A spatially resolved single-cell genomic atlas of the adult human breast.

The adult human breast is comprised of an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue1-3. Although most previous studies have focused on the breast epithelial system4-6, many of the non-epithelial cell types remain understudied. Here we constructed the comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics study profiled 714,331 cells from 126 women, and 117,346 nuclei from 20 women, identifying 12 major cell types and 58 biological cell states. These data reveal abundant perivascular, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Spatial mapping using four different technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide a reference of the adult normal breast tissue for studying mammary biology and diseases such as breast cancer.

Clinical Translation and Evaluation of a Handheld and Biocompatible Mass Spectrometry Probe for Surgical Use.

BACKGROUND: Intraoperative tissue analysis and identification are critical to guide surgical procedures and improve patient outcomes. Here, we describe the clinical translation and evaluation of the MasSpec Pen technology for molecular analysis of in vivo and freshly excised tissues in the operating room (OR). METHODS: An Orbitrap mass spectrometer equipped with a MasSpec Pen interface was installed in an OR. A "dual-path" MasSpec Pen interface was designed and programmed for the clinical studies with 2 parallel systems that facilitated the operation of the MasSpec Pen. The MasSpec Pen devices were autoclaved before each surgical procedure and were used by surgeons and surgical staff during 100 surgeries over a 12-month period. RESULTS: Detection of mass spectral profiles from 715 in vivo and ex vivo analyses performed on thyroid, parathyroid, lymph node, breast, pancreatic, and bile duct tissues during parathyroidectomies, thyroidectomies, breast, and pancreatic neoplasia surgeries was achieved. The MasSpec Pen enabled gentle extraction and sensitive detection of various molecular species including small metabolites and lipids using a droplet of sterile water without causing apparent tissue damage. Notably, effective molecular analysis was achieved while no limitations to sequential histologic tissue analysis were identified and no device-related complications were reported for any of the patients. CONCLUSIONS: This study shows that the MasSpec Pen system can be successfully incorporated into the OR, allowing direct detection of rich molecular profiles from tissues with a seconds-long turnaround time that could be used to inform surgical and clinical decisions without disrupting tissue analysis workflows.

The oral selective oestrogen receptor degrader (SERD) AZD9496 is comparable to fulvestrant in antagonising ER and circumventing endocrine resistance.

BACKGROUND: The oestrogen receptor (ER) is an important therapeutic target in ER-positive (ER+) breast cancer. The selective ER degrader (SERD), fulvestrant, is effective in patients with metastatic breast cancer, but its intramuscular route of administration and low bioavailability are major clinical limitations. METHODS: Here, we studied the pharmacology of a new oral SERD, AZD9496, in a panel of in vitro and in vivo endocrine-sensitive and -resistant breast cancer models. RESULTS: In endocrine-sensitive models, AZD9496 inhibited cell growth and blocked ER activity in the presence or absence of oestrogen. In vivo, in the presence of oestrogen, short-term AZD9496 treatment, like fulvestrant, resulted in tumour growth inhibition and reduced expression of ER-dependent genes. AZD9496 inhibited cell growth in oestrogen deprivation-resistant and tamoxifen-resistant cell lines and xenograft models that retain ER expression. AZD9496 effectively reduced ER levels and ER-induced transcription. Expression analysis of short-term treated tumours showed that AZD9496 potently inhibited classic oestrogen-induced gene transcription, while simultaneously increasing expression of genes negatively regulated by ER, including genes potentially involved in escape pathways of endocrine resistance. CONCLUSIONS: These data suggest that AZD9496 is a potent anti-oestrogen that antagonises and degrades ER with anti-tumour activity in both endocrine-sensitive and endocrine-resistant models.

Circulating tumor cell investigation in breast cancer patient-derived xenograft models by automated immunofluorescence staining, image acquisition, and single cell retrieval and analysis.

BACKGROUND: Breast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®-CyteFinder® system. METHODS: CTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation. RESULTS: The recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method. CONCLUSIONS: Analysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte-CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression.

Multicenter Study Using Desorption-Electrospray-Ionization-Mass-Spectrometry Imaging for Breast-Cancer Diagnosis.

The histological and molecular subtypes of breast cancer demand distinct therapeutic approaches. Invasive ductal carcinoma (IDC) is subtyped according to estrogen-receptor (ER), progesterone-receptor (PR), and HER2 status, among other markers. Desorption-electrospray-ionization-mass-spectrometry imaging (DESI-MSI) is an ambient-ionization MS technique that has been previously used to diagnose IDC. Aiming to investigate the robustness of ambient-ionization MS for IDC diagnosis and subtyping over diverse patient populations and interlaboratory use, we report a multicenter study using DESI-MSI to analyze samples from 103 patients independently analyzed in the United States and Brazil. The lipid profiles of IDC and normal breast tissues were consistent across different patient races and were unrelated to country of sample collection. Similar experimental parameters used in both laboratories yielded consistent mass-spectral data in mass-to-charge ratios ( m/ z) above 700, where complex lipids are observed. Statistical classifiers built using data acquired in the United States yielded 97.6% sensitivity, 96.7% specificity, and 97.6% accuracy for cancer diagnosis. Equivalent performance was observed for the intralaboratory validation set (99.2% accuracy) and, most remarkably, for the interlaboratory validation set independently acquired in Brazil (95.3% accuracy). Separate classification models built for ER and PR statuses as well as the status of their combined hormone receptor (HR) provided predictive accuracies (>89.0%), although low classification accuracies were achieved for HER2 status. Altogether, our multicenter study demonstrates that DESI-MSI is a robust and reproducible technology for rapid breast-cancer-tissue diagnosis and therefore is of value for clinical use.

Granulomatous mastitis: etiology, imaging, pathology, treatment, and clinical findings.

PURPOSE: To outline the demographics, clinical presentation, imaging features, and treatment modalities observed among a series of patients diagnosed with biopsy-proven granulomatous mastitis (GM). METHOD: Following approval by institutional review board, retrospective chart review was performed on patients with biopsy-proven granulomatous mastitis at our institution in the period from January 2013 until October 2017. RESULTS: A total of 90 patients were identified: 87 women and 3 men. The mean age was 35 years, mostly women in their reproductive age. In our study, patients with GM were more likely to be Hispanic compared to the general population. Sixty-three percent of patients were within 5 years of previous pregnancy. Painful palpable mass-like lesion was the most common physical finding. Breast ultrasound (US) was performed in all patients, and most commonly showed a hypoechoic irregular-shaped mass. Mammography (MG) showed asymmetry or irregular mass as the main finding. Definitive diagnosis was obtained by imaging-guided core needle biopsies in 94.4%. Conservative management was preferred, and only one patient underwent surgery. CONCLUSION: Although clinical and radiological findings of patients with GM may mimic those of breast carcinoma, our study showed that women of childbearing age, especially among Hispanic ethnicity with a recent history of pregnancy or high prolactin level and newly tender mass-like lesion, in addition to new focal asymmetry on mammogram and heterogeneous hypoechoic irregular-shaped mass on ultrasound exam, should raise concern for GM. Non-invasive approach and clinical follow-up were the preferred treatment method.

Breast pathology review: does it make a difference?

BACKGROUND: Breast pathology is a challenging field, and previous work has shown discrepancies in diagnoses, even among experts. We set out to determine whether mandatory pathology review changes the diagnosis or surgical management of breast disease. METHODS: Cases were referred for pathology review after patients presented for surgical opinion to the Dubin Breast Center at Mount Sinai Medical Center over the course of 2 years. Surgical pathologists with expertise in breast disease reviewed slides submitted from the primary institution and rendered a second opinion diagnosis. Comparison of these reports was performed for evaluation of major changes in diagnosis and definitive surgical management. RESULTS: A total of 306 patients with 430 biopsy specimens were reviewed. Change in diagnosis was documented in 72 (17 %) of 430 cases and change in surgical management in 41 (10 %). A change in diagnosis was more likely to occur in patients originally diagnosed with benign rather than malignant disease (31 vs. 7 %, p < 0.001). Twelve (7 %) of 169 specimens initially diagnosed as benign were reclassified as malignant. A malignant diagnosis was changed to benign in 4 (2 %) of 261 cases. Change in diagnosis was less common in specimens originating from commercial laboratories than community hospitals or university hospitals (8, 19, 21 %, p = 0.023). Change in management was not dependent on initial institution. Type of biopsy specimen (surgical or core) did not influence diagnostic or management changes. CONCLUSIONS: We recommend considering breast pathology review based on the individual clinical scenario, regardless of initial pathologic diagnosis or originating institution.

Intraoperative Evaluation of Breast Tissues During Breast Cancer Operations Using the MasSpec Pen.

Importance: Surgery with complete tumor resection remains the main treatment option for patients with breast cancer. Yet, current technologies are limited in providing accurate assessment of breast tissue in vivo, warranting development of new technologies for surgical guidance. Objective: To evaluate the performance of the MasSpec Pen for accurate intraoperative assessment of breast tissues and surgical margins based on metabolic and lipid information. Design, Setting, and Participants: In this diagnostic study conducted between February 23, 2017, and August 19, 2021, the mass spectrometry-based device was used to analyze healthy breast and invasive ductal carcinoma (IDC) banked tissue samples from adult patients undergoing breast surgery for ductal carcinomas or nonmalignant conditions. Fresh-frozen tissue samples and touch imprints were analyzed in a laboratory. Intraoperative in vivo and ex vivo breast tissue analyses were performed by surgical staff in operating rooms (ORs) within 2 different hospitals at the Texas Medical Center. Molecular data were used to build statistical classifiers. Main Outcomes and Measures: Prediction results of tissue analyses from classification models were compared with gross assessment, frozen section analysis, and/or final postoperative pathology to assess accuracy. Results: All data acquired from the 143 banked tissue samples, including 79 healthy breast and 64 IDC tissues, were included in the statistical analysis. Data presented rich molecular profiles of healthy and IDC banked tissue samples, with significant changes in relative abundances observed for several metabolic species. Statistical classifiers yielded accuracies of 95.6%, 95.5%, and 90.6% for training, validation, and independent test sets, respectively. A total of 25 participants enrolled in the clinical, intraoperative study; all were female, and the median age was 58 years (IQR, 44-66 years). Intraoperative testing of the technology was successfully performed by surgical staff during 25 breast operations. Of 273 intraoperative analyses performed during 25 surgical cases, 147 analyses from 22 cases were subjected to statistical classification. Testing of the classifiers on 147 intraoperative mass spectra yielded 95.9% agreement with postoperative pathology results. Conclusions and Relevance: The findings of this diagnostic study suggest that the mass spectrometry-based system could be clinically valuable to surgeons and patients by enabling fast molecular-based intraoperative assessment of in vivo and ex vivo breast tissue samples and surgical margins.

Localization of BRCA1 protein in breast cancer tissue and cell lines with mutations.

BACKGROUND: The breast and ovarian cancer susceptibility gene (BRCA1) encodes a tumor suppressor. The BRCA1 protein is found primarily in cell nuclei and plays an important role in the DNA damage response and transcriptional regulation. Deficiencies in DNA repair capabilities have been associated with higher histopathological grade and worse prognosis in breast cancer. METHODS: In order to investigate the subcellular distribution of BRCA1 in tumor tissue we randomly selected 22 breast carcinomas and tested BRCA1 protein localization in frozen and contiguous formalin-fixed, paraffin embedded (FFPE) tissue, using pressure cooker antigen-retrieval and the MS110 antibody staining. To assess the impact of BRCA1 germline mutations on protein localization, we retrospectively tested 16 of the tumor specimens to determine whether they contained the common Ashkenazi Jewish founder mutations in BRCA1 (185delAG, 5382insC), and BRCA2 (6174delT). We also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC). RESULTS: In FFPE tissue, with MS110 antibody staining, we frequently found reduced BRCA1 nuclear staining in breast tumor tissue compared to normal tissue, and less BRCA1 staining with higher histological grade in the tumors. However, in the frozen sections, BRCA1 antibody staining showed punctate, intra-nuclear granules in varying numbers of tumor, lactating, and normal cells. Two mutation carriers were identified and were confirmed by gene sequencing. We have also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC) and found altered sub-nuclear and nucleolar localization patterns consistent with a functional impact of the mutation on protein localization. CONCLUSIONS: The data presented here support a role for BRCA1 in the pathogenesis of sporadic and inherited breast cancers. The use of well-characterized reagents may lead to further insights into the function of BRCA1 and possibly the further development of targeted therapeutics.

Incidental intraductal papillomas (<2 mm) of the breast diagnosed on needle core biopsy do not need to be excised.

Most authors recommend excision of intraductal papillomas diagnosed on core needle biopsy. This leads to the question of whether or not excision is necessary for incidental intraductal papillomas on core needle biopsy as opposed to those corresponding to imaging findings. Using the pathology computerized data base we retrospectively identified 46 incidental intraductal papillomas diagnosed on core needle biopsy from 1/2000 to 12/2008. Clinical, radiologic, and pathologic information was gathered and correlated. All core needle biopsies were reviewed to confirm the diagnosis of incidental intraductal papillomas, and excision specimens reviewed when available. Of the 46 patients, follow-up information was available in only 38. The age of the patients ranged from 39 to 82 years (mean = 48 years). Most incidental intraductal papillomas were diagnosed by mammotome core needle biopsy (36 cases). A total of 33 cases were performed for calcifications with the following indications: clustered = 21, new = 4, pleomorphic = 3, increasing = 3, indeterminant = 2. The correlating diagnoses included the following: fibrocystic changes with calcium phosphate = 18 or calcium oxalate = 10, fibroadenoma with calcifications = 5. The three masses were: two cases of cystic papillary apocrine metaplasia (I Ultrasound and 1 MRI) and 1 fibroadenoma (Ultrasound). In all cases, the intraductal papillomas were ≤0.2 cm, were not associated with calcifications, and were incidental to them or the underlying mass. A total of 14 patients underwent excision, whereas the remaining 24 have remained radiologically stable for over 12 months. The excision specimen findings were: fibrocystic changes = 8 and intraductal papilloma = 6. With the exception of one case, all the intraductal papilloma remained incidental to imaging findings. In this solitary case, the calcifications were described as pleomorphic and corresponded to fibrocystic changes calcifications on core needle biopsy. However, on excision, residual pleomorphic calcifications on mammogram correlated with both fibrocystic changes and intraductal papilloma. No cases were upstaged on excision to atypical duct hyperplasia or intraductal or invasive carcinoma. With the exception of one case, all incidental intraductal papillomas diagnosed on core needle biopsy were either completely excised or remained incidental. The exception occurred due to sampling error and accounted for the change from an incidental intraductal papillomas on core needle biopsy to one that was associated with calcifications on excision. Given the complete lack of upstaging, it is difficult to recommend excision of incidental intraductal papillomas diagnosed on core needle biopsy provided the index lesion has been adequately sampled and radiologic follow-up is maintained.

Short-term PI3K Inhibition Prevents Breast Cancer in Preclinical Models.

Antiestrogen medication is the only chemoprevention currently available for women at a high risk of developing breast cancer; however, antiestrogen therapy requires years to achieve efficacy and has adverse side effects. Therefore, it is important to develop an efficacious chemoprevention strategy that requires only a short course of treatment. PIK3CA is commonly activated in breast atypical hyperplasia, the known precancerous precursor of breast cancer. Targeting PI3K signaling in these precancerous lesions may offer a new strategy for chemoprevention. Here, we first established a mouse model that mimics the progression from precancerous lesions to breast cancer. Next, we demonstrated that a short-course prophylactic treatment with the clinically approved PI3K inhibitor alpelisib slowed early lesion expansion and prevented cancer formation in this model. Furthermore, we showed that alpelisib suppressed ex vivo expansion of patient-derived atypical hyperplasia. Together, these data indicate that the progression of precancerous breast lesions heavily depends on the PI3K signaling, and that prophylactic targeting of PI3K activity can prevent breast cancer. PREVENTION RELEVANCE: PI3K protein is abnormally high in breast precancerous lesions. This preclinical study demonstrates that the FDA-approved anti-PI3K inhibitor alpelisib can prevent breast cancer and thus warrant future clinical trials in high-risk women.

MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming.

Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined 'macro-Bound Enhancers', that modulate enhancer activity. We find macroH2A variants localized at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and their repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling of normal mammary stem cells derived from mice, we show that macroH2A deficiency facilitates increased activity of transcription factors associated with stem cell activity.

A spatially resolved single cell genomic atlas of the adult human breast.

The adult human breast comprises an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue. While previous studies have mainly focused on the breast epithelial system, many of the non-epithelial cell types remain understudied. Here, we constructed a comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics data profiled 535,941 cells from 62 women, and 120,024 nuclei from 20 women, identifying 11 major cell types and 53 cell states. These data revealed abundant pericyte, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Our spatial mapping using three technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells in the ducts and lobules, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide an unprecedented reference of adult normal breast tissue for studying mammary biology and disease states such as breast cancer.

STAT5 confers lactogenic properties in breast tumorigenesis and restricts metastatic potential.

Signal transducer and activator of transcription 5 (STAT5) promotes cell survival and instigates breast tumor formation, and in the normal breast it also drives alveolar differentiation and lactogenesis. However, whether STAT5 drives a differentiated phenotype in breast tumorigenesis and therefore impacts cancer spread and metastasis is unclear. We found in two genetically engineered mouse models of breast cancer that constitutively activated Stat5a (Stat5aca) caused precancerous mammary epithelial cells to become lactogenic and evolve into tumors with diminished potential to metastasize. We also showed that STAT5aca reduced the migratory and invasive ability of human breast cancer cell lines in vitro. Furthermore, we demonstrated that STAT5aca overexpression in human breast cancer cells lowered their metastatic burden in xenografted mice. Moreover, RPPA, Western blotting, and studies of ChIPseq data identified several EMT drivers regulated by STAT5. In addition, bioinformatic studies detected a correlation between STAT5 activity and better prognosis of breast cancer patients. Together, we conclude that STAT5 activation during mammary tumorigenesis specifies a tumor phenotype of lactogenic differentiation, suppresses EMT, and diminishes potential for subsequent metastasis.

Benign mucocele-like lesions of the breast: revisited.

Mucocele-like lesions of the breast are ruptured ducts that discharge their contents into the stroma. They constitute a spectrum from benign to atypical to malignant. The current management of these lesions diagnosed on core biopsy is excision. The goal of our study was to evaluate the necessity of this practice for benign mucocele-like lesions. Retrospective review of the pathology database from 1 January 2000 to 1 June 2008 identified 61 cases, with follow-up information available in 50 cases. Clinical, radiological, and pathological information was correlated. Core biopsies were reviewed to confirm the diagnosis and verify previous biopsy site. In all, 45 patients underwent surgery, whereas 5 patients were followed for >1 year and remained stable. Patient's ages ranged from 44 to 76 years. Most benign mucoceles were diagnosed stereotactically while targeting calcifications (93.3%); rarely, the lesion was a sonographically detected mass. Most excisions had no residual mucocele (37/45=82%). In seven cases (15.6%), atypical duct hyperplasia was present, three with residual mucocele. In one case, the residual mucocele showed a continuum from florid to atypical duct hyperplasia at the core biopsy site. The other six cases showed atypical duct hyperplasia adjacent to but not directly at the core biopsy site. The sizes of the benign mucoceles ranged from incipient to 0.6 cm, all containing calcifications except one, which was incidental. Radiological-pathological correlation was concordant in all cases except one with suspicious calcification, which was ductal carcinoma in situ on excision. In this series, the largest of its kind, the upstage rate of benign mucoceles diagnosed on core biopsy was 17.8%. With the exception of the ductal carcinoma in situ case, no radiological or morphological features were predictive of atypia. Thus, because of associated atypical duct hyperplasia, sampling reasons, and intralesional heterogeneity, we continue to recommend excision of benign mucocele-like lesions diagnosed on core biopsy.

A Wnt-Independent LGR4-EGFR Signaling Axis in Cancer Metastasis.

Leucine-rich repeat-containing G protein-coupled receptors 4, 5, and 6 (LGR4/5/6) play critical roles in development and cancer. The widely accepted mechanism is that these proteins, together with their R-spondin ligands, stabilize Wnt receptors, thus potentiating Wnt signaling. Here we show that LGR4 enhanced breast cancer cell metastasis even when Wnt signaling was deactivated pharmacologically or genetically. Furthermore, LGR4 mutants that cannot potentiate Wnt signaling nevertheless promoted breast cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Multiomic screening identified EGFR as a crucial mediator of LGR4 activity in cancer progression. Mechanistically, LGR4 interacted with EGFR and blocked EGFR ubiquitination and degradation, resulting in persistent EGFR activation. Together, these data uncover a Wnt-independent LGR4-EGFR signaling axis with broad implications for cancer progression and targeted therapy. SIGNIFICANCE: This work demonstrates a Wnt-independent mechanism by which LGR4 promotes cancer metastasis.See related commentary by Stevens and Williams, p. 4397.

Neratinib plus trastuzumab is superior to pertuzumab plus trastuzumab in HER2-positive breast cancer xenograft models.

Lapatinib (L) plus trastuzumab (T), with endocrine therapy for estrogen receptor (ER)+ tumors, but without chemotherapy, yielded meaningful response in HER2+ breast cancer (BC) neoadjuvant trials. The irreversible/pan-HER inhibitor neratinib (N) has proven more potent than L. However, the efficacy of N+T in comparison to pertuzumab (P) + T or L + T (without chemotherapy) remains less studied. To address this, mice bearing HER2+ BT474-AZ (ER+) cell and BCM-3963 patient-derived BC xenografts were randomized to vehicle, N, T, P, N+T, or P+T, with simultaneous estrogen deprivation for BT474-AZ. Time to tumor regression/progression and incidence/time to complete response (CR) were determined. Changes in key HER pathway and proliferative markers were assessed by immunohistochemistry and western blot of short-term-treated tumors. In the BT474-AZ model, while all N, P, T, N + T, and P + T treated tumors regressed, N + T-treated tumors regressed faster than P, T, and P + T. Further, N + T was superior to N and T alone in accelerating CR. In the BCM-3963 model, which was refractory to T, P, and P + T, while N and N + T yielded 100% CR, N + T accelerated the CR compared to N. Ki67, phosphorylated (p) AKT, pS6, and pERK levels were largely inhibited by N and N + T, but not by T, P, or P + T. Phosphorylated HER receptor levels were also markedly inhibited by N and N + T, but not by P + T or L + T. Our findings establish the efficacy of combining N with T and support clinical testing to investigate the efficacy of N + T with or without chemotherapy in the neoadjuvant setting for HER2+ BC.

Correction: UDP-glucose 6-dehydrogenase regulates hyaluronic acid production and promotes breast cancer progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

UDP-glucose 6-dehydrogenase regulates hyaluronic acid production and promotes breast cancer progression.

An improved understanding of the biochemical alterations that accompany tumor progression and metastasis is necessary to inform the next generation of diagnostic tools and targeted therapies. Metabolic reprogramming is known to occur during the epithelial-mesenchymal transition (EMT), a process that promotes metastasis. Here, we identify metabolic enzymes involved in extracellular matrix remodeling that are upregulated during EMT and are highly expressed in patients with aggressive mesenchymal-like breast cancer. Activation of EMT significantly increases production of hyaluronic acid, which is enabled by the reprogramming of glucose metabolism. Using genetic and pharmacological approaches, we show that depletion of the hyaluronic acid precursor UDP-glucuronic acid is sufficient to inhibit several mesenchymal-like properties including cellular invasion and colony formation in vitro, as well as tumor growth and metastasis in vivo. We found that depletion of UDP-glucuronic acid altered the expression of PPAR-gamma target genes and increased PPAR-gamma DNA-binding activity. Taken together, our findings indicate that the disruption of EMT-induced metabolic reprogramming affects hyaluronic acid production, as well as associated extracellular matrix remodeling and represents pharmacologically actionable target for the inhibition of aggressive mesenchymal-like breast cancer progression.

Evaluation of the Predictive Role of Tumor Immune Infiltrate in Patients with HER2-Positive Breast Cancer Treated with Neoadjuvant Anti-HER2 Therapy without Chemotherapy.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are associated with benefit to trastuzumab and chemotherapy in patients with early-stage HER2+ breast cancer. The predictive value of TILs, TIL subsets, and other immune cells in patients receiving chemotherapy-sparing lapatinib plus trastuzumab treatment is unclear.Experimental Design: Hematoxylin and eosin-stained slides (n = 59) were used to score stromal (s-)TILs from pretreatment biopsies of patients enrolled in the neoadjuvant TBCRC006 trial of 12-week lapatinib plus trastuzumab therapy (plus endocrine therapy for ER+ tumors). A 60% threshold was used to define lymphocyte-predominant breast cancer (LPBC). Multiplexed immunofluorescence (m-IF) staining (CD4, CD8, CD20, CD68, and FoxP3) and multispectral imaging were performed to characterize immune infiltrates in single formalin-fixed paraffin-embedded slides (n = 33). RESULTS: The pathologic complete response (pCR) rate was numerically higher in patients with LPBC compared with patients with non-LPBC (50% vs. 19%, P = 0.057). Unsupervised hierarchical clustering of the five immune markers identified two patient clusters with different responses to lapatinib plus trastuzumab treatment (pCR = 7% vs. 50%, for cluster 1 vs. 2 respectively; P = 0.01). In multivariable analysis, cluster 2, characterized by high CD4+, CD8+, CD20+ s-TILs, and high CD20+ intratumoral TILs, was independently associated with a higher pCR rate (P = 0.03). Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by s-CD4, intratumoral (i-) CD4, and i-CD20+ TILs. CONCLUSIONS: LPBC was marginally associated with higher pCR rate than non-LPBC in patients with lapatinib plus trastuzumab treated HER2+ breast cancer. Quantitative assessment of the immune infiltrate by m-IF is feasible and may help correlate individual immune cell subpopulations and immune cell profiles with treatment response.