Jamie R. Kutasovic

Metastatic progression of breast cancer: insights from 50 years of autopsies

There remain no clear guidelines for the optimal management of patients with metastatic breast cancer. To better understand its natural history, we undertook a detailed examination of 197 autopsies performed on women who died of breast cancer. We reviewed clinical, treatment and pathological aspects of all cases and, additionally, pathological features and biomarker expression (ER, PgR, HER2, EGFR, p53, Ki67, c‐Kit, CK AE1/AE3) were assessed in detail for the primary tumour and matched metastases for 55 of the cases. Genomes of the primary tumour and multiple metastases were analysed by array‐based comparative genomic hybridization for six cases##. 945 metastatic deposits were identified, with a median of four/patient. The most common organs involved were lung/pleura (80%), bone (74%), liver (71%) and non‐axillary lymph nodes (55%). Major findings included: (a) patients with CNS metastases were more likely to have bone metastases (p < 0.013); (b) younger age was associated with metastasis to the liver (≤ 49 years; p < 0.001) and to gynaecological organs (≤ 49 years; p = 0.001); (c) surgical excision of the primary tumour was associated with metastasis to the liver (p = 0.002); and (d) ER and PgR showed down‐regulation during progression in a non‐random manner, particularly in lung/pleura (ER; p < 0.001), liver and bone metastases. Genomic analysis revealed DNA copy number variation between the primary tumour and metastases (e.g. amplification of 2q11.2–q12.1 and 10q22.2–q22.3) but little variation between metastases from the same patient. In summary, the association of CNS and bone metastases, liver and gynaecological metastases in young women and the risk of liver metastases following surgery have important implications for the management of patients with breast cancer. Clonal heterogeneity of the primary tumour is important in developing metastatic propensity and the change in tumour phenotype during progression/colonization highlights the importance of sampling metastatic disease for optimal treatment strategies.

Invasive lobular carcinoma of the breast: morphology, biomarkers and ’omics

Invasive lobular carcinoma of the breast is the most common ‘special’ morphological subtype of breast cancer, comprising up to 15% of all cases. Tumours are generally of a good prognostic phenotype, being low histological grade and low mitotic index, hormone receptor positive and HER2, p53 and basal marker negative, and with a generally good response to endocrine therapy. Despite this, clinicians face countless challenges in the diagnosis and long-term management of patients, as they encounter a tumour that can be difficult to detect through screening, elicits a very invasive nature, a propensity for widespread metastatic colonisation and, consequently, in some studies a worse long-term poor outcome compared with invasive carcinoma of no special type. Here we review the morphological and molecular features that underpin the disparate biological and clinical characteristics of this fascinating tumour type.

Meta-analysis of the global gene expression profile of triple-negative breast cancer identifies genes for the prognostication and treatment of aggressive breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking expression of estrogen and progesterone receptors (ER/PR) and HER2, thus limiting therapy options. We hypothesized that meta-analysis of TNBC gene expression profiles would illuminate mechanisms underlying the aggressive nature of this disease and identify therapeutic targets. Meta-analysis in the Oncomine database identified 206 genes that were recurrently deregulated in TNBC compared with non-TNBC and in tumors that metastasized or led to death within 5 years. This ‘aggressiveness gene list’ was enriched for two core functions/metagenes: chromosomal instability (CIN) and ER signaling metagenes. We calculated an ‘aggressiveness score’ as the ratio of the CIN metagene to the ER metagene, which identified aggressive tumors in breast cancer data sets regardless of subtype or other clinico-pathological indicators. A score calculated from six genes from the CIN metagene and two genes from the ER metagene recapitulated the aggressiveness score. By multivariate survival analysis, we show that our aggressiveness scores (from 206 genes or the 8 representative genes) outperformed several published prognostic signatures. Small interfering RNA screen revealed that the CIN metagene holds therapeutic targets against TNBC. Particularly, the inhibition of TTK significantly reduced the survival of TNBC cells and synergized with docetaxel in vitro. Importantly, mitosis-independent expression of TTK protein was associated with aggressive subgroups, poor survival and further stratified outcome within grade 3, lymph node-positive, HER2-positive and TNBC patients. In conclusion, we identified the core components of CIN and ER metagenes that identify aggressive breast tumors and have therapeutic potential in TNBC and aggressive breast tumors. Prognostication from these metagenes at the mRNA level was limited to ER-positive tumors. However, we provide evidence that mitosis-independent expression of TTK protein was prognostic in TNBC and other aggressive breast cancer subgroups, suggesting that protection of CIN/aneuploidy drives aggressiveness and treatment resistance.

Thrombospondin-4 expression is activated during the stromal response to invasive breast cancer

The thromobospondins are a family of extracellular glycoproteins that are activated during tissue remodeling processes such as embryogenesis, wound healing and cancer. Thrombospondin-4 (THBS4) is known to have roles in cellular migration, adhesion and attachment, as well as proliferation in different contexts. Data to support a role in cancer biology is increasing, including for gastrointestinal and prostate tumours. Here, using a combination of immunohistochemistry, immunofluorescence and analysis of publicly available genomic and expression data, we present the first study describing the pattern of expression of THBS4 in normal breast and breast cancer. THBS4 was located to the basement membrane of large ducts and vessels in normal breast tissue, but was absent from epithelium and extracellular matrix. There was a significant induction in expression in cancer-associated stroma relative to normal stroma (P = 0.0033), neoplastic epithelium (P < 0.0001) and normal epithelium (P < 0.0001). There was no difference in stromal expression of THBS4 between invasive ductal carcinomas (IDC) and invasive lobular carcinomas (ILC). The THBS4 mRNA levels were variable yet were generally highest in tumours typically rich in stromal content (ILC, ER positive low grade IDC; luminal A and normal-like subtypes). Genomic alterations of the THBS4 gene (somatic mutations and gene copy number) are rare suggesting this dramatic activation in expression is most likely dynamically regulated through the interaction between invading tumour cells and stromal fibroblasts in the local microenvironment. In summary, THBS4 expression in breast cancer-associated extracellular matrix contributes to the activated stromal response exhibited during tumour progression and this may facilitate invasion of tumour cells.

Evaluating the repair of DNA derived from formalin-fixed paraffin-embedded tissues prior to genomic profiling by SNP-CGH analysis

Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples. Owing to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromized, which means obtaining informative data regarding epigenetic, genomic, and expression alterations can be challenging. Here, we have investigated the utility of repairing damaged DNA derived from FFPE tumors prior to single-nucleotide polymorphism (SNP) arrays for whole-genome DNA copy number analysis. DNA was extracted from FFPE samples spanning five decades, involving tumor material obtained from surgical specimens and postmortems. Various aspects of the protocol were assessed, including the method of DNA extraction, the role of Quality Control quantitative PCR (qPCR) in predicting sample success, and the effect of DNA restoration on assay performance, data quality, and the prediction of copy number aberrations (CNAs). DNA that had undergone the repair process yielded higher SNP call rates, reduced log R ratio variance, and improved calling of CNAs compared with matched FFPE DNA not subjected to repair. Reproducible mapping of genomic break points and detection of focal CNAs representing high-level gains and homozygous deletions (HD) were possible, even on autopsy material obtained in 1974. For example, DNA amplifications at the ERBB2 and EGFR gene loci and a HD mapping to 13q14.2 were validated using immunohistochemistry, in situ hybridization, and qPCR. The power of SNP arrays lies in the detection of allele-specific aberrations; however, this aspect of the analysis remains challenging, particularly in the distinction between loss of heterozygosity (LOH) and copy neutral LOH. In summary, attempting to repair DNA that is damaged during fixation and storage may be a useful pretreatment step for genomic studies of large archival FFPE cohorts with long-term follow-up or for understanding rare cancer types, where fresh frozen material is scarce.

Novel highly specific anti-periostin antibodies uncover the functional importance of the fascilin 1-1 domain and highlight preferential expression of periostin in aggressive breast cancer

Periostin (POSTN), a secreted homodimeric protein that binds integrins αvβ3, αvβ5, and α6β4, was originally found to be expressed in fetal tissues and in the adult upon injury particularly bone fractures due to its role in remodelling and repair. Recently it was found to be over‐expressed in human breast cancer and a variety of other tumour types including head and neck squamous cell carcinoma, where its overexpression correlates with increased tumour invasion. Progress in studying its functional role in tumour pathogenesis has been hampered by the paucity of antibodies for its specific and sensitive detection. It has proven very difficult to obtain monoclonal antibodies (mAbs) against this highly conserved protein but we report here that combining infection of mice with lactate dehydrogenase elevating virus (LDV), a B cell activating arterivirus, with conjugation of human POSTN to ovalbumin as an immunogenic carrier, enabled us to develop six mAbs recognizing both human and mouse POSTN and inhibiting its binding to αvβ3 integrin. Two of the mAbs, MPB4B1 and MPC5B4, were tested and found to inhibit POSTN‐induced migration of human endothelial colony forming cells. All six mAbs recognized amino acids 136‐51 (APSNEAWDNLDSDIRR) within the POSTN fascilin (FAS) 1‐1 domain revealing the functional importance of this motif; this was further highlighted by the ability of aa 136–151 peptide to inhibit integrin‐mediated cell migration. Immunohistochemistry using MPC5B4, indicated that breast tumour cell POSTN expression was a strong prognostic indicator, along with tumour size, lymph node, and human epidermal growth factor receptor 2 (HER2) status.

An epithelial to mesenchymal transition programme does not usually drive the phenotype of invasive lobular carcinomas

Epithelial to mesenchymal transition (EMT) is a cellular phenotype switching phenomenon which occurs during normal development and is proposed to promote tumour cell invasive capabilities during tumour progression. Invasive lobular carcinoma (ILC) is a histological special type of breast cancer with a peculiar aetiology – the tumour cells display an invasive growth pattern, with detached, single cells or single files of cells, and a canonical feature is the loss of E‐cadherin expression. These characteristics are indicative of an EMT or at the very least that they represent some plasticity between phenotypes. While some gene expression profiling data support this view, the tumour cells remain epithelial and limited immunohistochemistry data suggest that EMT markers may not feature prominently in ILC. We assessed the expression of a panel of EMT markers (fibronectin, vimentin, N‐cadherin, smooth muscle actin, osteonectin, Snail, Twist) in 148 ILCs and performed a meta‐analysis of publically available molecular data from 154 ILCs. Three out of 148 (2%) ILCs demonstrated an early and coordinated alteration of multiple EMT markers (down‐regulation of E‐cadherin, nuclear TWIST, and up‐regulation of vimentin, osteonectin, and smooth muscle actin). However, the data overall do not support a role for EMT in defining the phenotypic peculiarities of the majority of ILCs. Copyright

Breast cancer metastasis to gynaecological organs: a clinico-pathological and molecular profiling study: Breast cancer metastasis to gynaecological organs

Breast cancer metastasis to gynaecological organs is an understudied pattern of tumour spread. We explored clinico‐pathological and molecular features of these metastases to better understand whether this pattern of dissemination is organotropic or a consequence of wider metastatic dissemination. Primary and metastatic tumours from 54 breast cancer patients with gynaecological metastases were analysed using immunohistochemistry, DNA copy‐number profiling, and targeted sequencing of 386 cancer‐related genes. The median age of primary tumour diagnosis amongst patients with gynaecological metastases was significantly younger compared to a general breast cancer population (46.5 versus 60 years; p < 0.0001). Median age at metastatic diagnosis was 54.4, time to progression was 4.8 years (range 0–20 years), and survival following a diagnosis of metastasis was 1.95 years (range 0–18 years). Patients had an average of five involved sites (most frequently ovary, fallopian tube, omentum/peritoneum), with fewer instances of spread to the lungs, liver, or brain. Invasive lobular histology and luminal A‐like phenotype were over‐represented in this group (42.8 and 87.5%, respectively) and most patients had involved axillary lymph nodes (p < 0.001). Primary tumours frequently co‐expressed oestrogen receptor cofactors (GATA3, FOXA1) and harboured amplifications at 8p12, 8q24, and 11q13. In terms of phenotype conversion, oestrogen receptor status was generally maintained in metastases, FOXA1 increased, and expression of progesterone receptor, androgen receptor, and GATA3 decreased. ESR1 and novel AR mutations were identified. Metastasis to gynaecological organs is a complication frequently affecting young women with invasive lobular carcinoma and luminal A‐like breast cancer, and hence may be driven by sustained hormonal signalling. Molecular analyses reveal a spectrum of factors that could contribute to de novo or acquired resistance to therapy and disease progression.

Mixed ductal-lobular carcinomas: evidence for progression from ductal to lobular morphology

Mixed ductal–lobular carcinomas (MDLs) show both ductal and lobular morphology, and constitute an archetypal example of intratumoural morphological heterogeneity. The mechanisms underlying the coexistence of these different morphological entities are poorly understood, although theories include that these components either represent ‘collision’ of independent tumours or evolve from a common ancestor. We performed comprehensive clinicopathological analysis of a cohort of 82 MDLs, and found that: (1) MDLs more frequently coexist with ductal carcinoma in situ (DCIS) than with lobular carcinoma in situ (LCIS); (2) the E‐cadherin–catenin complex was normal in the ductal component in 77.6% of tumours; and (3) in the lobular component, E‐cadherin was almost always aberrantly located in the cytoplasm, in contrast to invasive lobular carcinoma (ILC), where E‐cadherin is typically absent. Comparative genomic hybridization and multiregion whole exome sequencing of four representative cases revealed that all morphologically distinct components within an individual case were clonally related. The mutations identified varied between cases; those associated with a common clonal ancestry included BRCA2, TBX3, and TP53, whereas those associated with clonal divergence included CDH1 and ESR1. Together, these data support a model in which separate morphological components of MDLs arise from a common ancestor, and lobular morphology can arise via a ductal pathway of tumour progression. In MDLs that present with LCIS and DCIS, the clonal divergence probably occurs early, and is frequently associated with complete loss of E‐cadherin expression, as in ILC, whereas, in the majority of MDLs, which present with DCIS but not LCIS, direct clonal divergence from the ductal to the lobular phenotype occurs late in tumour evolution, and is associated with aberrant expression of E‐cadherin. The mechanisms driving the phenotypic change may involve E‐cadherin–catenin complex deregulation, but are yet to be fully elucidated, as there is significant intertumoural heterogeneity, and each case may have a unique molecular mechanism.

Phenotypic and molecular dissection of Metaplastic Breast Cancer and the prognostic implications: Prognostic features of Metaplastic breast cancer.

Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5‐year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology – the biological basis and clinical relevance of its seven sub‐categories are currently unclear. By establishing the Asia‐Pacific MBC (AP‐MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan‐cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for ‘mixed’ MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis‐1 (NF1) were also overrepresented [16.7% MBCs compared to ∼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed‐type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan‐cytokeratin expression are important prognostic markers. Copyright