The Role of ESRP1 in the Regulation of PHGDH in Estrogen Receptor-Positive Breast Cancer.

Resistance to hormone therapy leads to a recurrence of estrogen receptor-positive breast cancer. We have demonstrated that the epithelial splicing regulatory protein 1 (ESRP1) significantly affects cell/tumor growth and metabolism and is associated with a poor prognosis in this breast cancer subtype. In this study, we aimed to investigate the ESRP1 protein-messenger RNA (mRNA) interaction in hormone therapy-resistant breast cancer. RNA-binding protein immunoprecipitation (RIP) followed by Clariom D (Applied Biosystems/Thermo Fisher Scientific) transcriptomics microarray (RIP-Chip) was performed to identify mRNA-binding partners of ESRP1. The integration of RIP-Chip and immunoprecipitation-mass spectrometry analyses identified phosphoglycerate dehydrogenase (PHGDH), a key metabolic enzyme, as a binding partner of ESRP1 in hormone-resistant breast cancer. Bioinformatic analysis showed ESRP1 binding to the 5' untranslated region of PHGDH. RNA electrophoresis mobility shift assay and RIP-quantitative reverse transcription-polymerase chain reaction further validated the ESRP1-PHGDH binding. In addition, knockdown of ESRP1 decreased PHGDH mRNA stability significantly, suggesting the posttranscriptional regulation of PHGDH by ESRP1. The presence or absence of ESRP1 levels significantly affected the stability in tamoxifen-resistant LCC2 and fulvestrant-resistant LCC9 cells. PHGDH knockdown in tamoxifen-resistant cells further reduced the oxygen consumption rate (ranging from P = .005 and P = .02), mimicking the effects of ESRP1 knockdown. Glycolytic parameters were also altered (ranging P = .001 and P = .005). ESRP1 levels did not affect the stability of PHGDH in T-47D cells, although knockdown of PHGDH affected the growth of these cells. In conclusion, to our knowledge, this study, for the first time, reports that ESRP1 binds to the 5' untranslated region of PHGDH, increasing its mRNA stability in hormone therapy-resistant estrogen receptor-positive breast cancer. These findings provide evidence for a novel mechanism of action of RNA-binding proteins such as ESRP1. These new insights could assist in developing novel strategies for the treatment of hormone therapy-resistant breast cancer.

Intraoperative assessment of axillary sentinel lymph nodes by telepathology.

PURPOSE: Although axillary dissection is no longer indicated for many breast cancer patients with 1-2 positive axillary sentinel lymph nodes (ASLN), intraoperative ASLN assessment is still performed in many institutions for patients undergoing mastectomy or neoadjuvant therapy. With recent advancements in digital pathology, pathologists increasingly evaluate ASLN via remote telepathology. We aimed to compare the performance characteristics of remote telepathology and conventional on-site intraoperative ASLN assessment. METHODS: Data from ASLN evaluation for breast cancer patients performed at two sites between April 2021 and October 2022 was collated. Remote telepathology consultation was conducted via the Aperio eSlideManager system. RESULTS: A total of 385 patients were identified during the study period (83 telepathology, 302 on-site evaluations). Although not statistically significant (P = 0.20), the overall discrepancy rate between intraoperative and final diagnoses was slightly higher at 9.6% (8/83) for telepathology compared with 5.3% (16/302) for on-site assessment. Further comparison of performance characteristics of ASLN assessment between telepathology and conventional on-site evaluation revealed no statistically significant differences between deferral rates, discrepancy rates, interpretive or sampling errors, major or minor disagreements, false negative or false positive results as well as clinical impact and turn-around time (P ≥ 0.12). CONCLUSION: ASLN assessment via telepathology is not significantly different from conventional on-site evaluation, although it shows a slightly higher overall discrepancy rate between intraoperative and final diagnoses (9.6% vs. 5.3%). Further studies are warranted to ensure accuracy of ASLN assessment via telepathology.

High Interobserver Variability Among Pathologists Using Combined Positive Score to Evaluate PD-L1 Expression in Gastric, Gastroesophageal Junction, and Esophageal Adenocarcinoma.

Reliable, reproducible methods to interpret programmed death ligand-1 (PD-L1) expression on tumor cells (TC) and immune cells (IC) are needed for pathologists to inform decisions associated with checkpoint inhibitor therapies. Our international study compared interpathologist agreement of PD-L1 expression using the combined positive score (CPS) under standardized conditions on samples from patients with gastric/gastroesophageal junction/esophageal adenocarcinoma. Tissue sections from 100 adenocarcinoma pretreatment biopsies were stained in a single laboratory using the PD-L1 immunohistochemistry 28-8 and 22C3 (Agilent) pharmDx immunohistochemical assays. PD-L1 CPS was evaluated by 12 pathologists on scanned whole slide images of these biopsies before and after a 2-hour CPS training session by Agilent. Additionally, pathologists determined PD-L1-positive TC, IC, and total viable TC on a single tissue fragment from 35 of 100 biopsy samples. Scoring agreement among pathologists was assessed using the intraclass correlation coefficient (ICC). Interobserver variability for CPS for 100 biopsies was high, with only fair agreement among pathologists both pre- (range, 0.45-0.55) and posttraining (range, 0.56-0.57) for both assays. For the 35 single biopsy samples, poor/fair agreement was also observed for the total number of viable TC (ICC, 0.09), number of PD-L1-positive IC (ICC, 0.19), number of PD-L1-positive TC (ICC, 0.54), and calculated CPS (ICC, 0.14), whereas calculated TC score (positive TC/total TC) showed excellent agreement (ICC, 0.82). Retrospective histologic review of samples with the poorest interpathologist agreement revealed the following as possible confounding factors: (1) ambiguous identification of positively staining stromal cells, (2) faint or variable intensity of staining, (3) difficulty in distinguishing membranous from cytoplasmic tumor staining, and (4) cautery and crush artifacts. These results emphasize the need for objective techniques to standardize the interpretation of PD-L1 expression when using the CPS methodology on gastric/gastroesophageal junction cancer biopsies to accurately identify patients most likely to benefit from immune checkpoint inhibitor therapy.

Clinical and Genomic Risk to Guide the Use of Adjuvant Therapy for Breast Cancer.

BACKGROUND: The use of adjuvant chemotherapy in patients with breast cancer may be guided by clinicopathological factors and a score based on a 21-gene assay to determine the risk of recurrence. Whether the level of clinical risk of breast cancer recurrence adds prognostic information to the recurrence score is not known. METHODS: We performed a prospective trial involving 9427 women with hormone-receptor-positive, human epidermal growth factor receptor 2-negative, axillary node-negative breast cancer, in whom an assay of 21 genes had been performed, and we classified the clinical risk of recurrence of breast cancer as low or high on the basis of the tumor size and histologic grade. The effect of clinical risk was evaluated by calculating hazard ratios for distant recurrence with the use of Cox proportional-hazards models. The initial endocrine therapy was tamoxifen alone in the majority of the premenopausal women who were 50 years of age or younger. RESULTS: The level of clinical risk was prognostic of distant recurrence in women with an intermediate 21-gene recurrence score of 11 to 25 (on a scale of 0 to 100, with higher scores indicating a worse prognosis or a greater potential benefit from chemotherapy) who were randomly assigned to endocrine therapy (hazard ratio for the comparison of high vs. low clinical risk, 2.73; 95% confidence interval [CI], 1.93 to 3.87) or to chemotherapy plus endocrine (chemoendocrine) therapy (hazard ratio, 2.41; 95% CI, 1.66 to 3.48) and in women with a high recurrence score (a score of 26 to 100), all of whom were assigned to chemoendocrine therapy (hazard ratio, 3.17; 95% CI, 1.94 to 5.19). Among women who were 50 years of age or younger who had received endocrine therapy alone, the estimated (±SE) rate of distant recurrence at 9 years was less than 5% (≤1.8±0.9%) with a low recurrence score (a score of 0 to 10), irrespective of clinical risk, and 4.7±1.0% with an intermediate recurrence score and low clinical risk. In this age group, the estimated distant recurrence at 9 years exceeded 10% among women with a high clinical risk and an intermediate recurrence score who received endocrine therapy alone (12.3±2.4%) and among those with a high recurrence score who received chemoendocrine therapy (15.2±3.3%). CONCLUSIONS: Clinical-risk stratification provided prognostic information that, when added to the 21-gene recurrence score, could be used to identify premenopausal women who could benefit from more effective therapy. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00310180.).

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

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.

Multi-protein spatial signatures in ductal carcinoma in situ (DCIS) of breast.

BACKGROUND: There is limited knowledge about DCIS cellular composition and relationship with breast cancer events (BCE). METHODS: Immunofluorescence multiplexing (MxIF) was used to image and quantify 32 cellular biomarkers in FFPE DCIS tissue microarrays. Over 75,000 DCIS cells from 51 patients (median 9 years follow-up for non-BCE cases) were analysed for profiles predictive of BCE. K-means clustering was used to evaluate cellular co-expression of epithelial markers with ER and HER2. RESULTS: Only ER, PR and HER2 significantly correlated with BCE. Cluster analysis identified 6 distinct cell groups with different levels of ER, Her2, cMET and SLC7A5. Clusters 1 and 3 were not significant. Clusters 2 and 4 (high ER/low HER2 and SLC7A5/mixed cMET) significantly correlated with low BCE risk (P = 0.001 and P = 0.034), while cluster 6 (high HER2/low ER, cMET and SLC7A5) correlated with increased risk (P = 0.018). Cluster 5 (similar to cluster 6, except high SLC7A5) trended towards significance (P = 0.072). A continuous expression score (Escore) based on these 4 clusters predicted likelihood of BCE (AUC = 0.79, log-rank test P = 5E-05; LOOCV AUC = 0.74, log-rank test P = 0.006). CONCLUSION: Multiplexed spatial analysis of limited tissue is a novel method for biomarker analysis and predicting BCEs. Further validation of Escore is needed in a larger cohort.

Adjuvant Chemotherapy Guided by a 21-Gene Expression Assay in Breast Cancer.

BACKGROUND: The recurrence score based on the 21-gene breast cancer assay predicts chemotherapy benefit if it is high and a low risk of recurrence in the absence of chemotherapy if it is low; however, there is uncertainty about the benefit of chemotherapy for most patients, who have a midrange score. METHODS: We performed a prospective trial involving 10,273 women with hormone-receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative, axillary node-negative breast cancer. Of the 9719 eligible patients with follow-up information, 6711 (69%) had a midrange recurrence score of 11 to 25 and were randomly assigned to receive either chemoendocrine therapy or endocrine therapy alone. The trial was designed to show noninferiority of endocrine therapy alone for invasive disease-free survival (defined as freedom from invasive disease recurrence, second primary cancer, or death). RESULTS: Endocrine therapy was noninferior to chemoendocrine therapy in the analysis of invasive disease-free survival (hazard ratio for invasive disease recurrence, second primary cancer, or death [endocrine vs. chemoendocrine therapy], 1.08; 95% confidence interval, 0.94 to 1.24; P=0.26). At 9 years, the two treatment groups had similar rates of invasive disease-free survival (83.3% in the endocrine-therapy group and 84.3% in the chemoendocrine-therapy group), freedom from disease recurrence at a distant site (94.5% and 95.0%) or at a distant or local-regional site (92.2% and 92.9%), and overall survival (93.9% and 93.8%). The chemotherapy benefit for invasive disease-free survival varied with the combination of recurrence score and age (P=0.004), with some benefit of chemotherapy found in women 50 years of age or younger with a recurrence score of 16 to 25. CONCLUSIONS: Adjuvant endocrine therapy and chemoendocrine therapy had similar efficacy in women with hormone-receptor-positive, HER2-negative, axillary node-negative breast cancer who had a midrange 21-gene recurrence score, although some benefit of chemotherapy was found in some women 50 years of age or younger. (Funded by the National Cancer Institute and others; TAILORx ClinicalTrials.gov number, NCT00310180 .).

Preoperative Breast MRI for Newly Diagnosed Ductal Carcinoma in Situ: Imaging Features and Performance in a Multicenter Setting (ECOG-ACRIN E4112 Trial).

Background There are limited data from clinical trials describing preoperative MRI features and performance in the evaluation of mammographically detected ductal carcinoma in situ (DCIS). Purpose To report qualitative MRI features of DCIS, MRI performance in the identification of additional disease, and associations of imaging features with pathologic, genomic, and surgical outcomes from the Eastern Cooperative Oncology Group-American College of Radiology Imaging Network (ECOG-ACRIN) E4112 trial. Materials and Methods Secondary analyses of a multicenter prospective clinical trial from the ECOG-ACRIN Cancer Research Group included women with DCIS diagnosed with conventional imaging techniques (mammography and US), confirmed via core-needle biopsy (CNB), and enrolled between March 2015 and April 2016 who were candidates for wide local excision (WLE) based on conventional imaging and clinical examination results. DCIS MRI features and pathologic features from CNB and excision were recorded. Each woman without invasive upgrade of the index DCIS at WLE received a 12-gene DCIS score. MRI performance metrics were calculated. Associations of imaging features with invasive upgrade, dichotomized DCIS score (<39 vs ≥39), and single WLE success were estimated in uni- and multivariable analyses. Results Among 339 women (median age, 60 years; interquartile range, 51-66 years), most DCIS cases showed nonmass enhancement (NME) (195 of 339 [58%]) on MRI scans with larger median size than on mammograms (19 mm vs 12 mm; P < .001). Positive predictive value of MRI-prompted CNBs was 32% (21 of 66) (95% CI: 22, 44), yielding an additional cancer detection rate of 6.2% (21 of 339) (95% CI: 4.1, 9.3). MRI false-positive rate was 14.2% (45 of 318) (95% CI: 10.7, 18.4). No imaging features were associated with invasive upgrade or DCIS score (P = .05 to P = .95). Smaller size and focal NME distribution at MRI were linked to single WLE success (P < .001). Conclusion Preoperative MRI depicted ductal carcinoma in situ (DCIS) diagnosed with conventional imaging most commonly as nonmass enhancement, with larger median span than mammography, and additional cancer detection rate of 6.2%. MRI features of this subset of DCIS did not enable prediction of pathologic or genomic outcomes. Clinical trial registration no. NCT02352883 © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by Kuhl in this issue. An earlier incorrect version of this article appeared online. This article was corrected on August 4, 2021.

Splicing factor ESRP1 controls ER-positive breast cancer by altering metabolic pathways.

The epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) control the epithelial-to-mesenchymal transition (EMT) splicing program in cancer. However, their role in breast cancer recurrence is unclear. In this study, we report that high levels of ESRP1, but not ESRP2, are associated with poor prognosis in estrogen receptor positive (ER+) breast tumors. Knockdown of ESRP1 in endocrine-resistant breast cancer models decreases growth significantly and alters the EMT splicing signature, which we confirm using TCGA SpliceSeq data of ER+ BRCA tumors. However, these changes are not accompanied by the development of a mesenchymal phenotype or a change in key EMT-transcription factors. In tamoxifen-resistant cells, knockdown of ESRP1 affects lipid metabolism and oxidoreductase processes, resulting in the decreased expression of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), and phosphoglycerate dehydrogenase (PHGDH) at both the mRNA and protein levels. Furthermore, ESRP1 knockdown increases the basal respiration and spare respiration capacity. This study reports a novel role for ESRP1 that could form the basis for the prevention of tamoxifen resistance in ER+ breast cancer.

Tumour-infiltrating lymphocytes in ductal carcinoma in situ (DCIS)-assessment with three different methodologies and correlation with Oncotype DX DCIS Score.

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 < 5% (P = 0.031), and absent circumferential TILs (P = 0.002). In logistic regression analysis adjusted for necrosis and nuclear grade, touching TILs and circumferential TILs showed significant associations with the ODX DCIS Score, whereas stromal TILs did not. CONCLUSIONS: Our results suggest that both the presence of TILs and the spatial arrangement of TILs or close proximity of TILs to DCIS, and TILs touching or encircling DCIS, may be predictive of recurrence.

Profiling molecular regulators of recurrence in chemorefractory triple-negative breast cancers.

BACKGROUND: Approximately two thirds of patients with localized triple-negative breast cancer (TNBC) harbor residual disease (RD) after neoadjuvant chemotherapy (NAC) and have a high risk-of-recurrence. Targeted therapeutic development for TNBC is of primary significance as no targeted therapies are clinically indicated for this aggressive subset. In view of this, we conducted a comprehensive molecular analysis and correlated molecular features of chemorefractory RD tumors with recurrence for the purpose of guiding downstream therapeutic development. METHODS: We assembled DNA and RNA sequencing data from RD tumors as well as pre-operative biopsies, lymphocytic infiltrate, and survival data as part of a molecular correlative to a phase II post-neoadjuvant clinical trial. Matched somatic mutation, gene expression, and lymphocytic infiltrate were assessed before and after chemotherapy to understand how tumors evolve during chemotherapy. Kaplan-Meier survival analyses were conducted categorizing cancers with TP53 mutations by the degree of loss as well as by the copy number of a locus of 18q corresponding to the SMAD2, SMAD4, and SMAD7 genes. RESULTS: Analysis of matched somatic genomes pre-/post-NAC revealed chaotic acquisition of copy gains and losses including amplification of prominent oncogenes. In contrast, significant gains in deleterious point mutations and insertion/deletions were not observed. No trends between clonal evolution and recurrence were identified. Gene expression data from paired biopsies revealed enrichment of actionable regulators of stem cell-like behavior and depletion of immune signaling, which was corroborated by total lymphocytic infiltrate, but was not associated with recurrence. Novel characterization of TP53 mutation revealed prognostically relevant subgroups, which were linked to MYC-driven transcriptional amplification. Finally, somatic gains in 18q were associated with poor prognosis, likely driven by putative upregulation of TGFß signaling through the signal transducer SMAD2. CONCLUSIONS: We conclude TNBCs are dynamic during chemotherapy, demonstrating complex plasticity in subclonal diversity, stem-like qualities, and immune depletion, but somatic alterations of TP53/MYC and TGFß signaling in RD samples are prominent drivers of recurrence, representing high-yield targets for additional interrogation.

Analytical validation of a standardised scoring protocol for Ki67 immunohistochemistry on breast cancer excision whole sections: an international multicentre collaboration.

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.

Prospective Validation of a 21-Gene Expression Assay in Breast Cancer.

BACKGROUND: Prior studies with the use of a prospective-retrospective design including archival tumor samples have shown that gene-expression assays provide clinically useful prognostic information. However, a prospectively conducted study in a uniformly treated population provides the highest level of evidence supporting the clinical validity and usefulness of a biomarker. METHODS: We performed a prospective trial involving women with hormone-receptor-positive, human epidermal growth factor receptor type 2 (HER2)-negative, axillary node-negative breast cancer with tumors of 1.1 to 5.0 cm in the greatest dimension (or 0.6 to 1.0 cm in the greatest dimension and intermediate or high tumor grade) who met established guidelines for the consideration of adjuvant chemotherapy on the basis of clinicopathologic features. A reverse-transcriptase-polymerase-chain-reaction assay of 21 genes was performed on the paraffin-embedded tumor tissue, and the results were used to calculate a score indicating the risk of breast-cancer recurrence; patients were assigned to receive endocrine therapy without chemotherapy if they had a recurrence score of 0 to 10, indicating a very low risk of recurrence (on a scale of 0 to 100, with higher scores indicating a greater risk of recurrence). RESULTS: Of the 10,253 eligible women enrolled, 1626 women (15.9%) who had a recurrence score of 0 to 10 were assigned to receive endocrine therapy alone without chemotherapy. At 5 years, in this patient population, the rate of invasive disease-free survival was 93.8% (95% confidence interval [CI], 92.4 to 94.9), the rate of freedom from recurrence of breast cancer at a distant site was 99.3% (95% CI, 98.7 to 99.6), the rate of freedom from recurrence of breast cancer at a distant or local-regional site was 98.7% (95% CI, 97.9 to 99.2), and the rate of overall survival was 98.0% (95% CI, 97.1 to 98.6). CONCLUSIONS: Among patients with hormone-receptor-positive, HER2-negative, axillary node-negative breast cancer who met established guidelines for the recommendation of adjuvant chemotherapy on the basis of clinicopathologic features, those with tumors that had a favorable gene-expression profile had very low rates of recurrence at 5 years with endocrine therapy alone. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00310180.).

Single-cell heterogeneity in ductal carcinoma in situ of breast.

Heterogeneous patterns of mutations and RNA expression have been well documented in invasive cancers. However, technological challenges have limited the ability to study heterogeneity of protein expression. This is particularly true for pre-invasive lesions such as ductal carcinoma in situ of the breast. Cell-level heterogeneity in ductal carcinoma in situ was analyzed in a single 5 µm tissue section using a multiplexed immunofluorescence analysis of 11 disease-related markers (EGFR, HER2, HER4, S6, pmTOR, CD44v6, SLC7A5 and CD10, CD4, CD8 and CD20, plus pan-cytokeratin, pan-cadherin, DAPI, and Na+K+ATPase for cell segmentation). Expression was quantified at cell level using a single-cell segmentation algorithm. K-means clustering was used to determine co-expression patterns of epithelial cell markers and immune markers. We document for the first time the presence of epithelial cell heterogeneity within ducts, between ducts and between patients with ductal carcinoma in situ. There was moderate heterogeneity in a distribution of eight clusters within each duct (average Shannon index 0.76; range 0-1.61). Furthermore, within each patient, the average Shannon index across all ducts ranged from 0.33 to 1.02 (s.d. 0.09-0.38). As the distribution of clusters within ducts was uneven, the analysis of eight ducts might be sufficient to represent all the clusters ie within- and between-duct heterogeneity. The pattern of epithelial cell clustering was associated with the presence and type of immune infiltrates, indicating a complex interaction between the epithelial tumor and immune system for each patient. This analysis also provides the first evidence that simultaneous analysis of both the epithelial and immune/stromal components might be necessary to understand the complex milieu in ductal carcinoma in situ lesions.

A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas.

BACKGROUND: Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing. METHODS: The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used. RESULTS: Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability. CONCLUSIONS: A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (http://clinicaltrials.gov/ct2/show/NCT02220855).

Genetic events in the progression of adenoid cystic carcinoma of the breast to high-grade triple-negative breast cancer.

Adenoid cystic carcinoma of the breast is a rare histological type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Although the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intratumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by the MYB-NFIB fusion gene and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-negative breast cancer of no special type may involve the selection of neoplastic clones and/or the acquisition of additional genetic alterations.

An international study to increase concordance in Ki67 scoring.

Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90-0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes.