 days 1, 8, and 15 every 28 days. RP2D for Arm B (n = 12) was ipatasertib 400 mg daily and carboplatin AUC2 days 1, 8, and 15 every 28 days. RP2D for Arm C (n = 6) was likely ipatasertib 300 mg 21 days on 7 days off, capecitabine 750 mg m-2, twice a day, 7 days on 7 days off, and atezolizumab 840 mg days 1 and 15 every 28 days. The most common (≥10%) grade 3-4 AEs at RP2D for Arm A (N = 7 at RP2D) were neutropenia (29%), diarrhea (14%), oral mucositis (14%), and neuropathy (14%); Arm B had diarrhea (17%) and lymphopenia (25%); and Arm C had anemia, fatigue, cognitive disturbance, and maculopapular rash (17% each). Overall responses at RP2D were 29% Arm A, 25% Arm B, and 33% Arm C. PFS was 4.8, 3.9, and 8.2 months for patients on Arms A, B, and C, respectively. CONCLUSIONS: Continuous dosing of ipatasertib with chemotherapy was safe and well-tolerated. Further study is warranted in understanding the role of AKT inhibition in treatment of TNBCs. TRIAL REGISTRATION: NCT03853707.

Immunogenicity and efficacy of pembrolizumab and doxorubicin in a phase I trial for patients with metastatic triple-negative breast cancer.

Currently there is a limited understanding for the optimal combination of immune checkpoint inhibitor and chemotherapy for patients with metastatic triple-negative breast cancer (mTNBC). Here we evaluate the safety, efficacy, and immunogenicity of a phase I trial for patients with mTNBC treated with pembrolizumab plus doxorubicin. Patients without prior anthracycline use and 0-2 lines of prior systemic chemotherapies received pembrolizumab and doxorubicin every 3 weeks for 6 cycles followed by pembrolizumab maintenance until disease progression or intolerance. The primary objectives were safety and objective response rate per RECIST 1.1. Best responses included one complete response (CR), five partial responses (PR), two stable disease (SD), and one progression of disease (PD). Overall response rate was 67% (95% CI 13.7%, 78.8%) and clinical benefit rate at 6 months was 56% (95% CI 21.2%, 86.3%). Median PFS was 5.2 months (95% CI 4.7, NA); median OS was 15.6 months (95% CI 13.3, NA). Grade 3-4 AEs per CTCAE 4.0 were neutropenia n = 4/10 (40%), leukopenia n = 2/10 (20%), lymphopenia n = 2/10 (20%), fatigue n = 2/10 (20%), and oral mucositis n = 1/10 (10%). Immune correlates showed increased frequencies of circulating CD3 + T cells (p = 0.03) from pre-treatment to cycle 2 day 1 (C2D1). An expansion of a proliferative exhausted-like PD-1 + CD8 + T cell population was identified in 8/9 patients, and exhausted CD8 + T cells were significantly expanded from pre-treatment to C2D1 in the patient with CR (p = 0.01). In summary, anthracycline-naïve patients with mTNBC treated with the combination of pembrolizumab and doxorubicin showed an encouraging response rate and robust T cell response dynamics.Trial registration: NCT02648477.

Stressed out and fed up: The effect of stress on maternal feeding behaviors and the moderating role of executive function.

Stress is associated with a range of unhealthy eating habits, yet few studies have examined how stress may influence the intergenerational transmission of eating habits from parents to their children. Specifically, there is a lack of data regarding the role of stress on feeding practices. Moreover, most work investigating the associations between parental stress and their feeding behaviors has been correlational, limiting our understanding of causality. In the current study, we used an experimental design, induced high and low stress in mothers using a standard laboratory stressor, and observed mother-child interactions during a snack break. We also examined the potential role of maternal executive functioning (EF) for buffering the effects of stress on maternal feeding behaviors. Levels of maternal stress were manipulated with the Trier Social Stress Task (TSST) in a community sample (N = 80 dyads, Child Mage = 41.89 months, female = 43). We measured maternal EF with a series of computerized tasks. Maternal feeding behaviors were coded for controlling behaviors, which included pressuring and restricting behaviors. Results indicate a main effect of stress on controlling feeding behaviors, such that mothers in the high-stress condition exhibited higher levels of controlling behaviors. The effect of stress on controlling feeding behaviors was ameliorated among mothers with higher levels of EF after controlling for child age and income. Results provide causal evidence for the role of stress on feeding behaviors and suggest EF as a factor to be considered in the treatment and prevention of diet-related illnesses.

Physics approaches to the spatial distribution of immune cells in tumors.

The goal of immunotherapy is to mobilize the immune system to kill cancer cells. Immunotherapy is more effective and, in general, the prognosis is better, when more immune cells infiltrate the tumor. We explore the question of whether the spatial distribution rather than just the density of immune cells in the tumor is important in forecasting whether cancer recurs. After reviewing previous work on this issue, we introduce a novel application of maximum entropy to quantify the spatial distribution of discrete point-like objects. We apply our approach to B and T cells in images of tumor tissue taken from triple negative breast cancer patients. We find that the immune cells are more spatially dispersed in good clinical outcome (no recurrence of cancer within at least 5 years of diagnosis) compared to poor clinical outcome (recurrence within 3 years of diagnosis). Our results highlight the importance of spatial distribution of immune cells within tumors with regard to clinical outcome, and raise new questions on their role in cancer recurrence.

Phase II Trial of Neoadjuvant Carboplatin and Nab-Paclitaxel in Patients with Triple-Negative Breast Cancer.

BACKGROUND: In this phase II clinical trial, we evaluated the efficacy of the nonanthracycline combination of carboplatin and nab-paclitaxel in early stage triple-negative breast cancer (TNBC). PATIENTS AND METHODS: Patients with newly diagnosed stage II-III TNBC (n = 69) were treated with neoadjuvant carboplatin (area under the curve 6) every 28 days for four cycles plus nab-paclitaxel (100 mg/m2 ) weekly for 16 weeks. Pathological complete response (pCR) and residual cancer burden (RCB) were analyzed with germline mutation status, tumor-infiltrating lymphocytes (TILs), TNBC molecular subtype, and GeparSixto immune signature (GSIS). RESULTS: Sixty-seven patients were evaluable for safety and response. Fifty-three (79%) patients experienced grade 3/4 adverse events, including grade 3 anemia (43%), neutropenia (39%), leukopenia (15%), thrombocytopenia (12%), fatigue (7%), peripheral neuropathy (7%), neutropenia (16%), and leukopenia (1%). Twenty-four patients (35%) had at least one dose delay, and 50 patients (72%) required dose reduction. Sixty-three (94%) patients completed scheduled treatment. The responses were as follows: 32 of 67 patients (48%) had pCR (RCB 0), 10 of 67 (15%) had RCB I, 19 of 67 (28%) had RCB II, 5 of 67 (7%) had RCB III, and 1 of 67 (2%) progressed and had no surgery. Univariate analysis showed that immune-hot GSIS and DNA repair defect (DRD) were associated with higher pCR with odds ratios of 4.62 (p = .005) and 4.76 (p = .03), respectively, and with RCB 0/I versus RCB II/III with odds ratio 4.80 (p = .01). Immune-hot GSIS was highly correlated with DRD status (p = .03), TIL level (p < .001), and TNBC molecular subtype (p < .001). After adjusting for age, race, stage, and grade, GSIS remained associated with higher pCR and RCB class 0/I versus II/III with odds ratios 7.19 (95% confidence interval [CI], 2.01-25.68; p = .002) and 8.95 (95% CI, 2.09-38.23; p = .003), respectively. CONCLUSION: The combination of carboplatin and nab-paclitaxel for early stage high-risk TNBC showed manageable toxicity and encouraging antitumor activity. Immune-hot GSIS is associated with higher pCR rate and RCB class 0/1. This study provides an additional rationale for using nonanthracycline platinum-based therapy for future neoadjuvant trials in early stage TNBCs. Clinical trial identification number: NCT01525966 IMPLICATIONS FOR PRACTICE: Platinum is an important neoadjuvant chemotherapy agent for treatment of early stage triple-negative breast cancer (TNBC). In this study, carboplatin and nab-paclitaxel were well tolerated and highly effective in TNBC, resulting in pathological complete response of 48%. In univariate and multivariate analyses adjusting for age, race, tumor stage and grade, "immune-hot" GeparSixto immune signature (GSIS) and DNA repair defect (DRD) were associated with higher pathological complete response (pCR) and residual cancer burden class 0/1. The association of immune-hot GSIS with higher pCR holds promise for de-escalating neoadjuvant chemotherapy for patients with early stage TNBC. Although GSIS is not routinely used in clinic, further development of this immune signature into a clinically applicable assay is indicated.

A Phase II Clinical Trial of Pembrolizumab and Enobosarm in Patients with Androgen Receptor-Positive Metastatic Triple-Negative Breast Cancer.

LESSONS LEARNED: The combination of enobosarm and pembrolizumab was well tolerated and showed a modest clinical benefit rate of 25% at 16 weeks. Future trials investigating androgen receptor-targeted therapy in combination with immune checkpoint inhibitors are warranted. BACKGROUND: Luminal androgen receptor is a distinct molecular subtype of triple-negative breast cancer (TNBC) defined by overexpression of androgen receptor (AR). AR-targeted therapy has shown modest activity in AR-positive (AR+) TNBC. Enobosarm (GTx-024) is a nonsteroidal selective androgen receptor modulator (SARM) that demonstrates preclinical and clinical activity in AR+ breast cancer. The current study was designed to explore the safety and efficacy of the combination of enobosarm and pembrolizumab in patients with AR+ metastatic TNBC (mTNBC). METHODS: This study was an open-label phase II study for AR+ (≥10%, 1+ by immunohistochemistry [IHC]) mTNBC. Eligible patients received pembrolizumab 200 mg intravenous (IV) every 3 weeks and enobosarm 18 mg oral daily. The primary objective was to evaluate the safety of enobosarm plus pembrolizumab and determine the response rate. Peripheral blood, tumor biopsies, and stool samples were collected for correlative analysis. RESULTS: The trial was stopped early because of the withdrawal of GTx-024 drug supply. Eighteen patients were enrolled, and 16 were evaluable for responses. Median age was 64 (range 36-81) years. The combination was well tolerated, with only a few grade 3 adverse events: one dry skin, one diarrhea, and one musculoskeletal ache. The responses were 1 of 16 (6%) complete response (CR), 1 of 16 (6%) partial response (PR), 2 of 16 (13%) stable disease (SD), and 12 of 16 (75%) progressive disease (PD). Response rate (RR) was 2 of 16 (13%). Clinical benefit rate (CBR) at 16 weeks was 4 of 16 (25%). Median follow-up was 24.9 months (95% confidence interval [CI], 17.5-30.9). Progression-free survival (PFS) was 2.6 months (95% CI, 1.9-3.1) and overall survival (OS) was 25.5 months (95% CI, 10.4-not reached [NR]). CONCLUSION: The combination of enobosarm and pembrolizumab was well tolerated, with a modest clinical benefit rate of 25% at 16 weeks in heavily pretreated AR+ TNBC without preselected programmed death ligand-1 (PD-L1). Future clinical trials combining AR-targeted therapy with immune checkpoint inhibitor (ICI) for AR+ TNBC warrant investigation.

Phase I clinical trial of the combination of eribulin and everolimus in patients with metastatic triple-negative breast cancer.

BACKGROUND: Alteration of the PI3K/AKT/mTOR pathway is a common genomic abnormality detected in triple-negative breast cancer (TNBC). Everolimus acts synergistically with eribulin in TNBC cell lines and xenograft models. This phase I trial was designed to test the safety and tolerability of combining eribulin and everolimus in patients with metastatic TNBC. METHODS: The primary objective of this study was to evaluate the safety and toxicities of the combination. Patients with metastatic TNBC who had up to four lines of prior chemotherapies were enrolled. The combination of eribulin and everolimus was tested using three dosing levels: A1 (everolimus 5 mg daily; eribulin 1.4 mg/m2 days 1 and 8 every 3 weeks), A2 (everolimus 7.5 mg daily; eribulin 1.4 mg/m2, days 1 and 8 every 3 weeks), and B1 (everolimus 5 mg daily; eribulin 1.1 mg/m2 days 1 and 8 every 3 weeks). RESULTS: Twenty-seven patients with median age 55 years were enrolled. Among 8 evaluable patients who received dose level A1, 4 had dose-limiting toxicities (DLTs). Among 3 evaluable patients treated with dose level A2, 2 had DLTs. Among 12 evaluable patients who received dose level B1, 4 had DLTs. The DLTs were neutropenia, stomatitis, and hyperglycemia. Over the study period, 59% had a ≥ grade 3 toxicity, 44% had ≥ grade 3 hematologic toxicities, and 22% had grade 4 hematologic toxicities. The most common hematological toxicities were neutropenia, leukopenia, and lymphopenia. Thirty-three percent had grade 3 non-hematologic toxicities. The most common non-hematological toxicities were stomatitis, hyperglycemia, and fatigue. The median number of cycles completed was 4 (range 0-8). Among 25 eligible patients, 9 patients (36%) achieved the best response as partial response, 9 (36%) had stable disease, and 7 (28%) had progression. The median time to progression was 2.6 months (95% CI [2.1, 4.0]), and median overall survival (OS) was 8.3 months (95% CI [5.5, undefined]). CONCLUSION: Eribulin 1.1 mg/m2 days 1 and 8 every 3 weeks with everolimus 5 mg daily was defined as the highest dose with acceptable toxicity (RP2D). The combination is safe, and efficacy is modest. A post hoc analysis showed that participants that used dexamethasone mouthwash stayed on treatment for one additional cycle. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02120469. Registered 18 April 2014.

Pathway activity profiling of growth factor receptor network and stemness pathways differentiates metaplastic breast cancer histological subtypes.

BACKGROUND: Gene expression profiling of rare cancers has proven challenging due to limited access to patient materials and requirement of intact, non-degraded RNA for next-generation sequencing. We customized a gene expression panel compatible with degraded RNA from formalin-fixed, paraffin-embedded (FFPE) patient cancer samples and investigated its utility in pathway activity profiling in patients with metaplastic breast cancer (MpBC). METHODS: Activity of various biological pathways was profiled in samples from nineteen patients with MpBC and 8 patients with invasive ductal carcinoma with triple negative breast cancer (TNBC) phenotype using a custom gene expression-based assay of 345 genes. RESULTS: MpBC samples of mesenchymal (chondroid and/or osteoid) histology demonstrated increased SNAI1 and BCL2L11 pathway activity compared to samples with non-mesenchymal histology. Additionally, late cornified envelope and keratinization genes were downregulated in MpBC compared to TNBC, and epithelial-to-mesenchymal transition (EMT) and collagen genes were upregulated in MpBC. Patients with high activity of an invasiveness gene expression signature, as well as high expression of the mesenchymal marker and extracellular matrix glycoprotein gene SPARC, experienced worse outcomes than those with low invasiveness activity and low SPARC expression. CONCLUSIONS: This study demonstrates the utility of gene expression profiling of metaplastic breast cancer FFPE samples with a custom counts-based assay. Gene expression patterns identified by this assay suggest that, although often histologically triple negative, patients with MpBC have distinct pathway activation compared to patients with invasive ductal TNBC. Incorporation of targeted therapies may lead to improved outcome for MpBC patients, especially in those patients expressing increased activity of invasiveness pathways.

EMDomics: a robust and powerful method for the identification of genes differentially expressed between heterogeneous classes.

MOTIVATION: A major goal of biomedical research is to identify molecular features associated with a biological or clinical class of interest. Differential expression analysis has long been used for this purpose; however, conventional methods perform poorly when applied to data with high within class heterogeneity. RESULTS: To address this challenge, we developed EMDomics, a new method that uses the Earth mover's distance to measure the overall difference between the distributions of a gene's expression in two classes of samples and uses permutations to obtain q-values for each gene. We applied EMDomics to the challenging problem of identifying genes associated with drug resistance in ovarian cancer. We also used simulated data to evaluate the performance of EMDomics, in terms of sensitivity and specificity for identifying differentially expressed gene in classes with high within class heterogeneity. In both the simulated and real biological data, EMDomics outperformed competing approaches for the identification of differentially expressed genes, and EMDomics was significantly more powerful than conventional methods for the identification of drug resistance-associated gene sets. EMDomics represents a new approach for the identification of genes differentially expressed between heterogeneous classes and has utility in a wide range of complex biomedical conditions in which sample classes show within class heterogeneity. AVAILABILITY AND IMPLEMENTATION: The R package is available at http://www.bioconductor.org/packages/release/bioc/html/EMDomics.html.

Assessment of breast pathologies using nonlinear microscopy.

Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.

Association Between 18F-FDG PET Activity and HER2 Status in Breast Cancer Brain Metastases.

Purpose: The objective of this study was to evaluate whether uptake on 18F-fluorodeoxyglucose (18F-FDG) PET could help differentiate HER2-positive from HER2-negative breast cancer brain metastases. Methods: In this retrospective, cross-sectional study of a cohort of 14 histologically proven breast cancer brain metastases, we analyzed both preoperative 18F-FDG PET/CT and HER2 status of the resected/biopsied brain specimens. The maximum standardized uptake values (SUVmax) of the lesions were normalized to contralateral normal white matter and compared using Mann-Whitney U tests. Results: The study cohort was comprised of 12 women with breast cancer with a mean age of 59 years (range: 43-76 years) with a total of 14 distinct brain metastatic lesions. The SUVmax ratio of HER2-positive breast cancer brain metastases was significantly greater than that of HER2-negative lesions (3.98 vs 1.79, U = 38.00, p = 0.008). Conclusion: The SUVmax ratio may help to identify the HER2 status of breast cancer brain metastases, if validated prospectively.

Tumor heterogeneity and clinically invisible micrometastases in metastatic breast cancer-a call for enhanced surveillance strategies.

The biology of metastatic breast cancer (MBC) is understudied, primarily due to the difficulty of procuring multiple samples from patients with oligometastatic breast cancer. We developed a rapid postmortem tissue procurement program that allows the collection and analysis of numerous metastatic lesions, subclinical locations, and potential pre-metastatic niches that fall within this scope. We conducted a rapid postmortem tissue collection study on 9 patients with MBC. Patients and their families consented to donate tissues immediately after death in an IRB-approved study. Various disease subtypes, progression histories, organ involvement, and final causes of death are reported. In patients with hormone receptor-positive (HR+) disease, estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki-67 expression were heterogeneous across metastatic lesions within individual patients. Disease phenotype at the end of life trended toward complete loss of HR expression. Nearly all (n = 7) patients exhibited extensive tumor involvement of additional organs that had not been previously diagnosed clinically and were not retrospectively visible on recent imaging. Of these seven individuals, three included organs uncommonly associated with MBC: kidney, spleen, pancreas, and ovary. Finally, we identified clinically undetectable micrometastases in several organs uncommonly involved in MBC. Our findings raise several clinically relevant questions regarding the mechanisms of metastatic progression. Insights from this study argue for better surveillance strategies for monitoring MBC. We highlight the need to capture more accurate biomarker information in the context of heterogeneous disease and urge the consideration of treatment strategies that combine multiple targeted therapies.

Tumor draining lymph nodes connected to cold triple-negative breast cancers are characterized by Th2-associated microenvironment.

Tumor draining lymph nodes (TDLN) represent a key component of the tumor-immunity cycle. There are few studies describing how TDLNs impact lymphocyte infiltration into tumors. Here we directly compare tumor-free TDLNs draining "cold" and "hot" human triple negative breast cancers (TDLNCold and TDLNHot). Using machine-learning-based self-correlation analysis of immune gene expression, we find unbalanced intranodal regulations within TDLNCold. Two gene pairs (TBX21/GATA3-CXCR1) with opposite correlations suggest preferential priming of T helper 2 (Th2) cells by mature dendritic cells (DC) within TDLNCold. This is validated by multiplex immunofluorescent staining, identifying more mature-DC-Th2 spatial clusters within TDLNCold versus TDLNHot. Associated with this Th2 priming preference, more IL4 producing mast cells (MC) are found within sinus regions of TDLNCold. Downstream, Th2-associated fibrotic TME is found in paired cold tumors with increased Th2/T-helper-1-cell (Th1) ratio, upregulated fibrosis growth factors, and stromal enrichment of cancer associated fibroblasts. These findings are further confirmed in a validation cohort and public genomic data. Our results reveal a potential role of IL4+ MCs within TDLNs, associated with Th2 polarization and reduced immune infiltration into tumors.

Organ-specific immune setpoints underlie divergent immune profiles across metastatic sites in breast cancer.

Immune composition within the tumor microenvironment (TME) plays a central role in the propensity of cancer to metastasize and to respond to therapy. Previous studies suggested that the metastatic TME is immune suppressed. However, limited accessibility to multiple metastatic sites within patients has made assessment of the immune TME in the context of multi-organ metastases difficult. We utilized a rapid postmortem tissue collection protocol to assess immune composition in numerous sites of breast cancer metastasis and paired tumor-free tissues. Metastases were found to have comparable immune cell densities and composition to paired tumor-free tissues of the same organ type. In contrast, immune cell densities in both metastatic and tumor-free tissues were significantly different between organ types, with lung immune infiltration consistently greater than liver. These immune profiling results were consistent between both flow cytometry and multiplex immunofluorescence-based spatial analysis. Furthermore, we found granulocytes were a predominant tumor-infiltrating immune cell in both lung and liver metastases and these granulocytes made up the majority of PD-L1-expressing cells in many tissue sites. We also identified distinct potential mechanisms of immunosuppression in lung and liver metastases, with lung having increased expression of PD-L1+ antigen-presenting cells and liver having higher numbers of activated regulatory T cells and HLA-DRlow monocytes. Together these results demonstrate that immune contexture of metastases is dictated by organ type, and that immunotherapy strategies may benefit from unique tailoring to tissue-specific features of the immune TME.

Association of lesion contour and lesion composition on MR with HER2 status in breast cancer brain metastases.

BACKGROUND AND PURPOSE: With the development of HER2-directed therapies, identifying non-invasive imaging biomarkers of HER2 status in breast cancer brain metastases has become increasingly important, particularly given the risks of tissue sampling within the brain and the possibility of a change in receptor expression from the primary tumor to the brain metastasis. The purpose of this study was to evaluate whether lesion contour and composition on MR could help identify the HER2 status of breast cancer brain metastases. MATERIALS AND METHODS: We derived a cohort of 34 women with a mean age of 55 years (range: 31-81 years) with a total of 47 distinct histologically proven breast cancer brain metastases with preoperative contrast-enhanced brain MR and HER2 immunohistochemistry and/or fluorescent in-situ hybridization (FISH) of the resected/biopsied brain specimens from 2018 to 2021. Two fellowship-trained neuroradiologists evaluated the lesion contour and lesion composition of each lesion. Logistic regression analyses were performed. RESULTS: In a logistic regression model, an irregular contour had an odds ratio of 170 (p = 0.007) in differentiating HER2-positive from HER2-negative lesions. In a logistic regression model, when compared to a predominantly cystic lesion composition, a solid lesion composition had an odds ratio of 17 (p = 0.016) in differentiating HER2-positive from HER2-negative lesions. CONCLUSION: Lesion contour and lesion composition on MR were significantly associated with the HER2 status of breast cancer brain metastases. Current assessment of HER2 status requires tissue sampling and immunochemical and/or FISH analyses. A non-invasive imaging biomarker that may help predict HER2 status may be of great clinical value.

Association of Relative Cerebral Blood Volume from Dynamic Susceptibility Contrast-Enhanced Perfusion MR with HER2 Status in Breast Cancer Brain Metastases.

RATIONALE AND OBJECTIVES: With the development of HER2-directed therapies, identifying non-invasive imaging biomarkers of HER2 expression in breast cancer brain metastases has become increasingly important. The purpose of this study was to investigate whether relative cerebral blood volume (rCBV) from dynamic susceptibility contrast-enhanced (DSC) perfusion MR could help identify the HER2 status of breast cancer brain metastases. MATERIALS AND METHODS: With IRB approval for this HIPAA-compliant cross-sectional study and a waiver of informed consent, we queried our institution's electronic medical record to derive a cohort of 14 histologically proven breast cancer brain metastases with preoperative DSC perfusion MR and HER2 analyses of the resected/biopsied brain specimens from 2011-2021. The rCBV of the lesions was measured and compared using Mann-Whitney tests. Receiver operating characteristic analyses were performed to evaluate the performance of rCBV in identifying HER2 status. RESULTS: The study cohort was comprised of 14 women with a mean age of 56 years (range: 32-81 years) with a total of 14 distinct lesions. The rCBV of HER2-positive breast cancer brain metastases was significantly greater than the rCBV of HER2-negative lesions (8.02 vs 3.97, U=48.00, p=0.001). rCBV differentiated HER2-positive lesions from HER2-negative lesions with an area under the curve of 0.98 (standard error=0.032, p<0.001). The accuracy-maximizing rCBV threshold (4.8) was associated with an accuracy of 93% (13/14), a sensitivity of 100% (7/7), and a specificity of 86% (6/7). CONCLUSION: rCBV may assist in identifying the HER2 status of breast cancer brain metastases, if validated in a large prospective trial.