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Urine cytology is commonly used as a screening test for high-grade urothelial carcinoma for patients with risk factors or hematuria and is an essential step in longitudinal monitoring of patients with previous bladder cancer history. However, the semisubjective nature of current reporting systems for urine cytology (e.g., The Paris System) can hamper reproducibility. For instance, the incorporation of urothelial cell clusters into the classification schema is still an item of debate and perplexity among expert cytopathologists because several previous works have disputed their diagnostic relevance.In this work, an automated preprocessing tool for urothelial cell cluster assessment was developed that divides urothelial cell clusters into meaningful components for downstream assessment (ie, population-based studies, workflow automation).In this work, an automated preprocessing tool for urothelial cell cluster assessment was developed that divides urothelial cell clusters into meaningful components for downstream assessment (ie, population-based studies, workflow automation). Results indicate that cell cluster atypia (i.e., defined by whether the cell cluster harbored multiple atypical cells, thresholded by a minimum number of cells), cell border overlap and smoothness, and total number of clusters are important markers of specimen atypia when considering assessment of urothelial cell clusters.Markers established through techniques to separate cell clusters may have wider applicability for the design and implementation of machine learning approaches for urine cytology assessment.

Purpose: Strategies to implement estrogen therapy for advanced estrogen receptor (ER)-positive breast cancer are under-developed. Preclinical data suggest that cycling treatment with 17b-estradiol followed by estrogen deprivation can control tumor growth long-term. Experimental Design: Post-menopausal women with advanced ER+/HER2- breast cancer with recurrence or progression on ≥1 anti-estrogen- or aromatase inhibitor (AI)-based therapy were eligible. Patients received 17b-estradiol (2 mg orally TID) for 8 weeks followed by AI (physician’s choice) for 16 weeks, alternating treatments on an 8-week/16-week schedule until disease progression. Patients then optionally received continuous single-agent treatment until a second instance of disease progression. Endpoints included 24-week clinical benefit and objective response per RECIST, and tumor genetic alterations. Results: Of 19 evaluable patients, clinical benefit rate was 42.1% (95% CI: 23.1-63.9%) and objective response rate was 15.8% (95% CI: 5.7-37.9%). One subject experienced a grade 3 adverse event related to 17b-estradiol. Among patients who received continuous single-agent treatment until a second instance of disease progression, clinical benefit was observed in 5/12 (41.7%) cases. Tumor ER (ESR1) mutations were found by whole-exome profiling in 4/7 (57.1%) vs. 2/9 (22.2%) patients who did vs. did not experience clinical benefit from alternating 17b-estradiol/AI therapy. The only two patients to experience objective responses to initial 17b-estradiol had tumor ESR1 mutations. Conclusions: Alternating 17b-estradiol/aromatase inhibitor therapy may be a promising treatment for endocrine-refractory ER+ breast cancer, including following progression on CDK4/6 inhibitors or everolimus. Further study is warranted to determine whether the anti-tumor activity of 17b-estradiol differs according to ESR1 mutation status.

Introduction Molecular testing has helped clinicians and cytopathologists to further categorize indeterminate thyroid fine needle aspiration (FNA) specimens. The purpose of the present study was to evaluate the accuracy of commercially available molecular tests, review their effects on patient treatment, and correlate the molecular alterations with the histologic findings. Materials and methods A pathology laboratory information system search identified thyroid FNAs performed at our institution between January 1, 2015 and June 30, 2020. The results of surgical follow-up and ancillary molecular testing were collected. We evaluated the accuracy of these tests and whether they could reduce the number of surgeries performed. Results Our laboratory information system search identified 510 cases reported as atypia of undetermined significance, 94 as suspicious for follicular neoplasm, and 44 as suspicious for follicular neoplasm, Hurthle cell type. Of the specimens, 343 had no ancillary molecular testing, 146 were sent for ThyGenX/ThyraMIR, and 136 were sent for ThyroSeq. Of the patients without molecular testing, 50.4% had undergone follow-up surgery compared with 30.8% after ThyGenX/ThyraMIR and 38.2% after ThyroSeq testing, resulting in 38.9% and 24.2% fewer surgeries and an odds ratio of 0.04 (95% confidence interval, 0.00-0.33) and 0.14 (95% confidence interval, 0.01-0.95), respectively. For ThyGenX/ThyraMIR testing, the risk of malignancy for high and moderate to high risk alterations was 80%, 28.6% for moderate and low to moderate risk alterations, and 23.1% for low risk alterations. For ThyroSeq, the risk of malignancy was 87.5% for high risk alterations, 36.8% for intermediate to high risk alterations, 27.3% for intermediate risk alterations, and 0% for low risk alterations. The areas under the curve for ThyGenX/ThyraMIR and ThyroSeq testing were 0.65 and 0.85, respectively. Conclusions These findings suggest that, at our institution, both ThygenX/ThyraMIR and ThyroSeq can be used to effectively stratify cytology specimens based on the risk of malignancy and reduce the number of surgeries performed at our institution.

Supplementary Figure 1: Representative images of high and low sTILs.

Supplementary Methods; Table. S1- Pairwise comparisons of tumor growth patterns between treatment groups; Fig. S1- PI3K inhibition induces ER activation; Fig. S2- Transient, complete inhibition of PI3K more effectively suppresses cell growth than chronic, partial inhibition; Fig. S3- Short-term and long-term inhibition of PI3K and ER induce similar degrees of apoptosis in ER+ breast cancer cells; Fig. S4- Fulv inhibits ER in MCF-7 tumors; Fig. S5- Tumor GDC-0941 concentrations are inversely correlated with PI3K pathway activation; Fig. S6- Pharmacodynamic effects of GDC-0941 and fulv on MCF-7 tumors; Fig. S7- High-dose GDC-0941 in combination with fulv induces tumor cell apoptosis; Fig. S8- High-dose GDC-0941 in combination with fulv suppresses tumor cell proliferation as indicated by geminin IHC; Fig. S9- High-dose GDC-0941 in combination with fulv suppresses tumor cell proliferation as indicated by Ki67 IHC; Fig. S10- Individual MCF-7 tumor volumes; Fig. S11- Individual T47D/FR tumor volumes; Fig. S12- GDC-0941 inhibits PI3K in T47D/FR and HCI-003 tumors; Fig. S13- Individual HCI-003 tumor volumes; Fig. S14- Intermittent, longer-term inhibition of PI3K and ER induces a bolus of suppression of tumor cell proliferation indicated by Geminin and Ki67 staining, with proliferative rebound after recovery of PI3K activity; Fig. S15- Comparison of BCL-2 family members among HCI-003, MCF-7 and T47D FR tumors

Dataset 2: Metadata and clonotype data for TCRβ sequencing (Archer) in 4 patients (peripheral CD8+ PD-1HI and CD8+ PD-1NEG T cells and post-NAC tumor), before (n=3) and after (n=4) NAC.

Purpose: Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)–positive breast cancer in combination with antiestrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER+ breast cancer will provide a rationale for treatment scheduling to maximize therapeutic index.Experimental Design: Antiestrogen-sensitive and antiestrogen-resistant ER+ human breast cancer cell lines and mice bearing PIK3CA-mutant xenografts were treated with the antiestrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured.Results: Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than that with single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6 to 9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared with fulvestrant alone.Conclusions: Continuous and metronomic PI3K inhibition elicits robust anticancer effects in ER+, PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors with antiestrogens is warranted. Clin Cancer Res; 22(9); 2250–60. ©2016 AACR.See related commentary by Toska and Baselga, p. 2099

Supplementary Figure S1; Supplementary Table S1.

Supplementary Figure 5: The change in sTILs during NAC is not prognostic for outcome in TNBC patients with residual disease.

Dataset 1: Metadata and clonotype data for TCRβ sequencing (Adaptive) in 15 pairs of NAC-treated patients (tumor data).

Supplementary Figure 2: Pre-NAC sTILs have minimal prognostic value in breast cancer patients with residual disease.

Purpose:The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME).Experimental Design:We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood.Results:In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden.Conclusions:We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118–29. ©2016 AACR.

Document containing methods on whole exome and targeted massively parallel sequencing analysis, 2-hydroxyglutarate (2HG) assay of cell lines, methylation profiling, and supplementary references.

Supplementary Figure 7: Changes in immunologic signatures in response to NAC are not associated with outcome in non-TNBC tumors with residual disease.

Document containing figures illustrating results of Sanger sequencing, DNA methylation profiling of SPCRPs, the analysis of IDH2 expression in non-malignant breast epithelial cells and its downstream effects, and tables describing the antibodies used for immunohistochemical analysis, the alterations surveyed by the SNaPshot genotyping assay, Sanger sequencing primers, and statistics and results of the massively parallel sequencing analyses.

Supplementary Figure 3: The prognostic value of sTILs is primarily confined to the post-NAC specimen in TNBC patients with residual disease.

Supplementary Figure 6: Neither time of sample nor institution drive significant gene expression changes.

Supplementary Tables

Table S1 shows normalized mRNA counts from NanoString analysis of MCF-7 xenografts.

Supplementary Figure 4: sTILs are not prognostic in ER+ or HER2+ disease.

Purpose:Despite adjuvant endocrine therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies.Experimental Design:Mimicking the aromatase inhibitor–induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant endocrine therapy.Results:Dormant breast cancer cells exhibited upregulated 5′ adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the antidiabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival.Conclusions:AMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.

Legends for Supplementary Figures S1-S5

Legend for Table S1

Purpose: Androgen receptor (AR) is commonly expressed in breast cancers. However, the association between tumor AR status and breast cancer survival is uncertain. Hence, we examined the association between AR status and breast cancer survival in the Nurses' Health Study (NHS).Experimental Design: It was a prospective study of postmenopausal women enrolled in the Nurses' Health Study with stage I to III breast cancer diagnosed between 1976 and 1997 and followed from the date of diagnosis until January 1, 2008 or death. Analyses were conducted using Kaplan–Meier methods and Cox proportional hazard models, to determine the association of AR status with survival outcomes adjusting for covariates.Results: Among 1467 breast cancers, 78.7% were AR-positive (AR+). Among 1,164 estrogen receptor (ER)-positive cases, 88.0% were AR+. AR positivity was associated with a significant reduction in breast cancer mortality (HR, 0.68; 95% CI, 0.47–0.99) and overall mortality (HR, 0.70; 95% CI, 0.53–0.91) after adjustment for covariates. In contrast, among women with ER-negative tumors (303 cases), 42.9% were AR+. There was a nonsignificant association between AR status and breast cancer death (HR, 1.59; 95% CI, 0.94–2.68).Conclusions: The association of AR status and breast cancer survival is dependent on ER status. In particular, AR expression was associated with a more favorable prognosis among women with ER-positive tumors. Thus, determination of AR status may provide additional information on prognosis for postmenopausal women with breast cancer, and provide novel opportunities for targeted therapy. Clin Cancer Res; 17(7); 1867–74. ©2011 AACR.

Fig. S1 shows that dormant ER+ breast cancer cells retain tumor-initiating capacity. Fig. S2 shows individual tumor growth curves. Fig. S3 shows IHC images for Ki67, cleaved caspase-3 and ER in xenografts. Fig. S4 shows Gene Set Variation Analysis of RNA-seq data from xenografts. Fig. S5 shows overlap of altered genes from RNA-seq data. Fig. S6 shows IHC images for AMPK�2 and P-ACC in xenografts. Fig. S7 shows mTORC1 activation in MCF-7 tumors. Fig. S8 shows a transcriptional signature of FAO in human tumors. Fig. S9 shows mitochondrial content and activity in xenografts. Fig. S10 shows effects of AMPK knockdown on respiration. Fig. S11 shows that inhibition of FAO hastens tumor regression. Fig. S12 shows pharmacologic analysis of metformin. Fig. S13 shows metformin effects in MDA-MB-415 tumors. Fig. S14 shows that metformin prevents tumor regression. Fig. S15 shows that metformin does not alter fasting levels of blood glucose, serum insulin, or serum free fatty acids in mice. Fig. S16 show IHC images of P-ACC in tumors from mice treated with metformin. Fig. S17 shows AMPK effects on HCC-1500 cells and tumors. Fig. S18 shows the effects of high-fat diet on tumor regression. Fig. S19 shows IHC images of Ki67, CPT1α, and P-ACC in xenografts from mice treated with high-fat or low-fat diet.

Supplementary methods and supplementary references cited.

Supplementary Figure 8: Changes in immune-related genes in response to NAC are not associated with outcome in ER+ or HER2+ tumors with residual disease.

Urine cytology (UC) is generally considered the primary approach for screening for recurrence of bladder cancer. However, it is currently unclear how best to use cytological exams themselves for the assessment and early detection of recurrence, beyond identifying a positive finding which requires more invasive methods to confirm recurrence and decide on therapeutic options. As screening programs are frequent, and can be burdensome, finding quantitative means to reduce this burden for patients, cytopathologists and urologists is an important endeavor and can improve both the efficiency and reliability of findings. Additionally, identifying ways to risk-stratify patients is crucial for improving quality of life while reducing the risk of future recurrence or progression of the cancer. In this study, we leveraged a computational machine learning tool, AutoParis-X, to extract imaging features from UC exams longitudinally to study the predictive potential of urine cytology for assessing recurrence risk. This study examined how the significance of imaging predictors changes over time before and after surgery to determine which predictors and time periods are most relevant for assessing recurrence risk. Results indicate that imaging predictors extracted using AutoParis-X can predict recurrence as well or better than traditional cytological / histological assessments alone and that the predictiveness of these features is variable across time, with key differences in overall specimen atypia identified immediately before tumor recurrence. Further research will clarify how computational methods can be effectively utilized in high volume screening programs to improve recurrence detection and complement traditional modes of assessment.

Adopting a computational approach for the assessment of urine cytology specimens has the potential to improve the efficiency, accuracy and reliability of bladder cancer screening, which has heretofore relied on semi-subjective manual assessment methods. As rigorous, quantitative criteria and guidelines have been introduced for improving screening practices, e.g., The Paris System for Reporting Urinary Cytology (TPS), algorithms to emulate semi-autonomous diagnostic decision-making have lagged behind, in part due to the complex and nuanced nature of urine cytology reporting. In this study, we report on a deep learning tool, AutoParis-X, which can facilitate rapid semi-autonomous examination of urine cytology specimens. Through a large-scale retrospective validation study, results indicate that AutoParis-X can accurately determine urothelial cell atypia and aggregate a wide-variety of cell and cluster-related information across a slide to yield an Atypia Burden Score (ABS) that correlates closely with overall specimen atypia, predictive of TPS diagnostic categories. Importantly, this approach accounts for challenges associated with assessment of overlapping cell cluster borders, which improved the ability to predict specimen atypia and accurately estimate the nuclear-to-cytoplasm (NC) ratio for cells in these clusters. We developed an interactive web application that is publicly available and open-source, which features a simple, easy-to-use display for examining urine cytology whole-slide images (WSI) and determining the atypia level of specific cells, flagging the most abnormal cells for pathologist review. The accuracy of AutoParis-X (and other semi-automated digital pathology systems) indicates that these technologies are approaching clinical readiness and necessitates full evaluation of these algorithms via head-to-head clinical trials.

Introduction The College of American Pathologists mandates that telepathology services are included in laboratory quality management programs. The aim of this study was to assess a telecytology quality assurance (QA) process that we implemented in 2015. Materials and methods Each month, a cytotechnologist randomly selected 3 telecytology fine-needle aspiration (FNA) cases from each cytopathologist on the FNA service that month. Data were recorded in a monthly worksheet and included onsite telecytology adequacy, final adequacy, concordance, onsite operator, cytopathologist, and reason for discrepancy, if present. The worksheet was reviewed monthly, discordant cases were re-examined, and feedback to cytologists was provided. For this study, worksheets from October 2015 to December 2019 were retrospectively reviewed. Results The QA program captured 488 cases, representing 25% of total cases that utilized telecytology during the evaluation period (n = 1983). The telecytology onsite assessment was concordant with the final cytologic assessment in 84% (410 of 488) of cases. The majority of discordant cases (72 of 78, 92%) were the result of an “Inadequate" onsite telecytology assessment, but a final diagnosis was able to be rendered; 92% of these cases were attributed to diagnostic material being present in cytologic preparations not available during the onsite assessment. Nine telecytology onsite interpretation errors were identified, of which 7 were provided by cytopathologists with less than 2 years of experience. Conclusions Most telecytology cases with onsite assessment errors were evaluated by cytopathologists with less than 2 years of practice experience; therefore, careful monitoring of new staff should be considered when developing a telecytology QA program.

To investigate clinical and pathologic features of encapsulated papillary carcinomas (EPCs) that may be associated with invasive disease and characterize the axillary staging practices for EPCs at our institution. A pathology database search for cases containing "papillary carcinoma" was performed. Slides were reviewed by two pathologists. Clinicopathological features and axillary staging practices of EPCs with and without invasion were compared. Twenty-five cases of EPCs were identified. Fifteen cases contained a frank invasive tumor (60%), which were all pT1 (0.7 ± 0.56 cm), and the majority were ER-positive, HER2-negative, low-grade IDC-NST. Seventeen patients underwent sentinel lymph node biopsies (SLNB). No nodal metastases were identified. Follow-up was available for 24 patients (mean = 39 ± 29 months); 23 had no NED. Patients that presented with a self-palpated mass (versus screening) were more likely to have an invasive component; however, no pathologic or radiologic features differentiated EPCs with and without frank invasion. Pathologic and radiologic characteristics did not differentiate EPCs with and without frank invasion. EPCs have an excellent prognosis supported by the notable disease-free survival and negative nodal status in our cohort, which supports the notion that patients with EPCs may forgo axillary staging.

Urine cytology is used to screen for urothelial carcinoma in patients with hematuria or risk factors (e.g., smoking, industrial dye exposure) and is an essential clinical triage and longitudinal monitoring tool for patients with known bladder cancer. However, urine cytology is semi-subjective and thus susceptible to issues including specimen quality, inter-observer variability, and “hedging” towards equivocal (“atypical”) diagnoses. These factors limit the predictive value of urine cytology and increase reliance on invasive procedures (cystoscopy). The Paris System for Reporting Urine Cytology (TPS) was formulated to provide more quantitative/reproducible endpoints with well-defined criteria for urothelial atypia. TPS is often compared to other assessment techniques to justify its adoption. TPS results in decreased use of the atypical category and better reproducibility. Previous manuscripts comparing diagnoses pre- and post-TPS lacked a longitudinal component and thus failed to consider temporal differences between diagnoses made under prior systems and TPS. By aggregating across time, studies may underestimate the magnitude of differences between assessment methods. We conducted a large-scale longitudinal reassessment of urine cytology using TPS criteria from specimens collected from 2008 to 2018, prior to the mid-2018 adoption of TPS at an academic medical center. Findings indicate that differences in atypical assignment were largest at the start of the period and these differences progressively decreased towards insignificance just prior to TPS implementation. This finding suggests that cytopathologists had begun to utilize the quantitative TPS criteria prior to official adoption, which may more broadly inform adoption strategies, communication, and understanding for evolving classification systems in cytology.

Purpose: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME). Experimental Design: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood. Results: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden. Conclusions: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Purpose: Despite adjuvant endocrine therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Experimental Design: Mimicking the aromatase inhibitor–induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant endocrine therapy. Results: Dormant breast cancer cells exhibited upregulated 5′ adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the antidiabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. Conclusions: AMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.

PURPOSE: Circulating tumor DNA in plasma may present a minimally invasive opportunity to identify tumor-derived mutations to inform selection of targeted therapies for individual patients, particularly in cases of oligometastatic disease where biopsy of multiple tumors is impractical. To assess the utility of plasma DNA as a “liquid biopsy” for precision oncology, we tested whether sequencing of plasma DNA is a reliable surrogate for sequencing of tumor DNA to identify targetable genetic alterations. METHODS: Blood and biopsies of 1–3 tumors were obtained from 4 evaluable patients with advanced breast cancer. One patient provided samples from an additional 7 tumors post-mortem. DNA extracted from plasma, tumor tissues, and buffy coat of blood were used for probe-directed capture of all exons in 149 cancer-related genes and massively parallel sequencing. Somatic mutations in DNA from plasma and tumors were identified by comparison to buffy coat DNA. RESULTS: Sequencing of plasma DNA identified 27.94 +/− 11.81% (mean +/− SD) of mutations detected in a tumor(s) from the same patient; such mutations tended to be present at high allelic frequency. The majority of mutations found in plasma DNA were not found in tumor samples. Mutations were also found in plasma that matched clinically undetectable tumors found post-mortem. CONCLUSIONS: The incomplete overlap of genetic alteration profiles of plasma and tumors warrants caution in the sole reliance of plasma DNA to identify therapeutically targetable alterations in patients, and indicates that analysis of plasma DNA complements, but does not replace, tumor DNA profiling. TRIAL REGISTRATION: Subjects were prospectively enrolled in trial NCT01836640 (registered April 22, 2013).

The first genotype-phenotype relationship in breast pathology developed in 1994 with the discovery of the CDH1 gene. This finding eventually provided biological insight into the characteristic morphology of invasive lobular carcinoma. Subsequent investigative efforts have uncovered additional molecular alterations largely responsible for the histology of several breast neoplasms including secretory carcinoma, adenoid cystic carcinoma, tall cell carcinoma with reversed polarity, fibroepithelial lesions, and most recently, adenomyoepithelioma. Evaluation of the genomic landscape of other special types of breast cancer with distinctive growth patterns, such as invasive mucinous carcinoma, have yet to uncover recurring cytogenetic and/or molecular alterations. Despite the lack of a hallmark alteration in mucinous carcinoma, it is important to note the relative decrease in PIK3CA mutations compared with invasive carcinoma of no special type. In this review, we describe the clinical and pathologic features of breast tumors with recognized genotype-phenotype correlations and summarize the molecular alterations of mucinous carcinoma.

The recent approval of anti-PD-L1 immunotherapy in combination with nAB-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor-immune microenvironment (TIME). Patients with TNBC are routinely treated with neoadjuvant chemotherapy (NAC). Stromal tumor-infiltrating lymphocytes (sTILs) in the pre-treatment diagnostic biopsy are predictive of pathologic complete response (pCR). In patients with residual disease (RD) at surgery, sTILs confer good prognosis. However, the effect of chemotherapy on sTILs and how it influences the TIME are poorly understood. We examined immune-gene expression patterns before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. sTILs were enumerated by standardized guidelines. Gene expression patterns were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. In 4 patients undergoing NAC, PD-1-high and -negative CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNAseq and multiplexed cytokine secretion assays. Post-NAC sTILs (≥30%) were only predictive of outcome (RFS p=0.019; OS p=0.05) in TNBC patients, but not in non-TNBC patients (RFS p=0.28; OS p=0.78) confirming that the prognostic capacity of sTILs is confined to TNBC. Pre-NAC sTILs were not predictive of outcome in either group, likely due to exclusion of patients experiencing pCR. The change in sTILs during NAC did not prognosticate outcome in TNBC, suggesting that in the post-NAC setting, only the most proximal measurement of sTILs is meaningful. However, these results did suggest that NAC alters the TIME. To examine the interplay among NAC, the TIME, and clinical outcomes, we tested the change in expression of 770 immune-related genes during NAC in univariate cox-proportional hazards models. In non-TNBC, no change in expression of any single gene was associated with RFS or OS at a false-discovery rate (FDR) of 10%. In TNBC, individual changes in 12 genes and 204 genes were identified as associated with RFS and OS, respectively (FDR Citation Format: Justin M Balko, Mellissa Nixon, Paula I Gonzalez-Ericsson, Mark A Pilkinton, Wyatt J McDonnell, Violeta Sanchez, Susan R Opalenik, Sherene Loi, Brent Rexer, Vandana Abramson, Valerie Jansen, Simon Mallal, Jonathan D Marotti, Kevin Shee, Todd W Miller, Melinda E Sanders, Ingrid A Mayer, Roberto Salgado. Immunologic correlates of long-term outcome in the residual disease of triple-negative breast cancer 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 P3-08-15.

Background Breast cancer in young women is more likely to have higher risk features and be associated with germline BRCA1/BRCA2 mutations. We present the clinicopathologic features of breast cancers in a prospective cohort of young women, and associations between surrogate molecular subtype and BRCA1/BRCA2 mutation status. Methods Histopathological features, biomarker status, tumour stage and BRCA status were collected. Invasive tumours were categorised as luminal A-like (ER + and/or PR + , HER2-, grade 1/2), luminal B-like (ER + and/or PR + , HER2 + , or ER + and/or PR + , HER2-, and grade 3), HER2-enriched (ER/PR-, HER2 + ) or triple-negative. Results In all, 57.3% (654/1143) of invasive tumours were high grade. In total, 32.9% were luminal A-like, 42.4% luminal B-like, 8.3% HER2-enriched, and 16.4% triple-negative. Among different age groups, there were no differences in molecular phenotype, stage, grade or histopathology. 11% (131) of tumours were from BRCA mutation carriers; 64.1% BRCA1 (63.1% triple-negative), and 35.9% BRCA2 (55.3% luminal B-like). Discussion The opportunity to provide comparisons across young age groups, BRCA mutation status, surrogate molecular phenotype, and the identification of more aggressive hormone receptor-positive phenotypes in this population provides direction for future work to further understand and improve disparate outcomes for young women with luminal B-like cancers, particularly BRCA2-associated cancers, with potential implications for tailored prevention and treatment.

The molecular pathogenesis of breast fibroepithelial tumors continues to be elucidated. Recently, highly recurrent MED12 mutations arising in exon 2 at codon 44 were discovered in fibroadenomas and phyllodes tumors. In addition, a high prevalence of TERT promoter mutations in two hotspots (124 and 126 bp upstream from the translation start site) was discovered in up to 65% of phyllodes tumors. Breast periductal stromal tumors are a potentially distinct category of fibroepithelial lesions that are exceptionally rare with controversial classification and pathogenesis. Herein, we report the first comprehensive molecular genetic workup of a breast periductal stromal tumor that harbored a TERT promoter −124C > T mutation, supporting a relation to phyllodes tumors.

PURPOSE: This study evaluated epidemiologic and immune factors associated with pathologic complete response (pCR), breast cancer-specific survival (BCSS) and disease-free survival (DFS) outcomes in inflammatory (IBC) and locally advanced breast cancer (LABC) patients. METHODS: Tumor-infiltrating lymphocytes (TILs) and CD20(+) B-cell frequencies (CD20(+)), and PD-L1 expression on tumor (PD-L1(+)carcinoma cells) and immune (PD-L1(+)TILs) cells were analyzed by immunohistochemistry along with clinicopathologic factors as modifiers of pCR and outcomes in 221 IBC and 162 LABC patients. Analysis included Kaplan–Meier curves and Cox proportional hazard models. RESULTS: IBC and LABC display similar levels of TILs, CD20(+), and combined CD20(+) and PD-L1(+)TILs (CD20(+)PD-L1(+)TILs), while LABC contained more PD-L1(+)TILs and PD-L1(+) carcinoma cells. Absence of lymphovascular involvement, high TILs, PD-L1(+) carcinoma cells, and combined CD20(+) and PD-L1(+) carcinoma cells correlated with pCR in IBC and LABC patients. High PD-L1(+)TILs correlated with pCR only in LABC; less lymph node involvement at diagnosis, CD20(+) and CD20(+)PD-L1(+)TILs correlated with pCR only in IBC (P < 0.04, all comparisons). Achievement of pCR in IBC and LABC patients correlated with BCSS and DFS (P < 0.02). In multivariate analyses, pCR remained an independent prognostic factor of improved DFS in IBC and LABC patients, but of BCSS in only LABC. CD20(+)PD-L1(+)TILs remained an independent prognostic factor of improved DFS and BCSS only in IBC. CONCLUSION: CD20(+)PD-L1(+)TILs are an independent prognostic biomarker of improved outcomes in IBC, but not LABC. Selecting IBC patients by CD20 and PD-L1 status could stratify patients and potentially identify those in whom activating CD20 agents and anti-PD-1/PD-L1 therapy could be explored.

Historically, ductal carcinoma in situ (DCIS) of the breast has been managed aggressively with surgery and radiotherapy because of a risk of progression to invasive ductal carcinoma. However, this treatment paradigm has been challenged by overtreatment concerns and evidence that suggests that DCIS can be stratified according to risk of recurrence or risk of progression to invasive disease. Traditional methods of risk stratification include histologic grade and hormone receptor status. Recent technological advancements have enabled an era of precision medicine, where DCIS can be molecularly analyzed by tools, such as next-generation DNA and RNA sequencing, to identify molecular biomarkers for risk stratification. These findings have led to the development of tools such as the Oncotype DX Breast DCIS Score, a gene expression–based assay with the potential to prevent overtreatment in low-risk disease.

Benign papillary and sclerosing lesions of the breast (intraductal papillomas, complex sclerosing lesions, radial scars) are considered high-risk lesions due to the potential for upgrade to carcinoma on subsequent surgical excision. Optimal clinical management of such lesions remains unclear due to variable reported upgrade rates. Apocrine metaplasia is a common finding in breast tissue and its role in MRI enhancing lesions is increasingly being recognized. The purpose of this study was to investigate the MRI features of papillary and sclerosing lesions of the breast, evaluate the clinical management and upgrade rate of such lesions, and examine the contribution of apocrine metaplasia to the imaging findings. A 13-year retrospective review of MRI-guided biopsies identified 70 MRI-detected and -biopsied papillary and sclerosing lesions. Sixteen lesions without atypia underwent surgical excision; only one case (6%) was upgraded to pleomorphic lobular carcinoma in situ. The majority (64%) of biopsies contained apocrine metaplasia either within or adjacent to the targeted lesion. We found that half of MRI-detected lesions had T2 hyperintense foci (2-5 mm) or masses (>5 mm) adjacent to the lesion. Histologic correlation showed apocrine cysts were likely responsible for this imaging finding in 56% of these cases.

Objectives To review breast cancer with human epidermal growth factor receptor 2 (HER2) genetic heterogeneity (GH), with a focus on clinician interpretation of the results and subsequent management. Methods We retrospectively searched all consecutive invasive breast cancers that underwent HER2 fluorescent in situ hybridization testing and identified cases with HER2 GH. Results Eleven (0.4%) cases of primary breast tumors with reported HER2 GH were identified over 76 months. HER2-positive tumor cells comprised a mean ± SD of 20% ± 15% of the overall tumor population (range, 5%-50%) and exhibited high levels of amplification (HER2/CEP17 = 5.4 ± 2.9). Three cases had morphologic heterogeneity. Of the four patients who completed neoadjuvant chemotherapy, two had trastuzumab; all showed a partial pathologic response. We identified two cases in which there appeared to be clinical misinterpretation of the HER2 results. Conclusions HER2 GH is an infrequent event, may contain morphologic heterogeneity, could infer incomplete response to neoadjuvant therapy, and requires adequate communication with treating clinicians.

Solitary fibrous tumor (SFT) involving the pancreas is a rare entity often diagnosed on surgical specimens rather than cytology samples. Pancreatic SFT frequently mimics pancreatic neuroendocrine tumor radiographically, which presents unique challenges to the cytologist during rapid on-site assessment. We describe a case of pancreatic SFT with a focus on the cytologic pitfalls that might be encountered during transgastric procedures. We also review the available cytologic findings of prior pancreatic SFT cases.