The frequency and clinical significance of centromere enumeration probe 17 alterations in human epidermal growth factor receptor 2 immunohistochemistry-equivocal invasive breast cancer.

BACKGROUND AND AIMS: Chromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry-equivocal score. METHODS AND RESULTS: A large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in-situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2-amplified tumours with monosomy 17 (CEP17 copy number < 1.5 per nucleus), normal 17 (CEP17 1.5-< 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2-non-amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2-amplified and HER2-non-amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2-amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37-102.00; P = 0.02], and histological grade 3 in HER2 non-amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61-19.00; P = 0.007). CONCLUSION: The impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry-equivocal score who would benefit from NAT.

Transcriptome profiles of stem-like cells from primary breast cancers allow identification of ITGA7 as a predictive marker of chemotherapy response.

BACKGROUND: Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. METHODS: Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. RESULTS: Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. CONCLUSIONS: This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

Retrospective observational study of HER2 immunohistochemistry in borderline breast cancer patients undergoing neoadjuvant therapy, with an emphasis on Group 2 (HER2/CEP17 ratio ≥2.0, HER2 copy number <4.0 signals/cell) cases.

BACKGROUND: The ASCO/CAP guidance on HER2 testing in breast cancer (BC) has recently changed. Group 2 tumours with immunohistochemistry score 2+ and HER2/CEP17 ratio ≥2.0 and HER2 copy number <4.0 signals/cell were re-classified as HER2 negative. This study aims to examine the response of Group 2 tumours to neoadjuvant chemotherapy (NACT). METHODS: 749 BC cases were identified from 11 institutions. The association between HER2 groups and pathological complete response (pCR) was assessed. RESULTS: 54% of immunohistochemistry HER2 positive (score 3+) BCs showed pCR, compared to 19% of immunohistochemistry 2+ FISH amplified cases. 27% of Group 2 treated with HER2 targeted therapy achieved pCR, compared to 19 and 11% in the combined Groups 1 + 3 and Groups 4 + 5, respectively. No difference in pCR rates was identified between Group 2 and Group 1 or combined Groups 1 + 3. However, Group 2 response rate was higher than Groups 4 + 5 (p = 0.017). CONCLUSION: No difference in pCR was detected in tumours with a HER2/CEP17 ratio ≥2.0 and a HER2 score 2+ by IHC when stratified by HER2 gene copy number. Our data suggest that ASCO/CAP HER2 Group 2 carcinomas should be evaluated further with respect to eligibility for HER2 targeted therapy.

Predictors of pathological complete response to neoadjuvant treatment and changes to post-neoadjuvant HER2 status in HER2-positive invasive breast cancer.

The response of human epidermal growth factor receptor2 (HER2)- positive breast cancer (BC) patients to anti-HER2 targeted therapy is significant. However, the response is not uniform and a proportion of HER2-positive patients do not respond. This study aims to identify predictors of response in the neoadjuvant treatment and to assess the discordance rate of HER2 status between pre- and post-treatment specimens in HER2-positive BC patients. The study group comprised 500 BC patients treated with neoadjuvant chemotherapy (NACT) and/or neoadjuvant anti-HER2 therapy and surgery who had tumours that were 3+ or 2+ with HER2 immunohistochemistry (IHC). HER2 IHC 2+ tumours were classified into five groups by fluorescence in situ hybridisation (FISH) according to the 2018 ASCO/CAP guidelines of which Groups 1, 2 and 3 were considered HER2 amplified. Pathological complete response (pCR) was more frequent in HER2 IHC 3+ tumours than in HER2 IHC 2+/HER2 amplified tumours, when either in receipt of NACT alone (38% versus 13%; p = 0.22) or neoadjuvant anti-HER2 therapy (52% versus 20%; p < 0.001). Multivariate logistic regression analysis showed that HER2 IHC 3+ and histological grade 3 were independent predictors of pCR following neoadjuvant anti-HER2 therapy. In the HER2 IHC 2+/HER2 amplified tumours or ASCO/CAP FISH Group 1 alone, ER-negativity was an independent predictor of pCR following NACT and/or neoadjuvant anti-HER2 therapy. In the current study, 22% of HER2-positive tumours became HER2-negative by IHC and FISH following neoadjuvant treatment, the majority (74%) HER2 IHC 2+/HER2 amplified tumours. Repeat HER2 testing after neoadjuvant treatment should therefore be considered.

Digital pathology for primary diagnosis of screen-detected breast lesions - experimental data, validation and experience from four centres.

AIM: The rate of deployment of digital pathology (DP) systems for primary diagnosis in the UK is accelerating. The flexibility and resilience of digital versus standard glass slides could be of great benefit in the NHS breast screening programme (NHSBSP). This study aims to document the safety and benefits of DP for preoperative tissue diagnosis of screen-detected breast lesions. METHODS AND RESULTS: Concordance data for glass and digital slides of the same cases from four sites were subjected to detailed concordance-discordance analysis. A literature review of DP in the primary diagnosis of breast lesions is presented, making this the most comprehensive synthesis of digital breast cancer histopathological diagnostic data to date. Detailed concordance analysis of experimental data from two histopathology departments reveals clinical concordance rates for breast biopsies of 96% (216 of 225) and 99.6% (249 of 250). Data from direct comparison validation studies in two histopathology departments, utilising the protocol recommended by the Royal College of Pathologists, found concordance rates for breast histology cases of 99.4% (180 of 181) and 99.0% (887 of 896). An intraobserver variation study for glass versus digital slides for difficult cases from the NHSBSP yielded a kappa statistic of 0.80, indicating excellent agreement. Discordances encountered in the studies most frequently concerned discrepancies in grading attributable to mitotic count-scoring and identification of weddelite. CONCLUSIONS: The experience of four histopathology laboratories and our review of pre-existing literature suggests that DP is safe for the primary diagnosis of NHSBSP breast histology specimens, and does not increase the risk of misclassification.

Digital pathology for the primary diagnosis of breast histopathological specimens: an innovative validation and concordance study on digital pathology validation and training.

AIM: To train and individually validate a group of breast pathologists in specialty-specific digital primary diagnosis by using a novel protocol endorsed by the Royal College of Pathologists' new guideline for digital pathology. The protocol allows early exposure to live digital reporting, in a risk-mitigated environment, and focuses on patient safety and professional development. METHODS AND RESULTS: Three specialty breast pathologists completed training in the use of a digital microscopy system, and were exposed to a training set of 20 challenging cases, designed to help them identify personal digital diagnostic pitfalls. Following this, the three pathologists viewed a total of 694 live, entire breast cases. All primary diagnoses were made on digital slides, with immediate glass slide review and reconciliation before final case sign-out. There was complete clinical concordance between the glass and digital impression of the case in 98.8% of cases. Only 1.2% of cases had a clinically significant difference in diagnosis/prognosis on glass and digital slide reads. All pathologists elected to continue using the digital microscope as the standard for breast histopathology specimens, with deferral to glass for a limited number of clinical/histological scenarios as a safety net. CONCLUSION: Individual training and validation for digital primary diagnosis allows pathologists to develop competence and confidence in their digital diagnostic skills, and aids safe and responsible transition from the light microscope to the digital microscope.

Expression of phosphorylated eIF4E-binding protein 1, but not of eIF4E itself, predicts survival in male breast cancer.

BACKGROUND: Male breast cancer is rare and treatment is based on data from females. High expression/activity of eukaryotic initiation factor 4E (eIF4E) denotes a poor prognosis in female breast cancer, and the eIF4E pathway has been targeted therapeutically. Eukaryotic initiation factor 4E activity in female breast cancer is deregulated by eIF4E overexpression and by phosphorylation of its binding protein, 4E-BP1, which relieves inhibitory association between eIF4E and 4E-BP1. The relevance of the eIF4E pathway in male breast cancer is unknown. METHODS: We have assessed expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 (p4E-BP1) using immunohistochemistry in a large cohort of male breast cancers (n=337) and have examined correlations with prognostic factors and survival. RESULTS: Neither eIF4E expression nor estimated eIF4E activity were associated with prognosis. However, a highly significant correlation was found between p4E-BP1 expression and disease-free survival (DFS), linking any detectable p4E-BP1 with poor survival (univariate log rank P=0.001; multivariate HR 8.8, P=0.0001). CONCLUSIONS: Our data provide no support for direct therapeutic targeting of eIF4E in male breast cancer, unlike in females. However, as p4E-BP1 gives powerful prognostic insights that are unrelated to eIF4E function, p4E-BP1 may identify male breast cancers potentially suitable for therapies directed at the upstream kinase, mTOR.

Adding value to rare tissue samples donated to biobanks: characterisation of breast tissue and primary cell cultures obtained from a female-to-male transgender patient.

Biobanks provide a window of opportunity to store and add value to material from rare cases allowing their future use in biomedical research. One such example is the opportunityto obtain good quality tissue from patients undergoing gender re-assignment. Following patient agreement to donate tissue samples to our biobank we catalogued the histological appearance, defined the expression of the hormone receptors ERα, PR, AR and the proliferation marker Ki67, and generated and characterised primary cell cultures in a female to male (FTM) transgender patient referred to our unit for surgery. Immunohistochemistry was performed for ERα, PR and AR and the proliferation marker Ki67. Hormone receptor expression was confined to epithelial cells lining the breast ducts. Ki67 immunoreactivity was sparse indicating little proliferation of luminal epithelium, consistent with normal mammary gland. Cultures of epithelial cells and fibroblasts were derived from surplus tissue. The latter lacked expression of epithelial markers and hormone receptors but exhibited expression of vimentin. Culture of the former on Matrigel saw an outgrowth of more rounded "epithelial-like" cells. Immunofluoresence characterisation showed a mixed phenotype with expression of vimentin and both myoepithelial and luminal epithelial markers. Sporadic weak ERα expression and moderate PR expression was seen. In summary, as well as routinely collecting tissue and blood samples, we have characterised and stored tissue and cells from a FTM transgender patient, adding value to this resource which,available from the Breast Cancer Campaign Tissue Bank for those interested in further studying the biology of FTM transgender tissue.

PSMD9 expression predicts radiotherapy response in breast cancer.

BACKGROUND: More than 50% of cancer patients are recommended to receive radiotherapy. Recommendations are based mainly on clinical and pathological factors and not intrinsic tumour radio-sensitivity. Use of radiotherapy according to predictive markers would potentially reduce costly over-treatment, and improve the treatment risk-benefit ratio and cancer outcomes. Tumour expression of the 26S proteasome has been reported to predict radiotherapy response: low expression was associated with higher rates of local recurrence after radiotherapy, suggesting that low proteasome expression and activity was associated with radio-resistance. However, this conclusion is at odds with the emerging use of proteasome inhibitors as radio-sensitizers. Our aim was to further analyse the relevance of 26S proteasome expression, focussing specifically on the PSMD9 subunit, in the largest clinical cohort to date, and to investigate the functional role of PSMD9 in radio-sensitivity in breast cancer cell lines. METHODS: We examined expression of PSMD9 using immunohistochemistry in a cohort of 157 breast cancer patients, including 32 cases (20.4%) that subsequently developed local recurrences. The value of expression as a prognostic or radiotherapy predictive marker was tested using Kaplan-Meier and Cox regression analyses. PSMD9 function was examined in breast cancer cell lines MCF7 and MDA-MB-231 using siRNA knock-downs and colony forming assays after irradiation. RESULTS: Low tumour PSMD9 expression was significantly associated with a reduced incidence of local recurrence in patients receiving adjuvant radiotherapy (univariate log rank p = 0.02; multivariate regression p = 0.009), but not in those treated without radiotherapy, suggesting that low PSMD9 expression was associated with relative tumour radio-sensitivity. In support of this, reduction of PSMD9 expression using siRNA in breast cancer cell lines in vitro sensitized cells to radiotherapy. CONCLUSIONS: We conclude that PSMD9 expression may predict radiotherapy benefit, with low expression indicative of relative radio-sensitivity, the opposite of previous reports relating to 26S proteasome expression. Our conclusion is compatible with use of proteasome inhibitors as radio-sensitizers, and highlights PSMD9 as a potential target for radio-sensitizing drugs.

The sodium channel ß1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis.

Voltage-gated Na(+) channels (VGSCs) are heteromeric proteins composed of pore-forming α subunits and smaller ß subunits. The ß subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). ß1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. ß1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/ß1 protein were up-regulated in BCa specimens, compared with normal breast tissue. ß1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. ß1 over-expression also increased vascularization and reduced apoptosis in the primary tumours, and ß1 over-expressing tumour cells had an elongate morphology. In vitro, ß1 potentiated outgrowth of processes from BCa cells co-cultured with fibroblasts, via trans-homophilic adhesion. ß1-mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na(+) current, thus replicating the mechanism by which ß1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, ß1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for ß1 in tumour growth and metastasis in vivo. We propose that ß1 warrants further study as a potential biomarker and targeting ß1-mediated adhesion interactions may have value as a novel anti-cancer therapy.

Concordance of HER2-low scoring in breast carcinoma among expert pathologists in the United Kingdom and the republic of Ireland -on behalf of the UK national coordinating committee for breast pathology.

BACKGROUND: Recent clinical evidence showed that breast cancer with low HER2 expression levels responded to trastuzumab deruxtecan therapy. The HER2-low cancers comprise immunohistochemistry (IHC) score 1+ and 2+ ISH non-amplified tumours, currently classified as HER2 negative. Little data exists on the reproducibility of pathologists reporting of HER2-low cancer. PATIENT AND METHODS: Sixteen expert pathologists of the UK National Coordinating Committee for Breast Pathology scored 50 digitally scanned HER2 IHC slides. The overall level of agreement, Fleiss multiple-rater kappa statistics and Cohen's Kappa were calculated. Cases with low concordance were re-scored by the same pathologists after a washout period. RESULTS: Absolute agreement was achieved in 6% of cases, all of which scored 3+. Poor agreement was found in 5/50 (10%) of cases. This was due to heterogeneous HER2 expression, cytoplasmic staining and low expression spanning the 10% cut-off value. Highest concordance (86%) was achieved when scores were clustered as 0 versus others. Improvement in kappa of overall agreement was achieved when scores 1+ and 2+ were combined. Inter-observer agreement was moderate to substantial in the whole cohort but fair to moderate in the HER2-low group. Similarly, consensus-observer agreement was substantial to almost perfect in the whole cohort and moderate to substantial in the HER2-low group. CONCLUSION: HER2-low breast cancer suffers from lower concordance among expert pathologists. While most cases can reproducibly be classified, a small proportion (10%) remained challenging. Refining the criteria for reporting and consensus scoring will help select appropriate patients for targeted therapy.

Interobserver variation in the diagnosis of fibroepithelial lesions of the breast: a multicentre audit by digital pathology.

AIM: Fibroepithelial lesions (FELs) of the breast span a morphological continuum including lesions where distinction between cellular fibroadenoma (FA) and benign phyllodes tumour (PT) is difficult. The distinction is clinically important with FAs managed conservatively while equivocal lesions and PTs are managed with surgery. We sought to audit core biopsy diagnoses of equivocal FELs by digital pathology and to investigate whether digital point counting is useful in clarifying FEL diagnoses. METHOD: Scanned slide images from cores and subsequent excisions of 69 equivocal FELs were examined in a multicentre audit by eight pathologists to determine the agreement and accuracy of core needle biopsy (CNB) diagnoses and by digital point counting of stromal cellularity and expansion to determine if classification could be improved. RESULTS: Interobserver variation was high on CNB with a unanimous diagnosis from all pathologists in only eight cases of FA, diagnoses of both FA and PT on the same CNB in 15 and a 'weak' mean kappa agreement between pathologists (k=0.36). 'Moderate' agreement was observed on CNBs among breast specialists (k=0.44) and on excision samples (k=0.49). Up to 23% of lesions confidently diagnosed as FA on CNB were PT on excision and up to 30% of lesions confidently diagnosed as PT on CNB were FA on excision. Digital point counting did not aid in the classification of FELs. CONCLUSION: Accurate and reproducible diagnosis of equivocal FELs is difficult, particularly on CNB, resulting in poor interobserver agreement and suboptimal accuracy. Given the diagnostic difficulty, and surgical implications, equivocal FELs should be reported in consultation with experienced breast pathologists as a small number of benign FAs can be selected out from equivocal lesions.

MiR-19b non-canonical binding is directed by HuR and confers chemosensitivity through regulation of P-glycoprotein in breast cancer.

MicroRNAs and RNA-binding proteins exert regulation on >60% of coding genes, yet interplay between them is little studied. Canonical microRNA binding occurs by base-pairing of microRNA 3'-ends to complementary "seed regions" in mRNA 3'UTRs, resulting in translational repression. Similarly, regulatory RNA-binding proteins bind to mRNAs, modifying stability or translation. We investigated post-transcriptional regulation acting on the xenobiotic pump ABCB1/P-glycoprotein, which is implicated in cancer therapy resistance. We characterised the ABCB1 UTRs in primary breast cancer cells and identified UTR sequences that responded to miR-19b despite lacking a canonical binding site. Sequences did, however, contain consensus sites for the RNA-binding protein HuR. We demonstrated that a tripartite complex of HuR, miR-19b and UTR directs repression of ABCB1/P-glycoprotein expression, with HuR essential for non-canonical miR-19b binding thereby controlling chemosensitivity of breast cancer cells. This exemplifies a new cooperative model between RNA-binding proteins and microRNAs to expand the repertoire of mRNAs that can be regulated. This study suggests a novel therapeutic target to impair P-glycoprotein mediated drug efflux, and also indicates that current microRNA binding predictions that rely on seed regions alone may be too conservative.

Nav1.5 regulates breast tumor growth and metastatic dissemination in vivo.

Voltage-gated Na+ channels (VGSCs) mediate action potential firing and regulate adhesion and migration in excitable cells. VGSCs are also expressed in cancer cells. In metastatic breast cancer (BCa) cells, the Nav1.5 α subunit potentiates migration and invasion. In addition, the VGSC-inhibiting antiepileptic drug phenytoin inhibits tumor growth and metastasis. However, the functional activity of Nav1.5 and its specific contribution to tumor progression in vivo has not been delineated. Here, we found that Nav1.5 is up-regulated at the protein level in BCa compared with matched normal breast tissue. Na+ current, reversibly blocked by tetrodotoxin, was retained in cancer cells in tumor tissue slices, thus directly confirming functional VGSC activity in vivo. Stable down-regulation of Nav1.5 expression significantly reduced tumor growth, local invasion into surrounding tissue, and metastasis to liver, lungs and spleen in an orthotopic BCa model. Nav1.5 down-regulation had no effect on cell proliferation or angiogenesis within the in tumors, but increased apoptosis. In vitro, Nav1.5 down-regulation altered cell morphology and reduced CD44 expression, suggesting that VGSC activity may regulate cellular invasion via the CD44-src-cortactin signaling axis. We conclude that Nav1.5 is functionally active in cancer cells in breast tumors, enhancing growth and metastatic dissemination. These findings support the notion that compounds targeting Nav1.5 may be useful for reducing metastasis.

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis.

BACKGROUND: MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR-92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR-92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR-92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR-92 expression by qRT-PCR. Expression of ERß1, a direct miR-92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR-92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR-92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERß1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERß1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR-92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR-92 levels in NFs but not CAFs enhanced invasion of both MCF-7 and MDA-MB-231 breast cancer epithelial cells. CONCLUSIONS: miR-92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERß1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR-92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERß1 may not be the most important miR-92 target in breast cancer.

The practicalities of using tissue slices as preclinical organotypic breast cancer models.

Models considering breast cancer complexity cannot be easily or accurately replicated in routine cell line or animal models. We aimed to evaluate the practicality of organotypic tissue slice culture in breast cancer. Following ethical approval, 250 µm thick sections from surplus breast tumours (n=10) were prepared using a vibrating blade microtome. Triplicate tissue slices were placed in 6-well plates and cultured for up to 7 days ± tamoxifen (1 nM) or doxorubicin (1 µM). Tissue slices were fixed and embedded before sectioning for morphological evaluation and immunohistochemistry. H&E showed good preservation of tissue morphology. Collagen production was evident. Biomarkers of proliferation and apoptosis could be evaluated using immunohistochemistry and used as surrogates to quantify drug effects. In summary, breast cancer tissue slices can be cultured in vitro as organotypic models. Nevertheless, although simple in concept, the delicacy of the model with regard to handling makes subsequent analytical processes challenging.