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Effects of Akt3/-S472 knockout and Akt3/+S472 or Akt1 overexpression on apoptosis

Next generation sequencing of genomic DNA isolated from Akt3/+S472 and Akt3/-S472 knockout cell lines

A) Representative images of hematoxylin and eosin (H&E) staining of MCFDCIS, MCFDCIS-Î"4 and mixed (4:1 respectively) tumors that were injected in the mammary fat pad of SCID/Beige mice after two or five weeks. MCFDCIS-Î"4 tumors were not collected at two weeks due to small tumor size. Scale bar=50μm. B) Growth of subcutaneous tumors of DCIS or DCIS-Î"4-hy in athymic nude mice. n=5. **p

Supplementary Figure legends

Akt3/-S472 overexpression in MDA-MB-436 cells suppresses tumor growth by inducing Bim expression and tumor apoptosis

A list of differentially expressed genes in tumors and cells.

Supplementary Figure 1: Stained controls from IHC protocol development.

Purpose: HHLA2 (B7H7/B7-H5/B7y) is a newly identified B7 family member that regulates human T-cell functions. However, its protein expression in human organs and significance in human diseases are unknown. The objective of this study was to analyze HHLA2 protein expression in normal human tissues and cancers, as well as its prognostic significance, to explore mechanisms regulating HHLA2 expression, and to identify candidate HHLA2 receptors.Experimental Design: An immunohistochemistry protocol and a flow cytometry assay with newly generated monoclonal antibodies were developed to examine HHLA2 protein. HHLA2 gene copy-number variation was analyzed from cancer genomic data. The combination of bioinformatics analysis and immunologic approaches was established to explore HHLA2 receptors.Results: HHLA2 protein was detected in trophoblastic cells of the placenta and the epithelium of gut, kidney, gallbladder, and breast, but not in most other organs. In contrast, HHLA2 protein was widely expressed in human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, and esophagus. In a cohort of 50 patients with stage I–III triple-negative breast cancer, 56% of patients had aberrant expression of HHLA2 on their tumors, and high HHLA2 expression was significantly associated with regional lymph node metastasis and stage. The Cancer Genome Atlas revealed that HHLA2 copy-number gains were present in 29% of basal breast cancers, providing a potential mechanism for increased HHLA2 protein expression in breast cancer. Finally, Transmembrane and Immunoglobulin Domain Containing 2 (TMIGD2) was identified as one of the receptors for HHLA2.Conclusions: Wide expression of HHLA2 in human malignancies, together with its association with poor prognostic factors and its T-cell coinhibitory capability, suggests that the HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and an attractive target for human cancer therapy. Clin Cancer Res; 21(10); 2359–66. ©2014 AACR.See related commentary by Xiao and Freeman, p. 2201

Table S1. Expression of cell surface HHLA2 protein in various human cells lines determined by flow cytometry.

AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an “enabler” phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population.Significance:A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.

Next generation sequencing of genomic DNA isolated from Akt3/+S472 and Akt3/-S472 knockout 3475 tumors

Supplementary Methods

CRISPR mediated knockout of Akt3/-S472 in MDA-MB-231 cells increases mammary tumorigenesis

The Akt pathway is a well-known promoter of tumor malignancy. Akt3 is expressed as two alternatively spliced variants, one of which lacks the key regulatory serine 472 phosphorylation site. Whereas the function of full-length Akt3 isoform (Akt3/+S472) is well-characterized, that of Akt3/−S472 isoform remains unknown. Despite being expressed at a substantially lower level than Akt3/+S472 in triple-negative breast cancer cells, specific ablation of Akt3/−S472 enhanced, whereas overexpression, suppressed mammary tumor growth, consistent with a significant association with patient survival duration relative to Akt3/+S472. These effects were due to striking induction of apoptosis, which was mediated by Bim upregulation, leading to conformational activation of Bax and caspase-3 processing. Bim accumulation was caused by marked endocytosis of EGF receptors with concomitant ERK attenuation, which stabilizes BIM. These findings demonstrate an unexpected function of an endogenously expressed Akt isoform in promoting, as opposed to suppressing, apoptosis, underscoring that Akt isoforms may exert dissonant functions in malignancy.Significance: These results illuminate an unexpected function for an endogenously expressed Akt isoform in promoting apoptosis, underscoring the likelihood that different Akt isoforms exert distinct functions in human cancer. Cancer Res; 78(1); 103–14. ©2017 AACR.

Context.— Breast carcinoma grade, as determined by the Nottingham Grading System (NGS), is an important criterion for determining prognosis. The NGS is based on 3 parameters: tubule formation (TF), nuclear pleomorphism (NP), and mitotic count (MC). The advent of digital pathology and artificial intelligence (AI) have increased interest in virtual microscopy using digital whole slide imaging (WSI) more broadly. Objective.— To compare concordance in breast carcinoma grading between AI and a multi-institutional group of breast pathologists using digital WSI. Design.— We have developed an automated NGS framework using deep learning. Six pathologists and AI independently reviewed a digitally scanned slide from 137 invasive carcinomas and assigned a grade based on scoring of the TF, NP, and MC. Results.— Interobserver agreement for the pathologists and AI for overall grade was moderate (κ = 0.471). Agreement was good (κ = 0.681), moderate (κ = 0.442), and fair (κ = 0.368) for grades 1, 3, and 2, respectively. Observer pair concordance for AI and individual pathologists ranged from fair to good (κ = 0.313–0.606). Perfect agreement was observed in 25 cases (27.4%). Interobserver agreement for the individual components was best for TF (κ = 0.471 each) followed by NP (κ = 0.342) and was worst for MC (κ = 0.233). There were no observed differences in concordance amongst pathologists alone versus pathologists + AI. Conclusions.— Ours is the first study comparing concordance in breast carcinoma grading between a multi-institutional group of pathologists using virtual microscopy to a newly developed WSI AI methodology. Using explainable methods, AI demonstrated similar concordance to pathologists alone.

Neaodjuvant chemotherapy is used to treat high risk triple-negative breast cancer (TNBC). Residual cancer burden (RCB) is used to predict risk of relapse after neoadjuvant chemotherapy (NAC); however, it cannot predict disease recurrence with certainty. EZH2 is a targetable oncogenic protein overexpressed in TNBC and associated with metastasis and stem cell expansion. We quantified EZH2 protein expression in TNBC before NAC to examine potential utility as a predictive and prognostic biomarker.We retrospectively identified 63 patients with localized TNBC treated with NAC. We quantified EZH2 nuclear expression in pretherapy biopsies using a score which included intensity and percent of positive cells at each intensity. EZH2 expression was evaluated as a continuous variable and dichotomized at a score of 210. Logistic regression analysis was used to determine association between EZH2 expression and RCB, tumor-infiltrating lymphocytes, clinicopathologic features and disease-free survival.There was no significant association between EZH2 score and posttreatment RCB class evaluated as a continuous variable (P=0.831) or dichotomized at 210 (P=0.546). On multivariable logistic regression, adjusted for covariates including RCB, EZH2210 was associated with development of metastasis (odds ratio=14.35, 95% confidence interval: 2.69-76.66; P=0.002). Logistic regression was run with EZH2 scores as a continuous variable and increased EZH2 score was associated with metastasis (odds ratio=1.10, 95% confidence interval: 1.00-1.03; P=0.047).In our study of TNBC treated with NAC, high EZH2 expression in pretherapy core biopsies was significantly associated with metastatic recurrence independent of RCB. The potential value of EZH2 as a biomarker to improve stratification of outcome after NAC should be explored further.

Background: The incidence of breast cancer (BC) brain metastases (BM) is increasing as a result of both improved diagnostic techniques and longer survival due to better treatment approaches. However, the biological complexity of BCBMs is still poorly understood. We here evaluate the genomic profile of BCBMs and assess its prognostic implications. Methods: Clinical data and BM samples (FFPE) from BC patients undergoing neurosurgery (2003-2019) at three institutions were collected. Hormone receptor (HR) and HER2 status were evaluated on the BCBM. RNA extracted from BM samples was used to measure the expression of 758 BC–related genes and 18 housekeeping genes using the Breast Cancer 360 Panel on an nCounter platform (NanoString Technologies). Intrinsic molecular subtyping was determined using the previously reported PAM50 subtype predictor (Parker et al. JCO 2009). Median overall survival from neurosurgery (OS) was calculated using the Kaplan-Meier method. The correlations between expression of each gene/PAM50 signature, BC subtype and OS were studied using univariate and multivariate Cox-models. Results: Sixty-five BCBM samples were analyzed: 32% (N=21) were HR+/HER2-, 38% (N=25) HER2+ and 29% (N=19) HR-/HER2-. With a median follow-up of 33 months, no clinical variable was significantly associated with OS, despite a trend towards a shorter survival for patients with HR-/HER2- BMs, as compared to patients with HR+/HER2- and HER2+ subtypes (median OS 9.4 versus 22.1 and 20.0 months, respectively, log-rank p=NS). The intrinsic subtype distribution, as assessed by gene-expression profiling, was 37% Basal-like, 46% HER2-enriched (HER2-E), 15% Luminal B and 2% Normal-like. Non-luminal subtypes (basal-like and HER2-E) were extensively represented, both overall and in each BC subtype (52% in HR+/HER2- subgroup, 96% in HER2+ subgroup, see Table). The PAM50 basal-like signature was significantly associated with a worse OS (HR 2.7, 95% CI 1.0-7.2, p=0.045), even after correcting for BC subtype (HR 5.2, 95% CI 1.1-23.4, p=0.032). In fact, even within the subgroup of HR+/HER2- BCBMs, the PAM50 basal-like signature was strongly associated with a worse OS (HR 92.6, 95% CI 5.0-1860.1, p=0.003) and patients with basal-like HR+/HER2- BCBMs presented a median OS similar to patients with HR-/HER2- BCBMs (mOS 9.0 vs 9.4 months). We identified 36 genes whose high expression was significantly associated with a worse OS (p< 0.05) and one gene (LINC02381) whose high expression was significantly associated with better OS (p< 0.05); for 33 of these genes (BCL11A, BMP2, BNIP3, CAV1, CDH3, CDK6, CKB, CRYAB, CXCL12, EGFR, EYA4, FOXC1, FZD8, FZD9, GABRP, GAS1, GDF5, GPC4, IL6, KRT17, KRT5, KRT6B, KRT7, LAMB3, LINC02381, MYC, NOTCH1, PRKX, PSAT1, RUNX3, SNAI1, SPRY2, TTYH1), the association was confirmed even after correcting for BC subtype (p< 0.05). Conclusions: Non-luminal intrinsic subtypes are extensively represented in resected BCBMs, even if clinically classified as HR+/HER2-. Our data suggest that basal-like genomic features might be enriched in BCBMs and might be associated with worse survival. Distribution of PAM50 intrisic subtyping on the 65 brain metastases evaluated according to hormone receptor (HR) and HER2 status Citation Format: Gaia Griguolo, Maria Vittoria Dieci, Susan Fineberg, claudia Pinato, Michele Bottosso, Luc Bauchet, Federica Miglietta, Jack Jacob, Giovanni Zarrilli, Valérie Rigau, Maria Cristina Guarascio, Francesca Zanconato, Francesca Schiavi, Matteo Fassan, William Jacot, Stefano Piccolo, Amelie Darlix, Valentina Guarneri. Gene Expression Profiling of Breast Cancer Brain Metastasis shows enrichment for non-luminal subtypes with potential prognostic implications [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD7-08.

International audience; Abstract Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error ( p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections ( p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.; v2.5, 25 March 2021.

Babies born to severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) infected mothers are at greater risk for perinatal morbidity and more likely to receive a neurodevelopmental diagnosis in the first year of life. However, the effect of maternal infection on placental function and neonatal outcomes varies depending upon the patient population. We set out to test our hypothesis that maternal SARS-CoV-2 infection in our underserved, socioeconomically disadvantaged, predominantly African American and Latina population in the Bronx, NY would have effects evident at birth. Fifty-five SARS-CoV-2 positive and 61 negative third trimester patients were randomly selected from Montefiore Medical Center (MMC), Bronx, NY. In addition, two positive cases from Yale New Haven Hospital, CT were included as controls. All 55 placentas delivered by SARS-CoV-2 positive mothers were uninfected by the virus, based on immunohistochemistry, in-situ hybridization, and qPCR analysis. However, placental villous infarcts, mild preeclampsia, shortened gestational periods and lower Apgar scores were observed in the infected cases. These findings suggest that even without entering the placenta, SARS-CoV-2 can affect various systemic pathways culminating in altered placental development and function, which may adversely affect the fetus, especially in a high-risk patient population such as ours. These results underline the importance of vaccination among pregnant women, particularly in low resource areas.

Breast carcinoma grading is an important prognostic feature recently incorporated into the AJCC Cancer Staging Manual. There is increased interest in applying virtual microscopy (VM) using digital whole slide imaging (WSI) more broadly. Little is known regarding concordance in grading using VM and how such variability might affect AJCC prognostic staging (PS). We evaluated interobserver variability amongst a multi-institutional group of breast pathologists using digital WSI and how discrepancies in grading would affect PS. A digitally scanned slide from 143 invasive carcinomas was independently reviewed by 6 pathologists and assigned grades based on established criteria for tubule formation (TF), nuclear pleomorphism (NP), and mitotic count (MC). Statistical analysis was performed. Interobserver agreement for grade was moderate (κ = 0.497). Agreement was fair (κ = 0.375), moderate (κ = 0.491), and good (κ = 0.705) for grades 2, 3, and 1, respectively. Observer pair concordance ranged from fair to good (κ = 0.354-0.684) Perfect agreement was observed in 43 cases (30%). Interobserver agreement for the individual components was best for TF (κ = 0.503) and worst for MC (κ = 0.281). Seventeen of 86 (19.8%) discrepant cases would have resulted in changes in PS and discrepancies most frequently resulted in a PS change from IA to IB (n = 9). For two of these nine cases, Oncotype DX results would have led to a PS of 1A regardless of grade. Using VM, a multi-institutional cohort of pathologists showed moderate concordance for breast cancer grading, similar to studies using light microscopy. Agreement was the best at the extremes of grade and for evaluation of TF. Whether the higher variability noted for MC is a consequence of VM grading warrants further investigation. Discordance in grading infrequently leads to clinically meaningful changes in the prognostic stage.

Background Despite potential clinical implications, the complexity of breast cancer (BC) brain metastases (BM) immune microenvironment is poorly understood. Through multiplex immunofluorescence, we here describe the main features of BCBM immune microenvironment (density and spatial distribution) and evaluate its prognostic impact. Methods Sixty BCBM from patients undergoing neurosurgery at three institutions (2003-2018) were comprehensively assessed using two multiplex immunofluorescence panels (CD4, CD8, Granzyme B, FoxP3, CD68, pan-cytokeratin, DAPI; CD3, PD-1, PD-L1, LAG-3, TIM-3, CD163, pan-cytokeratin, DAPI). The prognostic impact of immune subpopulations and cell-to-cell spatial interactions was evaluated. Results Subtype-related differences in BCBM immune microenvironment and its prognostic impact were observed. While in HR−/HER2− BM and HER2+ BM, higher densities of intra-tumoral CD8+ lymphocytes were associated with significantly longer OS (HR 0.16 and 0.20, respectively), in HR+/HER2− BCBMs a higher CD4+FoxP3+/CD8+ cell ratio in the stroma was associated with worse OS (HR 5.4). Moreover, a higher density of intra-tumoral CD163+ M2-polarized microglia/macrophages in BCBMs was significantly associated with worse OS in HR−/HER2− and HR+/HER2− BCBMs (HR 6.56 and 4.68, respectively), but not in HER2+ BCBMs. In HER2+ BCBMs, multiplex immunofluorescence highlighted a negative prognostic role of PD-1/PD-L1 interaction: patients with a higher percentage of PD-L1+ cells spatially interacting with (within a 20 µm radius) PD-1+ cells presented a significantly worse OS (HR 4.60). Conclusions Our results highlight subtype-related differences in BCBM immune microenvironment and identify two potential therapeutic targets, M2 microglia/macrophage polarization in HER2− and PD-1/PD-L1 interaction in HER2+ BCBMs, which warrant future exploration in clinical trials.

Neoadjuvant endocrine therapy (NET) can be used to treat estrogen receptor positive (ER+) invasive breast cancer (IBC). Tumors with Ki67gt;10% after 2 to 4 weeks of NET are considered resistant to endocrine therapy. Enhancer of Zeste Homolog 2 (EZH2) is a targetable oncoprotein and overexpression in ER+ IBC has been linked to resistance to endocrine therapy. We examined whether EZH2 expression levels in ER+ IBC could be used to predict response to NET.We retrospectively identified 46 patients with localized ER+ HER2/neu negative IBC treated with a minimum of 4 weeks of NET. We quantified EZH2 nuclear expression in pretherapy core biopsies using a score that included intensity and percent of cells staining. Ki67 was evaluated in both pretherapy core biopsies and posttherapy tumor resections and scored according to the guidelines of the International Ki67 Working Groups, with a global weighted score. Ki67≤10% after NET was considered endocrine responsive. Logistic regression analysis was performed to determine the association between EZH2 expression and response to NET.We found significant associations of tumor grade ( P =0.011), pretherapy Ki67 ( P =0.003), and EZH2 ( Plt;0.001), with response to NET. On logistic regression adjusted for tumor grade and pretherapy Ki67, increased EZH2 scores were associated with decreased odds of endocrine responsiveness, defined as posttreatment Ki67≤10% (odds ratio=0.976, 95% CI, 0.956 to 0.997; P =0.026). In addition, with EZH2 score in the model, associations of tumor grade and pretreatment Ki67 with posttreatment Ki67≤10% response to NET became not significant.Our results suggest that EZH2 might be a useful biomarker to predict response to NET.

AIMS Tumour-infiltrating lymphocytes (TILs) are prognostic in invasive breast cancer; however, their prognostic significance in ductal carcinoma in situ (DCIS) has not been established. The Oncotype DX (ODX) Breast DCIS Score test is a genomic assay used to predict the local recurrence risk. The aims of this study were to quantify TILs in DCIS by the use of three methodologies, and correlate them with the ODX DCIS Score. METHODS AND RESULTS We studied 97 DCIS cases, all with an ODX DCIS Score. Cases with a low ODX DCIS Score were considered as one group, and those with an intermediate/high ODX Score were considered together. TILs were quantified on haematoxylin and eosin-stained slides. The methodologies used to quantify TILS included assessment of stromal TILs, assessment of touching TILs, and assessment of circumferential TILS. In cases with >5% stromal TILS, the percentage of stromal TILS was considered to be high. In cases with a mean number of more than five touching TILs per DCIS duct, TILs were considered to be present. The ODX DCIS Score was intermediate/high in 27 (28%) cases and low in 70 (72%) cases. There were >5% stromal TILs in 33 (34%) cases, and more than five touching TILs per DCIS duct in 15 (15%) cases; circumferential TILs were present in nine (9%) cases. In univariate analysis, a low ODX DCIS Score showed significant associations with absent touching TILS (P = 0.027), stromal TILs

Background: Ductal carcinoma in situ (DCIS) is considered a non-obligate precursor of invasive breast cancer (IBC). It is estimated that 5-14% of women diagnosed with DCIS experience an ipsilateral IBC. Current clinical criteria do not discriminate well between women with DCIS who will and those who will not develop ipsilateral IBC or a DCIS recurrence. The Oncotype DX DCIS assay (12 genes) was derived from a multigene expression array (Oncotype DX, 21 genes), and includes 5 proliferation genes, PGR, GSTM1, and 5 reference genes. This assay has been shown to predict risk of subsequent ipsilateral IBC and DCIS, and is used by clinicians to evaluate both the risk of local recurrence and need for radiation therapy. Since abnormal expression of microRNAs (miRNAs) is thought to contribute to the development of IBC, and that very few studies have evaluated miRNA expression deregulation in DCIS lesions, we sought to determine if specific miRNA expression changes may help identify DCIS lesions with different Oncotype DX DCIS scores. Methods: For this study, we selected archived specimens from 52 women diagnosed with DCIS between 2012 and 2018, whose DCIS lesions were tested with the Oncotype DX DCIS assay (low (n=35), intermediate (n=11), high (n=6) risk scores). Total RNA was extracted by macrodissection of the DCIS lesions. MiRNA expression differences between DCIS lesions were evaluated using small-RNA next-generation sequencing. Quantitative PCR validations were performed using Taqman miRNA assays on the top 15 differentially expressed miRNAs Results: Small-RNA expression data obtained by next-generation sequencing of 43 DCIS RNA specimens identified miRNAs differentially expressed between the risk score groups. Fifteen differentially expressed miRNAs were selected for their association based on Oncotype DX DCIS score differences (miR-142-3p, miR-142-5p, miR-744, miR-155, miR-30c, miR-150, miR-146a, miR-190b, miR-135b, miR-193b) or on age of the patients (miR-19b, miR-19a, miR-135a, miR-132). QPCR validation of these 15 miRNAs, using RNA from 30 DCIS lesions with known Oncotypes DX DCIS scores, of which 21 were samples that had been sequenced (low (n=8), intermediate (n=9), and high (n=4)), and 9 were not sequenced (low (n=7), intermediate (n=1), high (n=1)) and identified 5 miRNAs (miR-135a (p= Citation Format: Olivier Loudig, Iddo Ben-Dov, Christina Liu, Miglena Komforti, Susan Fineberg. Ductal carcinoma in situ (DCIS) of the breast: miRNA deregulation correlates with the Oncotype DX risk score [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 P6-10-20.

Neoadjuvant chemotherapy is increasingly being used to treat primary invasive breast carcinoma. Response to neoadjuvant chemotherapy is an important determinant of prognosis. A multidisciplinary group published recommendations for standardization of pathologic reporting of postneoadjuvant chemotherapy specimens. Based on these recommendations, we sent a survey to 26 pathologists currently practicing breast pathology in academic centers across the United States. The survey consisted of six questions with yes/no answers. The pathologists were encouraged to add comments. We received responses from 23 breast pathologists from 19 centers. The questions and responses were as follows: 1. Do you grade tumors after neoadjuvant chemotherapy?-17 (74%) responded yes and 6 (26%) responded no. 2. Do you routinely repeat hormone receptors, HER2/Neu results after neoadjuvant chemotherapy?-15 (65%) responded yes and 8 (35%) responded no. 3. If there are features of tumor regression/tumor bed at the margin but no actual tumor at the margin do you report this?-11 (48%) responded yes and 8 (35%) responded no and 4 (17%) reported a variable practice. 4. Do you report number of nodes with fibrosis/changes of regression?-17 (74%) responded yes and 6 (26%) responded no. 5. Do you report residual cancer burden score on your report or at least provide information on your report so clinicians can calculate residual cancer burden?-17 (74%) responded yes and 6 (26%) responded no. 6. Do you have a specific synoptic for cases after neoadjuvant chemotherapy?-5 (22%) responded yes and 18 (78%) responded no. The major reasons provided for nonadherence to recommended guidelines included pathologists were unaware of prognostic importance of providing the information, reporting practices were clinician driven and some pathologists were unaware of the recommendation. We document that academic breast pathology practices show significant variability in reporting of postneoadjuvant chemotherapy cases. We document barriers to standard practice and provide recommendations we hope will contribute to a more uniform reporting practice for these complex specimens.

AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an “enabler” phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population. Significance: A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.

Aims: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. Methods and results: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot-spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799–0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot-spot method (0.83; 95% CI = 0.74–0.90) did not. Visually, interobserver concordance in location of selected hot-spots varies between cases. The median times for scoring were 9 and 6 min for global and hot-spot methods, respectively. Conclusions: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further.

Background A ductal carcinoma in situ (DCIS) Nomogram integrating 10 clinicopathologic/treatment factors and a Refined DCIS Score (RDS) that incorporates a genomic assay and three clinicopathologic factors (Oncotype DX DCIS Score) are available to estimate DCIS 10-year local recurrence risk (LRR). This study compared these estimates. Methods Patients 50 years of age or older with DCIS size 2.5 cm or smaller and a genomic assay available were identified. An RDS within 1-2% of the range of Nomogram LRR estimates obtained by assuming use and non-use of endocrine therapy (Nomogram ± ET) was defined as concordant. Assuming a 10-year risk threshold of 10% for recommending radiation, Nomogram ± ET and RDS estimates were compared, and threshold concordance was determined. Results For 54 (92%) of 59 patients, the RDS and Nomogram ± ET LRR estimates were concordant. For the remaining 5 (8%) of the 59 patients, the RDS LRR estimates were lower than the Nomogram + ET estimates, with an absolute difference of 3-8%, and thus were discordant. For these five patients, the RDS estimates of 10-year LRR were lower than 10% (range 5-8%) and the Nomogram + ET estimates were 10% or higher (range 11-14%). These five patients with both discordant and threshold-discordant estimates all had close margins (≤ 2 mm). Conclusions Among 92% of women 50 years of age or older with DCIS size 2.5 cm or smaller, free-of-charge online Nomogram 10-year LRR estimates were concordant with those obtained using the commercially available RDS (> $4600). Among the 8% with discordant risk estimates, the RDS appeared to underestimate the LRR and may lead to inappropriate omission of radiotherapy. Unless other data show a clinically significant advantage of the RDS (Oncotype DX DCIS Score), the study data suggest that for women 50 years of age or older with DCIS size 2.5 cm or smaller, its use is not warranted.

Ki67 immunohistochemistry (IHC), commonly used as a proliferation marker in breast cancer, has limited value for treatment decisions due to questionable analytical validity. The International Ki67 in Breast Cancer Working Group (IKWG) consensus meeting, held in October 2019, assessed the current evidence for Ki67 IHC analytical validity and clinical utility in breast cancer, including the series of scoring studies the IKWG conducted on centrally stained tissues. Consensus observations and recommendations are: 1) as for estrogen receptor and HER2 testing, preanalytical handling considerations are critical; 2) a standardized visual scoring method has been established and is recommended for adoption; 3) participation in and evaluation of quality assurance and quality control programs is recommended to maintain analytical validity; and 4) the IKWG accepted that Ki67 IHC as a prognostic marker in breast cancer has clinical validity but concluded that clinical utility is evident only for prognosis estimation in anatomically favorable estrogen receptor–positive and HER2-negative patients to identify those who do not need adjuvant chemotherapy. In this T1-2, N0-1 patient group, the IKWG consensus is that Ki67 5% or less, or 30% or more, can be used to estimate prognosis. In conclusion, analytical validity of Ki67 IHC can be reached with careful attention to preanalytical issues and calibrated standardized visual scoring. Currently, clinical utility of Ki67 IHC in breast cancer care remains limited to prognosis assessment in stage I or II breast cancer. Further development of automated scoring might help to overcome some current limitations.

Background The effects of diet in cancer, in general, and breast cancer in particular, are not well understood. Insulin inhibition in ketogenic, high fat diets, modulate downstream signaling molecules and are postulated to have therapeutic benefits. Obesity and diabetes have been associated with higher incidence of breast cancer. Addition of anti-cancer drugs together with diet is also not well studied. Methods Two diets, one ketogenic, the other standard mouse chow, were tested in a spontaneous breast cancer model in 34 mice. Subgroups of 3–9 mice were assigned, in which the diet were implemented either with or without added rapamycin, an mTOR inhibitor and potential anti-cancer drug. Results Blood glucose and insulin concentrations in mice ingesting the ketogenic diet (KD) were significantly lower, whereas beta hydroxybutyrate (BHB) levels were significantly higher, respectively, than in mice on the standard diet (SD). Growth of primary breast tumors and lung metastases were inhibited, and lifespans were longer in the KD mice compared to mice on the SD (p Conclusions The study provides proof of principle that a ketogenic diet a) results in serum insulin reduction and ketosis in a spontaneous breast cancer mouse model; b) can serve as a therapeutic anti-cancer agent; and c) can enhance the effects of rapamycin, an anti-cancer drug, permitting dose reduction for comparable effect. Further, the ketogenic diet in this model produces superior cancer control than standard mouse chow whether with or without added rapamycin.

Introduction Pleomorphic lobular carcinoma in situ (PLCIS) is a variant of LCIS with high-grade morphologic features. The number of case series studying PLCIS is limited, and clinical management of patients with PLCIS is controversial. We report a breast core biopsy (BCBx) series of PLCIS. Materials and Methods We reviewed 37 cases of PLCIS with or without microinvasion diagnosed by BCBx. PLCIS was defined as dyscohesive cells showing acinar expansion and loss of immunohistochemical membranous expression of e-cadherin or beta-catenin with nuclear pleomorphism with at least 2- to 3-fold variation in nuclear size, membrane irregularities, and variably prominent nucleoli. Clinical information and findings on excision were evaluated. Results Thirty-one (84%) patients presented with mammographic calcifications, 4 (11%) presented with ultrasound findings, 1 (3%) presented with magnetic resonance imaging enhancement, and 1 (3%) with combined imaging abnormality. The mean patient age was 62.3 years. Nineteen patients (51%) had a family history of breast cancer. Microinvasion was present on BCBx in 9 (24%) of the 37 patients. Excision, available in 34 patients, demonstrated invasive carcinoma in 24 (65%), which was multifocal in 11 (46%). Twenty-three patients with PLCIS without microinvasion on BCBx, and without known history of ipsilateral invasive cancer, underwent excision; 14 of these patients demonstrated invasive carcinoma, representing an upgrade to invasive carcinoma of 60%. Conclusion We report the largest BCBx series of PLCIS and confirm its aggressive biology and frequent association with multifocal invasive lobular carcinoma, as well as frequent presentation in patients with a family history of breast cancer. Our results support excision to negative margins.

AIMS Neoadjuvant chemotherapy (NAC) is often used to treat localised invasive breast cancer. Invasive mucinous carcinoma (IMC) is considered to be an indolent form of invasive breast cancer, and is rarely treated with NAC. We report the largest series of IMCs treated with NAC, and report a characteristic, but not well recognised, pattern of pathological response. METHODS AND RESULTS Our series included seven patients with IMC treated with NAC. Three patients presented with locally advanced disease, three patients had tumours that were HER-2/neu-positive, and four patients had tumours with admixed mucinous and micropapillary features. Clinical and imaging assessment of response showed persistent and, in some cases, progressive disease, despite evidence of significant pathological response in these cases. Pathological assessment after NAC demonstrated marked reduction in tumour cellularity, but persistent space-occupying mucin pools, showing acellular mucin in one case

The Akt pathway is a well-known promoter of tumor malignancy. Akt3 is expressed as two alternatively spliced variants, one of which lacks the key regulatory serine 472 phosphorylation site. Whereas the function of full-length Akt3 isoform (Akt3/+S472) is well-characterized, that of Akt3/−S472 isoform remains unknown. Despite being expressed at a substantially lower level than Akt3/+S472 in triple-negative breast cancer cells, specific ablation of Akt3/−S472 enhanced, whereas overexpression, suppressed mammary tumor growth, consistent with a significant association with patient survival duration relative to Akt3/+S472. These effects were due to striking induction of apoptosis, which was mediated by Bim upregulation, leading to conformational activation of Bax and caspase-3 processing. Bim accumulation was caused by marked endocytosis of EGF receptors with concomitant ERK attenuation, which stabilizes BIM. These findings demonstrate an unexpected function of an endogenously expressed Akt isoform in promoting, as opposed to suppressing, apoptosis, underscoring that Akt isoforms may exert dissonant functions in malignancy. Significance: These results illuminate an unexpected function for an endogenously expressed Akt isoform in promoting apoptosis, underscoring the likelihood that different Akt isoforms exert distinct functions in human cancer. Cancer Res; 78(1); 103–14. ©2017 AACR.

2021 Background: Despite potential clinical implications, the complexity of immune microenvironment in breast cancer (BC) brain metastases (BM) is still poorly understood. Multiplex immunofluorescence (mIF) allows simultaneous visualization of several IF labeled proteins while maintaining spatial information. This novel technique can be used to comprehensively describe BCBM immune microenvironment, potentially providing useful information to guide novel therapeutic approaches. Methods: Clinical data and archival BM samples from 60 BC patients undergoing neurosurgery (2003-2018) at three institutions were collected. BCBMs were characterized using a custom mIF panel, including immune checkpoint and co-inhibitory molecules (CD3, PD1, PD-L1, TIM3, LAG3, CD163) and localization (keratin for tumor recognition) markers. Mean marker density was determined by digital image analysis (positive cells/mm2) and classified in tumor and stroma areas. Associations between immune marker densities, BC subtype and overall survival from BM diagnosis (OS) were studied. Results: Sixty BCBM samples were analyzed; 32% HR+/HER2-, 38% HER2+, 30% HR-/HER2-. At a median follow-up of 43 months, the only clinical variable associated with OS was BC subtype (shortest for HR-/HER2- and longest for HER2+, p=0.02). In the total sample area and tumor area, no significant difference in marker density was observed according to BC subtype. In the stroma area, a significant difference in TIM3+ cell density was observed according to BC subtype (highest density in HR+/HER2- and lowest density in HER2+ tumors, Kruskal-Wallis p=0.017). Higher CD163 density (a marker of M2 macrophage polarization), both in the tumor and in the stroma area, was significantly associated with worse OS, even after correction by BC subtype. In the subgroup of patients with HR+/HER2- BCBM, high TIM3+ cell density in the stroma area was significantly associated with longer OS (median OS 54.1 versus 23 months respectively for TIM3+ density above and below median value; p=0.01). Conclusions: In BCBM, stromal TIM3+ immune infiltrate differs according to BC subtype. M2 macrophage polarization is consistently associated with worse OS across all BC subtypes and might represent a potential therapeutic target for these patients.[Table: see text]

2526 Background: Despite clinical implications, the complexity of brain metastases (BM) immune microenvironment in breast cancer (BC) patients is poorly understood. Multiplex immunofluorescence (mIF), a novel imaging technique allowing simultaneous visualization and quantification of several IF labeled proteins while maintaining spatial information, holds promise to comprehensively describe BCBM immune microenvironment, potentially providing valuable information to improve treatment. Methods: Clinical data and archival BM samples were collected for 60 BC patients undergoing neurosurgery (2003-2018) at three institutions. BM immune contexture was characterized using a custom mIF panel, including cell subtyping (CD4, CD8, FOXP3, CD68), activation (Granzyme B) and localization (keratin for tumor recognition) markers. Mean immune cell density (cells/mm2) for each sample was determined by digital image analysis and classified in tumor and stroma areas. Associations between immune subpopulations, BC subtype and overall survival from BM diagnosis (OS) were studied. Results: Up to date, 30 BCBM samples have been analyzed; 33% HR+/HER2-, 20% HR-/HER2+, 10% HR+/HER2+, 37% HR-/HER2-. At a median follow-up of 46 months, BC subtype was the only clinical variable associated with OS (longest for HER2+ and shortest for HR-/HER2-, log-rank p = 0.002). In the total sample area, no significant difference in immune cell densities was observed according to BC subtype. In the tumor area, HR+/HER2- tumors showed higher densities of CD8+ and CD68+ cells compared to other subtypes (p = 0.036 and p = 0.016, respectively). In stroma, HR-/HER2- tumors presented numerically higher densities of CD4+ and FOXP3+ cells and higher ratio of CD4/CD8 and FOXP3/CD8 ratio (not statistically significant). Higher CD4/CD8 and FOXP3/CD8 ratio in the stroma was significantly associated with worse OS, even after correction by BC subtype (Table). Conclusions: In BCBM, immune infiltrate differs according to BC subtype. Preliminary results suggest that a more tolerogenic immune microenvironment is associated with worse OS and might represent a target for optimization of immunotherapy for these patients. Updated results for all 60 patients will be presented. [Table: see text]

The Oncotype DX Breast Cancer Assay for ductal carcinoma in situ is used to determine local recurrence risk in patients with ductal carcinoma in situ. The results help select patients with low-risk ductal carcinoma in situ who could forgo radiation therapy after conservative surgery. The genes assessed include five proliferation genes, progesterone receptor (PR), and GSTM-1. Our objective was to determine if PR, mitotic counting, or any other pathologic feature of ductal carcinoma in situ could predict the Oncotype DX DCIS Score. We identified 46 cases of ductal carcinoma in situ with a Oncotype DX DCIS Score. In addition to information obtained from routine pathology, we counted mitotic figures in the ductal carcinoma in situ and noted presence of dense chronic inflammatory infiltrate surrounding ductal carcinoma in situ. We found that PR ≥ 90% (P = 0.004), mitotic count ≤ 1 (P = 0.045), estrogen receptor ≥ 90% (P = 0.046), and low nuclear grade (P0.0001) were associated with a low score. Dense chronic inflammation surrounding ductal carcinoma in situ was associated with a high score (P = 0.034).All 13 cases with PR ≥ 90%, ≤ 1 mitotic figure and absence of dense chronic inflammation around ductal carcinoma in situ had a low score (100% specificity). A low score was not observed in any case with at least two of the following--negative PR,1 mitotic figure, and/or presence of dense chronic inflammation around ductal carcinoma in situ (100% specificity). Our study suggests using a combination of PR (≥ 90% vs negative) with mitotic count in ductal carcinoma in situ (≤ 1 vs1) and dense chronic inflammation around ductal carcinoma in situ one could predict the Oncotype DX DCIS score. Mitotic counting and evaluation of immune response might provide prognostic information in ductal carcinoma in situ.