Search

Tumour-associated macrophages (TAMs) play an important role in tumour progression, which is facilitated by their ability to respond to environmental cues. Here we report, using murine models of breast cancer, that TAMs expressing fibroblast activation protein alpha (FAP) and haem oxygenase-1 (HO-1), which are also found in human breast cancer, represent a macrophage phenotype similar to that observed during the wound healing response. Importantly, the expression of a wound-like cytokine response within the tumour is clinically associated with poor prognosis in a variety of cancers. We show that co-expression of FAP and HO-1 in macrophages results from an innate early regenerative response driven by IL-6, which both directly regulates HO-1 expression and licenses FAP expression in a skin-like collagen-rich environment. We show that tumours can exploit this response to facilitate transendothelial migration and metastatic spread of the disease, which can be pharmacologically targeted using a clinically relevant HO-1 inhibitor. The relationship between wound healing and cancer is intricate and complex. Here, the authors show that in breast cancer models an IL-6 driven co-expression of FAP and HO-1 in tumour-associated macrophages, similar to the wound healing response, facilitates migration and metastatic spread. [ABSTRACT FROM AUTHOR]

Background: MED12 and TERT promoter mutations have been shown to be the most common somatic mutations in phyllodes tumours (PTs). The aims of this study were to determine the frequency of these mutations in recurrent PTs, assess whether TERT promoter mutations could be helpful in distinguishing fibroadenomas (FAs) from PTs and identify novel mutations that may be driving malignant progression.Methods: MED12 and the TERT promoter were Sanger sequenced in 75 primary PTs, 21 recurrences, 19 single FAs and 2 cases of multiple FAs with benign PTs. Whole-exome sequencing was performed on one borderline PT.Results: Recurrent PTs and multiple FAs showed temporal discordance in MED12 but not TERT. Recurrent samples did acquire TERT mutations, with recurrent benign PTs more likely to have mutations in both genes. TERT mutations were not helpful in differentiating between benign PTs and FAs in cases of multiple FAs/PTs. Exome sequencing revealed a nonsense mutation in RBM15 and Sanger sequencing revealed another three RBM15 mutations in malignant/borderline PTs.Conclusions: This study has shown that MED12 mutations can be heterogeneous in both synchronous and recurrent PTs unlike TERT mutations. We have also shown that RBM15 mutations may be important in the pathogenesis of borderline/malignant PTs. [ABSTRACT FROM AUTHOR]

Background: The HOX genes are a family of homeodomain-containing transcription factors that determine cellular identity during development and which are dys-regulated in some cancers. In this study we examined the expression and oncogenic function of HOX genes in mesothelioma, a cancer arising from the pleura or peritoneum which is associated with exposure to asbestos.Methods: We tested the sensitivity of the mesothelioma-derived lines MSTO-211H, NCI-H28, NCI-H2052, and NCI-H226 to HXR9, a peptide antagonist of HOX protein binding to its PBX co-factor. Apoptosis was measured using a FACS-based assay with Annexin, and HOX gene expression profiles were established using RT-QPCR on RNA extracted from cell lines and primary mesotheliomas. The in vivo efficacy of HXR9 was tested in a mouse MSTO-211H flank tumor xenograft model.Results: We show that HOX genes are significantly dysregulated in malignant mesothelioma. Targeting HOX genes with HXR9 caused apoptotic cell death in all of the mesothelioma-derived cell lines, and prevented the growth of mesothelioma tumors in a mouse xenograft model. Furthermore, the sensitivity of these lines to HXR9 correlated with the relative expression of HOX genes that have either an oncogenic or tumor suppressive function in cancer. The analysis of HOX expression in primary mesothelioma tumors indicated that these cells could also be sensitive to the disruption of HOX activity by HXR9, and that the expression of HOXB4 is strongly associated with overall survival.Conclusion: HOX genes are a potential therapeutic target in mesothelioma, and HOXB4 expression correlates with overall survival. [ABSTRACT FROM AUTHOR]

Background Integrin αvβ6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer. Methods Protein expression of integrin subunit beta6 (β6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student’s t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni’s Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided. Results High expression of either the mRNA or protein for the integrin subunit β6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of β6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts. Conclusions Targeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients. [ABSTRACT FROM PUBLISHER]

Introduction: Outcome predictors in use today are prognostic only for hormone receptor-positive (HRpos) breast cancer. Although microarray-derived multigene predictors of hormone receptor-negative (HRneg) and/or triple negative (Tneg) breast cancer recurrence risk are emerging, to date none have been transferred to clinically suitable assay platforms (for example, RT-PCR) or validated against formalin-fixed paraffin-embedded (FFPE) HRneg/Tneg samples.Methods: Multiplexed RT-PCR was used to assay two microarray-derived HRneg/Tneg prognostic signatures IR-7 and Buck-4) in a pooled FFPE collection of 139 chemotherapy-naïve HRneg breast cancers. The prognostic value of the RTPCR measured gene signatures were evaluated as continuous and dichotomous variables, and in conditional risk models incorporating clinical parameters. An optimized five-gene index was derived by evaluating gene combinations from both signatures.Results: RT-PCR measured IR-7 and Buck-4 signatures proved prognostic as continuous variables; and conditional risk modeling chose nodal status, the IR-7 signature, and tumor grade as significant predictors of distant recurrence (DR). From the Buck-4 and IR-7 signatures, an optimized five-gene (TNFRSF17, CLIC5, HLA-F, CXCL13, XCL2) predictor was generated, referred to as the Integrated Cytokine Score (ICS) based on its functional pathway linkage through interferon-γ and IL-10. Across all FFPE cases, the ICS was prognostic as either a continuous or dichotomous variable, and conditional risk modeling selected nodal status and ICS as DR predictors. Further dichotomization of node-negative/ICS-low FFPE cases identified a subset of low-grade HRneg tumors with <10% 5-year DR risk. The prognostic value of ICS was reaffirmed in two previously studied microarray assayed cohorts containing 274 node-negative and chemotherapy naive HRneg breast cancers, including 95 Tneg cases where it proved prognostically independent of Tneg molecular subtyping. In additional HRneg/Tneg microarray assayed cohorts, the five-gene ICS also proved prognostic irrespective of primary tumor nodal status and adjuvant chemotherapy intervention.Conclusion: We advanced the measurement of two previously reported microarray-derived HRneg/Tneg breast cancer prognostic signatures for use in FFPE samples, and derived an optimized five-gene Integrated Cytokine Score (ICS) with multi-platform capability of predicting metastatic outcome from primary HRneg/Tneg tumors independent of nodal status, adjuvant chemotherapy use, and Tneg molecular subtype. [ABSTRACT FROM AUTHOR]

Background: Invadopodia, actin-rich structures that release metallo-proteases at the interface with extra-cellular matrix, in a punctate manner are thought to be important drivers of tumour invasion. Invadopodia formation has been observed in-vitro and in-vivo in numerous metastatic cell lines derived from multiple tumour types. However, prostate cancer cell lines have not been routinely reported to generate invadopodia and the few instances have always required external stimulation.Methods: In this study, the invasive potential of primary prostate adenocarcinoma cell lines, which have never been fully characterised before, was investigated both in-vitro invadopodia assays and in-vivo zebrafish dissemination assay. Subsequently, circulating tumour cells from prostate cancer patients were isolated and tested in the invadopodia assay.Results: Retention of E-cadherin and N-cadherin expression indicated a transitional state of EMT progression, consistent with the idea of partial EMT that has been frequently observed in aggressive prostate cancer. All cell lines tested were capable of spontaneous invadopodia formation and possess a significant degradative ability in-vitro under basal conditions. These cell lines were invasive in-vivo and produced visible metastasis in the zebrafish dissemination assay. Importantly we have proceeded to demonstrate that circulating tumour cells isolated from prostate cancer patients exhibit invadopodia-like structures and degrade matrix with visible puncta. This work supports a role for invadopodia activity as one of the mechanisms of dissemination employed by prostate cancer cells.Conclusion: The combination of studies presented here provide clear evidence that invadopodia activity can play a role in prostate cancer progression. [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular "nest" structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1+ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1+ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8+ T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy. [Display omitted] • LYVE-1+ TAMs co-express HO-1 and form multi-cellular nests proximal to blood vessels • LYVE-1+ TAMs develop in response to IL-6 within the tumor • CCR5 drives LYVE-1+ TAMs' cross-communication to form dynamic nest arrangements • The LYVE-1+ TAM niche facilitates CD8+ T cell exclusion and resistance to chemotherapy Lymphatic vessel endothelial hyaluronan receptor-1+ (LYVE-1+) tumor-associated macrophages (TAMs) form coordinated multi-cellular "nests" proximal to blood vasculature in cancer. In a murine breast cancer model, Anstee et al. show that LYVE-1+ TAMs develop in response to IL-6, promoting a CCR5-dependent signaling axis that guides their formation into a collaborative niche. [ABSTRACT FROM AUTHOR]

Invasive lobular breast cancer (ILC) accounts for 10–15% of all invasive breast carcinomas. It is generally ER positive (ER+) and often associated with lobular carcinoma in situ (LCIS). Genome-wide association studies have identified more than 70 common polymorphisms that predispose to breast cancer, but these studies included predominantly ductal (IDC) carcinomas. To identify novel common polymorphisms that predispose to ILC and LCIS, we pooled data from 6,023 cases (5,622 ILC, 401 pure LCIS) and 34,271 controls from 36 studies genotyped using the iCOGS chip. Six novel SNPs most strongly associated with ILC/LCIS in the pooled analysis were genotyped in a further 516 lobular cases (482 ILC, 36 LCIS) and 1,467 controls. These analyses identified a lobular-specific SNP at 7q34 (rs11977670, OR (95%CI) for ILC = 1.13 (1.09–1.18), P = 6.0×10−10; P-het for ILC vs IDC ER+ tumors = 1.8×10−4). Of the 75 known breast cancer polymorphisms that were genotyped, 56 were associated with ILC and 15 with LCIS at P<0.05. Two SNPs showed significantly stronger associations for ILC than LCIS (rs2981579/10q26/FGFR2, P-het = 0.04 and rs889312/5q11/MAP3K1, P-het = 0.03); and two showed stronger associations for LCIS than ILC (rs6678914/1q32/LGR6, P-het = 0.001 and rs1752911/6q14, P-het = 0.04). In addition, seven of the 75 known loci showed significant differences between ER+ tumors with IDC and ILC histology, three of these showing stronger associations for ILC (rs11249433/1p11, rs2981579/10q26/FGFR2 and rs10995190/10q21/ZNF365) and four associated only with IDC (5p12/rs10941679; rs2588809/14q24/RAD51L1, rs6472903/8q21 and rs1550623/2q31/CDCA7). In conclusion, we have identified one novel lobular breast cancer specific predisposition polymorphism at 7q34, and shown for the first time that common breast cancer polymorphisms predispose to LCIS. We have shown that many of the ER+ breast cancer predisposition loci also predispose to ILC, although there is some heterogeneity between ER+ lobular and ER+ IDC tumors. These data provide evidence for overlapping, but distinct etiological pathways within ER+ breast cancer between morphological subtypes. [ABSTRACT FROM AUTHOR]

Purpose: In many centers, supraclavicular fossa radiotherapy (SCF RT) is not routinely offered to breast cancer patients with one to three positive lymph nodes. We aimed to identify a subgroup of these patients who are at high risk of supra or infraclavicular fossa relapse (SCFR) such that they can be offered SCFRT at the time of diagnosis to improve long term locoregional control. Methods and Materials: We performed a retrospective analysis of the pathological features of 1,065 cases of invasive breast cancer with one to three positive axillary lymph nodes. Patients underwent radical breast conserving surgery or mastectomy. A total of 45% of patients received adjuvant chest wall/breast RT. No patients received adjuvant SCFRT. The primary outcome was SCFR. Secondary outcomes were chest wall/breast recurrence, distant metastasis, all death, and breast-cancer specific death. Kaplan-Meier estimates were used to calculate actuarial event rates and survival functions compared using log–rank tests. Multivariate analyses (MVA) of factors associated with outcome were conducted using Cox proportional hazards models. Results: Median follow-up was 9.7 years. SCFR rate was 9.2%. Median time from primary diagnosis to SCFR was 3.4 years (range, 0.7–14.4 years). SCFR was associated with significantly lower 10-year survival (18% vs. 65%; p < 0.001). Higher grade and number of positive lymph nodes were the most significant predictors of SCFR on MVA (p < 0.001). 10 year SCFR rates were less than 1% in all patients with Grade 1 cancers compared with 30% in those having Grade 3 cancers with three positive lymph nodes. Additional factors associated with SCFR on univariate analysis but not on MVA included larger nodal deposits (p = 0.002) and proportion of positive nodes (p = 0.003). Conclusions: Breast cancer patients with one to three positive lymph nodes have a heterogenous risk of SCFR. Patients with two to three positive axillary nodes and/or high-grade disease may warrant consideration of SCFRT. [ABSTRACT FROM AUTHOR]

PIK3 CA mutations are reported to be present in approximately 25% of breast cancer (BC), particularly the estrogen receptor-positive (ER+) and HER2-overexpressing (HER2+) subtypes, making them one of the most common genetic aberrations in BC. In experimental models, these mutations have been shown to activate AKTand induce oncogenic transformation, and hence these lesions have been hypothesized to render tumors highly sensitive to therapeutic P13K! mTOR inhibition. By analyzing gene expression and protein data from nearly 1,800 human BCs, we report that a PIK3CA mutationassociated gene signature (PIK3CA-GS) derived from exon 20 (kinase domain) mutations was able to predict PIK3CA mutation status in two independent datasets, strongly suggesting a characteristic set of gene expression-induced changes. However, in ER+/HER2BC despite pathway activation, PIK3CA mutations were associated with a phenotype of relatively low mTORC1 signaling and a good prognosis with tamoxifen monotherapy. The relationship between clinical outcome and the PIK3CA-GS was also assessed. Although the PIK3C.4-G5 was not associated with prognosis in ERand HER2+ BC, it could identify better clinical outcomes in ER+/HER2disease. In ER+ BC cell lines, PIK3CA mutations were also associated with sensitivity to tamoxifen. These findings could have important implications for the treatment of PIK3CA-mutant BC5 and the development of PI3K/mTOR inhibitors. [ABSTRACT FROM AUTHOR]

Checkpoint inhibition (CPI), particularly that targeting the inhibitory coreceptor programmed cell death protein 1 (PD-1), has transformed oncology. Although CPI can derepress cancer (neo)antigen-specific αβ T cells that ordinarily show PD-1-dependent exhaustion, it can also be efficacious against cancers evading αβ T cell recognition. In such settings, γδ T cells have been implicated, but the functional relevance of PD-1 expression by these cells is unclear. Here we demonstrate that intratumoral TRDV1 transcripts (encoding the TCRδ chain of Vδ1 + γδ T cells) predict anti-PD-1 CPI response in patients with melanoma, particularly those harboring below average neoantigens. Moreover, using a protocol yielding substantial numbers of tissue-derived Vδ1 + cells, we show that PD-1 + Vδ1 + cells display a transcriptomic program similar to, but distinct from, the canonical exhaustion program of colocated PD-1 + CD8 + αβ T cells. In particular, PD-1 + Vδ1 + cells retained effector responses to TCR signaling that were inhibitable by PD-1 engagement and derepressed by CPI., (© 2024. The Author(s).)

Purpose: The Graham Roberts Study was initiated in 2018 and is the first Trials Within Cohorts (TwiCs) study for bladder cancer. Its purpose is to provide an infrastructure for answering a breadth of research questions, including clinical, mechanistic, and supportive care centred questions for bladder cancer patients., Participants: All consented patients are those aged 18 or older, able to provide signed informedconsent and have a diagnosis of new or recurrent bladder cancer. All patients are required to have completed a series of baseline questionnaires. The questionnaires are then sent out every 12 months and include information on demographics and medical history as well as questionnaires to collect information on quality of life, fatigue, depression, overall health, physical activity, and dietary habits. Clinical information such as tumor stage, grade and treatment has also been extracted for each patient., Findings to Date: To date, a total of 125 bladder cancer patients have been consented onto the study with 106 filling in the baseline questionnaire. The cohort is made up of 75% newly diagnosed bladder cancer patients and 66% non-muscle invasive bladder cancer cases. At present, there is 1-year follow-up information for 70 patients, 2-year follow-up for 57 patients, 3-year follow-up for 47 patients and 4-year follow-up for 19 patients., Future Plans: We plan to continue recruiting further patients into the cohort study. Using the data collected within the study, we hope to carry out independent research studies with a focus on quality of life. We are also committed to utilizing the Roberts Study Cohort to set up and commence an intervention. The future studies and trials carried out using the Roberts Cohort have the potential to identify and develop interventions that could improve the prevention, diagnosis, and treatment of bladder cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Russell, Leech, Wylie, Moss, Haire, Enting, Amery, Chatterton, Khan, Thurairaja, Nair, Malde, Smith, Gillett, Josephs, Pintus, Rudman, Hughes, Relton and Van Hemelrijck.)

The suggestion that the systemic immune response in lymph nodes (LNs) conveys prognostic value for triple-negative breast cancer (TNBC) patients has not previously been investigated in large cohorts. We used a deep learning (DL) framework to quantify morphological features in haematoxylin and eosin-stained LNs on digitised whole slide images. From 345 breast cancer patients, 5,228 axillary LNs, cancer-free and involved, were assessed. Generalisable multiscale DL frameworks were developed to capture and quantify germinal centres (GCs) and sinuses. Cox regression proportional hazard models tested the association between smuLymphNet-captured GC and sinus quantifications and distant metastasis-free survival (DMFS). smuLymphNet achieved a Dice coefficient of 0.86 and 0.74 for capturing GCs and sinuses, respectively, and was comparable to an interpathologist Dice coefficient of 0.66 (GC) and 0.60 (sinus). smuLymphNet-captured sinuses were increased in LNs harbouring GCs (p < 0.001). smuLymphNet-captured GCs retained clinical relevance in LN-positive TNBC patients whose cancer-free LNs had on average ≥2 GCs, had longer DMFS (hazard ratio [HR] = 0.28, p = 0.02) and extended GCs' prognostic value to LN-negative TNBC patients (HR = 0.14, p = 0.002). Enlarged smuLymphNet-captured sinuses in involved LNs were associated with superior DMFS in LN-positive TNBC patients in a cohort from Guy's Hospital (multivariate HR = 0.39, p = 0.039) and with distant recurrence-free survival in 95 LN-positive TNBC patients of the Dutch-N4plus trial (HR = 0.44, p = 0.024). Heuristic scoring of subcapsular sinuses in LNs of LN-positive Tianjin TNBC patients (n = 85) cross-validated the association of enlarged sinuses with shorter DMFS (involved LNs: HR = 0.33, p = 0.029 and cancer-free LNs: HR = 0.21 p = 0.01). Morphological LN features reflective of cancer-associated responses are robustly quantifiable by smuLymphNet. Our findings further strengthen the value of assessment of LN properties beyond the detection of metastatic deposits for prognostication of TNBC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

All antibodies approved for cancer therapy are monoclonal IgGs but the biology of IgE, supported by comparative preclinical data, offers the potential for enhanced effector cell potency. Here we report a Phase I dose escalation trial (NCT02546921) with the primary objective of exploring the safety and tolerability of MOv18 IgE, a chimeric first-in-class IgE antibody, in patients with tumours expressing the relevant antigen, folate receptor-alpha. The trial incorporated skin prick and basophil activation tests (BAT) to select patients at lowest risk of allergic toxicity. Secondary objectives were exploration of anti-tumour activity, recommended Phase II dose, and pharmacokinetics. Dose escalation ranged from 70 μg-12 mg. The most common toxicity of MOv18 IgE is transient urticaria. A single patient experienced anaphylaxis, likely explained by detection of circulating basophils at baseline that could be activated by MOv18 IgE. The BAT assay was used to avoid enrolling further patients with reactive basophils. The safety profile is tolerable and maximum tolerated dose has not been reached, with evidence of anti-tumour activity observed in a patient with ovarian cancer. These results demonstrate the potential of IgE therapy for cancer., (© 2023. The Author(s).)

Background: Survival rates for ovarian cancer remain poor, and monitoring and prediction of therapeutic response may benefit from additional markers. Ovarian cancers frequently overexpress Folate Receptor alpha (FRα) and the soluble receptor (sFRα) is measurable in blood. Here we investigated sFRα as a potential biomarker., Methods: We evaluated sFRα longitudinally, before and during neo-adjuvant, adjuvant and palliative therapies, and tumour FRα expression status by immunohistrochemistry. The impact of free FRα on the efficacy of anti-FRα treatments was evaluated by an antibody-dependent cellular cytotoxicity assay., Results: Membrane and/or cytoplasmic FRα staining were observed in 52.7% tumours from 316 ovarian cancer patients with diverse histotypes. Circulating sFRα levels were significantly higher in patients, compared to healthy volunteers, specifically in patients sampled prior to neoadjuvant and palliative treatments. sFRα was associated with FRα cell membrane expression in the tumour. sFRα levels decreased alongside concurrent tumour burden in patients receiving standard therapies. High concentrations of sFRα partly reduced anti-FRα antibody tumour cell killing, an effect overcome by increased antibody doses., Conclusions: sFRα may present a non-invasive marker for tumour FRα expression, with the potential for monitoring patient response to treatment. Larger, prospective studies should evaluate FRα for assessing disease burden and response to systemic treatments., (© 2022. The Author(s).)

Purpose: To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)-resistant triple-negative breast cancer (TNBC) and ER+HER2- breast cancer disease., Experimental Design: Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2-n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2- cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC., Results: RCB II/III TNBC and ER+HER2- breast cancer were immunologically "cold" at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16-) within both the local TIME and peripheral white cell populations., Conclusions: This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients., (©2022 The Authors; Published by the American Association for Cancer Research.)

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20 + CD27 + IgD - isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers., (©2021 The Authors; Published by the American Association for Cancer Research.)

In breast cancer (BC), detecting low volumes of axillary lymph node (ALN) metastasis pre-operatively is difficult and novel biomarkers are needed. We recently showed that patient-derived ALNs can be sustained ex-vivo using normothermic perfusion. We now compare reactive (tumour-free; n = 5) and macrometastatic (containing tumour deposits >2 mm; n = 4) ALNs by combining whole section multiplex immunofluorescence with TMT-labelled LC-MS/MS of the circulating perfusate. Macrometastases contained significantly fewer B cells and T cells (CD4 + /CD8 + /regulatory) than reactive nodes (p = 0.02). Similarly, pathway analysis of the perfusate proteome (119/1453 proteins significantly differentially expressed) showed that immune function was diminished in macrometastases in favour of 'extracellular matrix degradation'; only 'neutrophil degranulation' was preserved. Qualitative comparison of the perfusate proteome to that of node-positive pancreatic and prostatic adenocarcinoma also highlighted 'neutrophil degranulation' as a contributing factor to nodal metastasis. Thus, metastasis-induced changes in the REPLICANT perfusate proteome are detectable, and could facilitate biomarker discovery.

Background: Major advances in breast cancer treatment have led to a reducuction in mortality. However, there are still women who are not cured. We hypothesize there is a sub-group of women with treatment-resistant cancers causing early death., Methods: Between 1975 and 2006, 5392 women with invasive breast cancer underwent surgery at Guy's Hospital, London. Data on patient demographics, tumour characteristics, treatment regimens, local recurrence, secondary metastasis, and death were prospectively recorded. We considered four time periods (1975-1982, 1983-1990, 1991-1998, 1999-2006). Risks and time to event analysis were performed with Cox proportional hazards model and Kaplan-Meier estimation., Results: Unadjusted hazard ratios for developing metastasis and overall mortality relative to the 1975-1982 cohort decreased steadily to 0.23 and 0.63, respectively in 1999-2006. However, metastasis-free interval shortened, with the proportion of women developing metastasis ≤5 years increasing from 73.9% to 83.0%. Furthermore, median post-metastatic survival decreased from 1.49 years to 0.94 years. Applying our risk criteria identified the presence of ±200 patients in each cohort who developed metastasis early and died within a much shorter time frame., Conclusions: Advances in treatment have decreased the risk of metastasis and improved survival in women with invasive breast cancer over the last 40 years. Despite this, a subpopulation with shorter metastasis-free and post-metastatic survival who are unresponsive to available treatment remains. This may be due to the ATRESS phenomenon (adjuvant therapy-related shortening of survival) secondary to preselection inherent in adjuvant therapy, successful treatment of less malignant tumour cells and treatment-induced resistance in the remaining tumour clones., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The rates and routes of lethal systemic spread in breast cancer are poorly understood owing to a lack of molecularly characterized patient cohorts with long-term, detailed follow-up data. Long-term follow-up is especially important for those with oestrogen-receptor (ER)-positive breast cancers, which can recur up to two decades after initial diagnosis 1-6 . It is therefore essential to identify patients who have a high risk of late relapse 7-9 . Here we present a statistical framework that models distinct disease stages (locoregional recurrence, distant recurrence, breast-cancer-related death and death from other causes) and competing risks of mortality from breast cancer, while yielding individual risk-of-recurrence predictions. We apply this model to 3,240 patients with breast cancer, including 1,980 for whom molecular data are available, and delineate spatiotemporal patterns of relapse across different categories of molecular information (namely immunohistochemical subtypes; PAM50 subtypes, which are based on gene-expression patterns 10,11 ; and integrative or IntClust subtypes, which are based on patterns of genomic copy-number alterations and gene expression 12,13 ). We identify four late-recurring integrative subtypes, comprising about one quarter (26%) of tumours that are both positive for ER and negative for human epidermal growth factor receptor 2, each with characteristic tumour-driving alterations in genomic copy number and a high risk of recurrence (mean 47-62%) up to 20 years after diagnosis. We also define a subgroup of triple-negative breast cancers in which cancer rarely recurs after five years, and a separate subgroup in which patients remain at risk. Use of the integrative subtypes improves the prediction of late, distant relapse beyond what is possible with clinical covariates (nodal status, tumour size, tumour grade and immunohistochemical subtype). These findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials.

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative 'BRCAness' subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected population (376 subjects; 188 carboplatin, 188 docetaxel), carboplatin was not more active than docetaxel (ORR, 31.4% versus 34.0%, respectively; P = 0.66). In contrast, in subjects with gBRCA-BC, carboplatin had double the ORR of docetaxel (68% versus 33%, respectively; biomarker, treatment interaction P = 0.01). Such benefit was not observed for subjects with BRCA1 methylation, BRCA1 mRNA-low tumors or a high score in a Myriad HRD assay. Significant interaction between treatment and the basal-like subtype was driven by high docetaxel response in the nonbasal subgroup. We conclude that patients with advanced TNBC benefit from characterization of BRCA1/2 mutations, but not BRCA1 methylation or Myriad HRD analyses, to inform choices on platinum-based chemotherapy. Additionally, gene expression analysis of basal-like cancers may also influence treatment selection.

The prognostic importance of lymph node (LN) status and tumour-infiltrating lymphocytes (TILs), is well established, particularly TILs in triple negative breast cancers (TNBCs). So far, few studies have interrogated changes in involved and uninvolved LNs and evaluated if their morphological patterns add valuable information for the prediction of disease progression in breast cancer. In a cohort of 309 patients enriched for TNBCs (170/309), we histologically characterised immune and stromal features in primary tumours and associated involved and uninvolved axillary LNs on routine haematoxylin and eosin stained sections. Of the 309 patients, 143 had LN-positive disease. Twenty-five histopathological features were assessed, including the degree of TIL presence, quantitative and qualitative assessment of germinal centres (GCs) and sinus histiocytosis. Multivariate and cross-validated proportional hazard regression analyses were used to identify optimal covariate sets for prediction of distant metastasis-free survival (DMFS). The degree of intratumoural and peritumoural immune infiltrate was associated with architectural changes in both uninvolved and involved LNs. By including clinicopathological characteristics as well as tumour and LN histopathological features in L2-regularised proportional hazard models, the prediction of 5-year DMFS was improved by 3-15% over the baseline in all cancers and in TNBCs. In LN-positive cancers, the combination of Salgado's classification, lymphocytic lobulitis, size and number of GCs in the uninvolved LNs and location of GCs in the involved LNs carried significant prognostic information. From these features, a multivariate cross-validation-stable risk signature was constructed, which identified low-risk groups within both LN-positive breast cancers and the LN-positive TNBCs group with a 10-year DMFS probability of 78 and 87%, respectively. This study illustrates that, by incorporating histopathological patterns of involved and uninvolved LNs combined with primary tumour immune and stromal features, the prediction of developing distant metastasis in LN-positive breast cancers can be estimated more accurately.

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt + group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3-stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt + ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083-96. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Unlabelled: Triple-negative breast cancers (TNBC) are characterized by a wide spectrum of genomic alterations, some of which might be caused by defects in DNA repair processes such as homologous recombination (HR). Despite this understanding, associating particular patterns of genomic instability with response to therapy has been challenging. Here, we show that allelic-imbalanced copy-number aberrations (AiCNA) are more prevalent in TNBCs that respond to platinum-based chemotherapy, thus providing a candidate predictive biomarker for this disease. Furthermore, we show that a high level of AiCNA is linked with elevated expression of a meiosis-associated gene, HORMAD1. Elevated HORMAD1 expression suppresses RAD51-dependent HR and drives the use of alternative forms of DNA repair, the generation of AiCNAs, as well as sensitizing cancer cells to HR-targeting therapies. Our data therefore provide a mechanistic association between HORMAD1 expression, a specific pattern of genomic instability, and an association with response to platinum-based chemotherapy in TNBC., Significance: Previous studies have shown correlation between mutational "scars" and sensitivity to platinums extending beyond associations with BRCA1/2 mutation, but do not elucidate the mechanism. Here, a novel allele-specific copy-number characterization of genome instability identifies and functionally validates the inappropriate expression of the meiotic gene HORMAD1 as a driver of HR deficiency in TNBC, acting to induce allelic imbalance and moderate platinum and PARP inhibitor sensitivity with implications for the use of such "scars" and expression of meiotic genes as predictive biomarkers., (©2015 American Association for Cancer Research.)

Background: Integrin αvβ6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer., Methods: Protein expression of integrin subunit beta6 (β6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student's t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni's Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided., Results: High expression of either the mRNA or protein for the integrin subunit β6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of β6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts., Conclusions: Targeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients., (© The Author 2014. Published by Oxford University Press.)

The HOX genes are a family of closely related transcription factors that help to define the identity of cells and tissues during embryonic development and which are also frequently deregulated in a number of malignancies, including breast cancer. While relatively little is known about the roles that individual HOX genes play in cancer, it is however clear that these roles can be both contradictory, with some members acting as oncogenes and some as tumor suppressors, and also redundant, with several genes essentially having the same function. Here, we have attempted to address this complexity using the HXR9 peptide to target the interaction between HOX proteins and PBX, a second transcription factor that serves as a common co-factor for many HOX proteins. We show that HXR9 causes apoptosis in a number of breast cancer-derived cell lines and that sensitivity to HXR9 is directly related to the averaged expression of HOX genes HOXB1 through to HOXB9, providing a potential biomarker to predict the sensitivity of breast tumors to HXR9 or its derivatives. Measuring the expression of HOX genes HOXB1-HOXB9 in primary tumors revealed that a subset of tumors show highly elevated expression indicating that these might be potentially very sensitive to killing by HXR9. Furthermore, we show that while HXR9 blocks the oncogenic activity of HOX genes, it does not affect the known tumor-suppressor properties of a subset of HOX genes in breast cancer.

Studies comparing the prognosis after contralateral breast cancer (CBC) with that after unilateral breast cancer (UBC) shows conflicting results. We assessed the risk of breast cancer-specific death for women with metachronous CBC compared to those with a UBC in 8,478 women with invasive primary breast cancer registered in the Guy's and St. Thomas' Breast Cancer Tissue and Data Bank. Risk factors associated with breast cancer-specific death for women with CBC were estimated using Cox proportional hazards modelling. Prognoses after UBC and CBC were compared, with survival time for women with CBC calculated: (i) from CBC, (ii) from the initial cancer with CBC as a time-dependent covariate. Women diagnosed with CBC within 5 years after the initial primary breast cancer had a worse prognosis than those with CBC after 5 years and those with UBC. Women with CBC who had positive lymph nodes at the initial breast cancer diagnosis were at an increased risk of dying from breast cancer compared to those without [HR 2.5 (95% CI 1.5-4.0)]. For all stages of the initial breast cancer, a worse prognosis was observed after CBC. CBC increased the hazard originating from the initial cancer at any follow-up time, but the highest hazards were associated with a short interval to CBC. Metachronous CBC adds to the risk of dying from breast cancer. The risk increases substantially when it occurs shortly after the initial cancer, indicating a CBC in some instances may be an indicator of active distant disease. The occurrence of CBC implies a new surveillance and therapeutic situation.

Breast cancer has a long natural history. Established and emerging biologic markers address overall risk but not necessarily timing of recurrence. 346 adjuvant naïve breast cancer cases from Guy's Hospital with 23 years minimum follow-up and archival blocks were recut and reassessed for hormone-receptors (HR), HER2-receptor and grade. Disease-specific survival (DSS) was analyzed by recursive partitioning. To validate insights from this analysis, gene-signatures (proliferative and HR-negative) were evaluated for their ability to predict early versus late metastatic risk in 683 node-negative, adjuvant naïve breast cancers annotated with expression microarray data. Risk partitioning showed that adjuvant naïve node-negative outcome risk was primarily partitioned by tumor receptor status and grade but not tumor size. HR-positive and HER2-negative (HRpos) risk was partitioned by tumor grade; low grade cases have very low early risk but a 20% fall-off in DSS 10 or more years after diagnosis. Higher grade HRpos cases have risk over >20 years. Triple-negative (Tneg) and HER2-positive (HER2pos) cases DSS events occurred primarily within the first 5 years. Among node-positive cases, only low grade conferred late risk, suggesting that proliferative gene signatures that identify proliferation would be important for predicting early but not late recurrence. Using pooled data from four publicly available data sets for node-negative tumors annotated with gene expression and outcome data, we evaluated four prognostic gene signatures: two proliferation-based and two immune function-based. Tumor proliferative capacity predicted early but not late metastatic risk for HRpos cases. The immune function or HRneg specific signatures predicted only early metastatic risk in Tneg and HER2pos cases. Breast cancer prognostic signatures need to inform both risk and timing of metastatic events and may best be applied within subsets. Current signatures predict for outcome risk within 5 years of diagnosis. Predictors of late risk for HR positive disease are needed.

Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individual's high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients.

Background: Given the large number of genes purported to be prognostic for breast cancer, it would be optimal if the genes identified are not confounded by the continuously changing systemic therapies. The aim of this study was to discover and validate a breast cancer prognostic expression signature for distant metastasis in untreated, early stage, lymph node-negative (N-) estrogen receptor-positive (ER+) patients with extensive follow-up times., Methods: 197 genes previously associated with metastasis and ER status were profiled from 142 untreated breast cancer subjects. A "metastasis score" (MS) representing fourteen differentially expressed genes was developed and evaluated for its association with distant-metastasis-free survival (DMFS). Categorical risk classification was established from the continuous MS and further evaluated on an independent set of 279 untreated subjects. A third set of 45 subjects was tested to determine the prognostic performance of the MS in tamoxifen-treated women., Results: A 14-gene signature was found to be significantly associated (p < 0.05) with distant metastasis in a training set and subsequently in an independent validation set. In the validation set, the hazard ratios (HR) of the high risk compared to low risk groups were 4.02 (95% CI 1.91-8.44) for the endpoint of DMFS and 1.97 (95% CI 1.28 to 3.04) for overall survival after adjustment for age, tumor size and grade. The low and high MS risk groups had 10-year estimates (95% CI) of 96% (90-99%) and 72% (64-78%) respectively, for DMFS and 91% (84-95%) and 68% (61-75%), respectively for overall survival. Performance characteristics of the signature in the two sets were similar. Ki-67 labeling index (LI) was predictive for recurrent disease in the training set, but lost significance after adjustment for the expression signature. In a study of tamoxifen-treated patients, the HR for DMFS in high compared to low risk groups was 3.61 (95% CI 0.86-15.14)., Conclusion: The 14-gene signature is significantly associated with risk of distant metastasis. The signature has a predominance of proliferation genes which have prognostic significance above that of Ki-67 LI and may aid in prioritizing future mechanistic studies and therapeutic interventions.

Immunohistochemistry allows specific proteins to be visualized while retaining cellular or tissue structure. Sensitivity of the technique has improved over the years from a single layer of labeled antibody to amplified, labeled polymer-based systems, which enable low levels of antigen to be detected in different types of tissue preparation. However, alongside selecting the most appropriate methods of antigen recovery and enhanced signal amplification is the need to show specificity. As described in this chapter, pertinent and reliable control material must be used to both optimize and monitor performance of novel breast-associated antibodies.

Tissue microarrays have been used effectively to study representative tissue from large groups of patients, with minimal technical and reagent costs. The construction of these arrays may appear complex, but with the use of a semiautomated tissue arrayer and a degree of manual dexterity, symmetrical, high-density arrays can be produced. Here, we highlight where problems in the construction, cutting, and evaluation of tissue microarrays can occur and how these can be prevented.

The aim of this study was to assess the expression of IGF-I and IGF-II in phyllodes tumours and fibroadenomas and to see if there is any correlation between nuclear beta-catenin expression and IGF-I and IGF-II expression in these tumours. In a previous study, it has been shown that Wnt signalling is important in the pathogenesis of phyllodes tumours of the breast. Epithelial Wnt5a overexpression and stromal Wnt2 overexpression were associated with abnormal, nuclear localization of beta-catenin in the stromal cells of these tumours. However, not all tumours with beta-catenin accumulation showed Wnt overexpression. One other possible cause of beta-catenin accumulation is overexpression of insulin-like growth factors (IGFs), as both IGF-I and IGF-II have been shown to stabilize beta-catenin. In this study, 30 fibroadenomas of the breast were assessed for beta-catenin expression using immunohistochemistry and the results were compared with previous data from 119 phyllodes tumours. In situ hybridization was used to assess IGF-I and IGF-II expression in 23 phyllodes tumours and 16 fibroadenomas. Nineteen phyllodes tumours (83%) showed widespread overexpression of IGF-II and 5/23 (22%) showed widespread overexpression of IGF-I. IGF-I expression correlated with nuclear beta-catenin staining in phyllodes tumours. Malignant phyllodes tumours showed no or little IGF-I expression. There was a degree of nuclear beta-catenin expression in the stroma (weak in 33%, moderate in 27%, and strong in 40%) in all fibroadenomas and nuclear beta-catenin staining correlated with IGF-I overexpression. Extensive IGF-II overexpression was also found in the majority of fibroadenomas (12/16). These results support the hypothesis that IGF-I and IGF-II overexpression may be important in the pathogenesis of fibroepithelial neoplasms of the breast and that IGF-I overexpression is likely to be contributing to the nuclear beta-catenin localization observed in the tumours., (Copyright 2003 John Wiley & Sons, Ltd.)