Stromal lymphocytes are associated with upgrade of B3 breast lesions.

Various histopathological, clinical and imaging parameters have been evaluated to identify a subset of women diagnosed with lesions with uncertain malignant potential (B3 or BIRADS 3/4A lesions) who could safely be observed rather than being treated with surgical excision, with little impact on clinical practice. The primary reason for surgery is to rule out an upgrade to either ductal carcinoma in situ or invasive breast cancer, which occurs in up to 30% of patients. We hypothesised that the stromal immune microenvironment could indicate the presence of carcinoma associated with a ductal B3 lesion and that this could be detected in biopsies by counting lymphocytes as a predictive biomarker for upgrade. A higher number of lymphocytes in the surrounding specialised stroma was observed in upgraded ductal and papillary B3 lesions than non-upgraded (p < 0.01, negative binomial model, n = 307). We developed a model using lymphocytes combined with age and the type of lesion, which was predictive of upgrade with an area under the curve of 0.82 [95% confidence interval 0.77-0.87]. The model can identify some patients at risk of upgrade with high sensitivity, but with limited specificity. Assessing the tumour microenvironment including stromal lymphocytes may contribute to reducing unnecessary surgeries in the clinic, but additional predictive features are needed.

Spatial transcriptomics and the anatomical pathologist: Molecular meets morphology.

In recent years anatomical pathology has been revolutionised by the incorporation of molecular findings into routine diagnostic practice, and in some diseases the presence of specific molecular alterations are now essential for diagnosis. Spatial transcriptomics describes a group of technologies that provide up to transcriptome-wide expression profiling while preserving the spatial origin of the data, with many of these technologies able to provide these data using a single tissue section. Spatial transcriptomics allows expression profiling of highly specific areas within a tissue section potentially to subcellular resolution, and allows correlation of expression data with morphology, tissue type and location relative to other structures. While largely still research laboratory-based, several spatial transcriptomics methods have now achieved compatibility with formalin-fixed paraffin-embedded tissue (FFPE), allowing their use in diagnostic tissue samples, and with further development potentially leading to their incorporation in routine anatomical pathology practice. This mini review provides an overview of spatial transcriptomics methods, with an emphasis on platforms compatible with FFPE tissue, approaches to assess the data and potential applications in anatomical pathology practice.

Infiltrative pattern of invasion is independently associated with shorter survival and desmoplastic stroma markers FAP and THBS2 in mucinous ovarian carcinoma.

AIMS: Mucinous ovarian carcinoma (MOC) is a rare ovarian cancer histotype with generally good prognosis when diagnosed at an early stage. However, MOC with the infiltrative pattern of invasion has a worse prognosis, although to date studies have not been large enough to control for covariables. Data on reproducibility of classifying the invasion pattern are limited, as are molecular correlates for infiltrative invasion. We hypothesized that the invasion pattern would be associated with an aberrant tumour microenvironment. METHODS AND RESULTS: Four subspecialty pathologists assessed interobserver reproducibility of the pattern of invasion in 134 MOC. Immunohistochemistry on fibroblast activation protein (FAP) and THBS2 was performed on 98 cases. Association with survival was tested using Cox regression. The average interobserver agreement for the infiltrative pattern was moderate (kappa 0.60, agreement 86.3%). After reproducibility review, 24/134 MOC (18%) were determined to have the infiltrative pattern and this was associated with a higher risk of death, independent of FIGO stage, grade, and patient age in a time-dependent manner (hazard ratio [HR] = 10.2, 95% confidence interval [CI] 3.0-34.5). High stromal expression of FAP and THBS2 was more common in infiltrative MOC (FAP: 60%, THBS2: 58%, both P < 0.001) and associated with survival (multivariate HR for FAP: 1.5 [95% CI 1.1-2.1] and THBS2: 1.91 [95% CI 1.1-3.2]). CONCLUSIONS: The pattern of invasion should be included in reporting for MOC due to the strong prognostic implications. We highlight the histological features that should be considered to improve reproducibility. FAP and THBS2 are associated with infiltrative invasion in MOC.

TP53 as a Diagnostic Aid in the Distinction of Ovarian Mucinous Borderline Tumors From Mucinous Carcinoma.

Ovarian mucinous borderline tumors (MBTs) are clinically managed as benign neoplasms while the management of ovarian mucinous carcinomas (MC) is dependent on tumor stage. Despite the standardization of sampling of ovarian mucinous neoplasms, limited interobserver reproducibility between MBT and MC persists. Based on our recent finding that abnormal TP53 expression is associated with unfavorable outcome in MBT, we hypothesized that TP53 status might improve the reproducible distinction of MBT from MC. A virtual slide set of 85 consecutive ovarian mucinous neoplasms received at a single institution, with each case represented by 3 full sections, were reviewed by 3 pathologists in 2 iterations. The initial assessment was based solely on morphologic review, while the second iteration was performed with knowledge of TP53 status. The reproducibility of a trinary categorization (MBT, MBT with intraepithelial carcinoma [IEC], MC) significantly improved from a κ of 0.60 based on the initial morphologic assessment to a κ of 0.76 (t-test, P =0.0042) after consideration of TP53 immunohistochemistry (IHC) results. Six out of 85 patients died of disease, and in 2 of them, at least 1 pathologist assessed MBT with IEC and not MC even after integration of TP53 IHC. With the integration of TP53 IHC, substantial interobserver agreement for MBT and MC can be reached, particularly in cases with an uncertain degree of confluent growth. TP53 IHC can also be used to highlight and support the presence of IEC in MBT, however, discordances remained in 2 cases with adverse outcome.

Mucinous ovarian carcinoma: A survey of practice in Australia and New Zealand.

BACKGROUND: Mucinous ovarian carcinoma (MOC) is a rare ovarian cancer with limited evidence to support clinical care. AIMS: We undertook a clinician survey to better understand current practice in treating MOC in Australia and New Zealand, and to determine any features associated with variation in care. In addition, we aimed to understand future research priorities. METHODS: A RedCap survey was distributed to clinician members of the Australia New Zealand Gynaecological Oncology Group (ANZGOG). Questions included respondent demographics, three case studies and future research priorities. Clinicians were asked questions specific to their speciality. RESULTS: Respondents (n = 47) were commonly experienced gynae-oncology specialists, most often surgical (38%) or medical (30%) oncologists. There was good consensus for surgical approaches for stage I disease; however, variation in practice was noted for advanced or recurrent MOC. Variation was also observed for medical oncologists; in early-stage disease there was no clear consensus on whether to offer chemotherapy, or which regimen to recommend. For advanced and recurrent disease a wide range of chemotherapy options was considered, with a trend away from an ovarian-type toward gastrointestinal (GI)-type regimens in advanced MOC. This practice was reflected in future research priorities, with 'Is a GI chemotherapy regimen better than an ovarian regimen?' the most highly ranked option, followed by 'Should stage 1C patients receive chemotherapy?' CONCLUSIONS: Although the number of respondents limited the analyses, it was clear that chemotherapy selection was a key point of divergence for medical oncologists. Future research is needed to establish well-evidenced guidelines for clinical care of MOC.

Identifying recurrences and metastasis after ductal carcinoma in situ (DCIS) of the breast.

Ductal carcinoma in situ (DCIS) of the breast is a non-invasive tumour that has the potential to progress to invasive ductal carcinoma (IDC). Thus, it represents a treatment dilemma: alone it does not present a risk to life, however, left untreated it may progress to a life-threatening condition. Current clinico-pathological features cannot accurately predict which patients with DCIS have invasive potential, and therefore clinicians are unable to quantify the risk of progression for an individual patient. This leads to many women being over-treated, while others may not receive sufficient treatment to prevent invasive recurrence. A better understanding of the molecular features of DCIS, both tumour-intrinsic and the microenvironment, could offer the ability to better predict which women need aggressive treatment, and which can avoid therapies carrying significant side-effects and such as radiotherapy. In this review, we summarise the current knowledge of DCIS, and consider future research directions.

Validated biomarker assays confirm that ARID1A loss is confounded with MMR deficiency, CD8+ TIL infiltration, and provides no independent prognostic value in endometriosis-associated ovarian carcinomas.

ARID1A (BAF250a) is a component of the SWI/SNF chromatin modifying complex, plays an important tumour suppressor role, and is considered prognostic in several malignancies. However, in ovarian carcinomas there are contradictory reports on its relationship to outcome, immune response, and correlation with clinicopathological features. We assembled a series of 1623 endometriosis-associated ovarian carcinomas, including 1078 endometrioid (ENOC) and 545 clear cell (CCOC) ovarian carcinomas, through combining resources of the Ovarian Tumor Tissue Analysis (OTTA) Consortium, the Canadian Ovarian Unified Experimental Resource (COEUR), local, and collaborative networks. Validated immunohistochemical surrogate assays for ARID1A mutations were applied to all samples. We investigated associations between ARID1A loss/mutation, clinical features, outcome, CD8+ tumour-infiltrating lymphocytes (CD8+ TILs), and DNA mismatch repair deficiency (MMRd). ARID1A loss was observed in 42% of CCOCs and 25% of ENOCs. We found no associations between ARID1A loss and outcomes, stage, age, or CD8+ TIL status in CCOC. Similarly, we found no association with outcome or stage in endometrioid cases. In ENOC, ARID1A loss was more prevalent in younger patients (p = 0.012) and was associated with MMRd (p < 0.001) and the presence of CD8+ TILs (p = 0.008). Consistent with MMRd being causative of ARID1A mutations, in a subset of ENOCs we also observed an association with ARID1A loss-of-function mutation as a result of small indels (p = 0.035, versus single nucleotide variants). In ENOC, the association with ARID1A loss, CD8+ TILs, and age appears confounded by MMRd status. Although this observation does not explicitly rule out a role for ARID1A influence on CD8+ TIL infiltration in ENOC, given current knowledge regarding MMRd, it seems more likely that effects are dominated by the hypermutation phenotype. This large dataset with consistently applied biomarker assessment now provides a benchmark for the prevalence of ARID1A loss-of-function mutations in endometriosis-associated ovarian cancers and brings clarity to the prognostic significance. © 2021 The Pathological Society of Great Britain and Ireland.

Targeted therapy for mucinous ovarian carcinoma: evidence from clinical trials.

Mucinous ovarian carcinoma is a rare subtype of epithelial ovarian cancer. Despite being a chemoresistant tumour type, surgical resection and chemotherapy are still the current standard for management. This narrative review aims to explore the current evidence for targeted therapies in mucinous ovarian carcinoma. A review of the literature was performed to identify clinical trials and case reports of targeted therapy in patients with mucinous ovarian carcinoma. The databases and registers (PubMed, MEDLINE, Embase, Europe PMC, Cochrane Central Register of Clinical Trials, clinicaltrials.gov) were searched for articles published between January 2009 to June 2021 using keywords specific for mucinous ovarian carcinoma and targeted therapy. Records were screened and assessed for eligibility based on inclusion and exclusion criteria. From 684 records, 21 studies met the criteria to be included in the review. A total of 11 different targeted therapies were identified, each demonstrating varying degrees of clinical evidence supporting further investigation in patients with mucinous ovarian carcinoma. Targeted therapies identified in this review that warrant further investigations are bevacizumab, trastuzumab, nintedanib, AZD1775, sunitinib, cediranib and pazopanib. Many of the therapeutic agents may be investigated further in combination with other targeted therapies or chemotherapy. More clinical trials focusing on targeted therapy specifically in patients with mucinous ovarian cancer are required to inform clinical use. Multinational efforts are likely to be required to successfully conduct trials in this rare tumor type.

Aurora Kinase A Is an Independent Predictor of Invasive Recurrence in Breast Ductal Carcinoma in situ.

INTRODUCTION: Aurora Kinase A (AURKA/STK15) has a role in centrosome duplication and is a regulator of mitotic cell proliferation. It is over-expressed in breast cancer and other cancers, however; its role in ductal carcinoma in situ (DCIS) remains to be defined. This study aims to characterize AURKA protein expression in DCIS and evaluate its prognostic significance. METHODS: AURKA was assessed immunohistochemically in a large well-characterized cohort of DCIS (n = 776 pure DCIS and 239 DCIS associated with invasive breast cancer [DCIS-mixed]) with long-term follow-up data (median = 105 months) and basic molecular characterization. RESULTS: High AURKA expression was observed in 15% of DCIS cases and was associated with features of aggressiveness including larger tumour size, high nuclear grade, hormone receptor negativity, HER2 positivity, and high Ki67 proliferation index. AURKA expression was higher in DCIS associated with invasive breast cancer than in pure DCIS (p < 0.0001). In the DCIS-mixed cohort, the invasive component showed higher AURKA expression than the DCIS component (p < 0.0001). Outcome analysis revealed that AURKA was a predictor of invasive recurrence (p = 0.002). CONCLUSION: High AURKA expression is associated with poor prognosis in DCIS and might be a potential marker to predict DCIS progression to invasive disease.

Genomic analysis of low-grade serous ovarian carcinoma to identify key drivers and therapeutic vulnerabilities.

Low-grade serous ovarian carcinoma (LGSOC) is associated with a poor response to existing chemotherapy, highlighting the need to perform comprehensive genomic analysis and identify new therapeutic vulnerabilities. The data presented here represent the largest genetic study of LGSOCs to date (n = 71), analysing 127 candidate genes derived from whole exome sequencing cohorts to generate mutation and copy-number variation data. Additionally, immunohistochemistry was performed on our LGSOC cohort assessing oestrogen receptor, progesterone receptor, TP53, and CDKN2A status. Targeted sequencing identified 47% of cases with mutations in key RAS/RAF pathway genes (KRAS, BRAF, and NRAS), as well as mutations in putative novel driver genes including USP9X (27%), MACF1 (11%), ARID1A (9%), NF2 (4%), DOT1L (6%), and ASH1L (4%). Immunohistochemistry evaluation revealed frequent oestrogen/progesterone receptor positivity (85%), along with CDKN2A protein loss (10%) and CDKN2A protein overexpression (6%), which were linked to shorter disease outcomes. Indeed, 90% of LGSOC samples harboured at least one potentially actionable alteration, which in 19/71 (27%) cases were predictive of clinical benefit from a standard treatment, either in another cancer's indication or in LGSOC specifically. In addition, we validated ubiquitin-specific protease 9X (USP9X), which is a chromosome X-linked substrate-specific deubiquitinase and tumour suppressor, as a relevant therapeutic target for LGSOC. Our comprehensive genomic study highlighted that there is an addiction to a limited number of unique 'driver' aberrations that could be translated into improved therapeutic paths. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Refined cut-off for TP53 immunohistochemistry improves prediction of TP53 mutation status in ovarian mucinous tumors: implications for outcome analyses.

TP53 mutations are implicated in the progression of mucinous borderline tumors (MBOT) to mucinous ovarian carcinomas (MOC). Optimized immunohistochemistry (IHC) for TP53 has been established as a proxy for the TP53 mutation status in other ovarian tumor types. We aimed to confirm the ability of TP53 IHC to predict TP53 mutation status in ovarian mucinous tumors and to evaluate the association of TP53 mutation status with survival among patients with MBOT and MOC. Tumor tissue from an initial cohort of 113 women with MBOT/MOC was stained with optimized IHC for TP53 using tissue microarrays (75.2%) or full sections (24.8%) and interpreted using established criteria as normal or abnormal (overexpression, complete absence, or cytoplasmic). Cases were considered concordant if abnormal IHC staining predicted deleterious TP53 mutations. Discordant tissue microarray cases were re-evaluated on full sections and interpretational criteria were refined. The initial cohort was expanded to a total of 165 MBOT and 424 MOC for the examination of the association of survival with TP53 mutation status, assessed either by TP53 IHC and/or sequencing. Initially, 82/113 (72.6%) cases were concordant using the established criteria. Refined criteria for overexpression to account for intratumoral heterogeneity and terminal differentiation improved concordance to 93.8% (106/113). In the expanded cohort, 19.4% (32/165) of MBOT showed evidence for TP53 mutation and this was associated with a higher risk of recurrence, disease-specific death, and all-cause mortality (overall survival: HR = 4.6, 95% CI 1.5-14.3, p = 0.0087). Within MOC, 61.1% (259/424) harbored a TP53 mutation, but this was not associated with survival (overall survival, p = 0.77). TP53 IHC is an accurate proxy for TP53 mutation status with refined interpretation criteria accounting for intratumoral heterogeneity and terminal differentiation in ovarian mucinous tumors. TP53 mutation status is an important biomarker to identify MBOT with a higher risk of mortality.

Options for the Treatment of Mucinous Ovarian Carcinoma.

OPINION STATEMENT: Complete surgical resection is the gold-standard treatment for all mucinous ovarian carcinoma (MOC) cases. Advanced-stage disease is often additionally treated with adjuvant platinum-based chemotherapy; however, these were developed largely against the more common high-grade serous ovarian carcinoma and have low efficacy in treating MOC. More effective therapeutics are needed to treat late-stage and platinum-resistant tumors; however, traditional drug development and clinical trial paradigms are a major challenge for such a rare disease. New approaches to support evidence-based treatment decisions are required, such as registry trials. Recently, a number of targeted therapies have emerged as viable treatment options in other cancer types, and for some of these, the actionable tumor mutations are also seen in MOC. Thus, a promising alternative approach to provide benefit to current MOC patients involves DNA sequencing to identify a tumor's unique mutational profile and allow matching to available targeted agents. Such a pipeline can involve special approval to administer a drug already approved for clinical use in other cancer types to a given MOC patient, or their inclusion in existing ongoing clinical trials, such as basket trials encompassing patients with tumors from a range of anatomical sites. Implementation of such personalized medicine can be boosted using improved pre-clinical models, where through a clinical research collaboration a patient's own tumor cells can be used to a test a range of putative therapies prior to administration in the clinic, enabling selection of the available pharmaceutical/s that give any given patient the best possible chance of cancer remission.

The prognostic significance of immune microenvironment in breast ductal carcinoma in situ.

BACKGROUND: The role of different subtypes of tumour infiltrating lymphocytes (TILs) in breast ductal carcinoma in situ (DCIS) is still poorly defined. This study aimed to assess the prognostic significance of B and T lymphocytes and immune checkpoint proteins expression in DCIS. METHODS: A well characterised DCIS cohort (n = 700) with long-term follow-up comprising pure DCIS (n = 508) and DCIS mixed with invasive carcinoma (IBC; n = 192) were stained immunohistochemically for CD20, CD3, CD4, CD8, FOXP3, PD1 and PDL1. Copy number variation and TP53 mutation status were assessed in a subset of cases (n = 58). RESULTS: CD3+ lymphocytes were the predominant cell subtype in the pure DCIS cohort, while FOXP3 showed the lowest levels. PDL1 expression was mainly seen in the stromal TILs. Higher abundance of TILs subtypes was associated with higher tumour grade, hormone receptor negativity and HER2 positivity. Mutant TP53 variants were associated with higher levels of stromal CD3+, CD4+ and FOXP3+ cells. DCIS coexisting with invasive carcinoma harboured denser stromal infiltrates of all immune cells and checkpoint proteins apart from CD4+ cells. Stromal PD1 was the most differentially expressed protein between DCIS and invasive carcinoma (Z = 5.8, p < 0.0001). Dense TILs, stromal FOXP3 and PDL1 were poor prognostic factors for DCIS recurrence, while dense TILs were independently associated with poor outcome for all recurrences (HR = 7.0; p = 0.024), and invasive recurrence (HR = 2.1; p = 0.029). CONCLUSIONS: Immunosuppressive proteins are potential markers for high risk DCIS and disease progression. Different stromal and intratumoural lymphocyte composition between pure DCIS, DCIS associated with IBC and invasive carcinoma play a potential role in their prognostic significance and related to the underlying genomic instability. Assessment of overall TILs provides a promising tool for evaluation of the DCIS immune microenvironment.

Germline whole exome sequencing of a family with appendiceal mucinous tumours presenting with pseudomyxoma peritonei.

BACKGROUND: Familial cases of appendiceal mucinous tumours (AMTs) are extremely rare and the underlying genetic aetiology uncertain. We identified potential predisposing germline genetic variants in a father and daughter with AMTs presenting with pseudomyxoma peritonei (PMP) and correlated these with regions of loss of heterozygosity (LOH) in the tumours. METHODS: Through germline whole exome sequencing, we identified novel heterozygous loss-of-function (LoF) (i.e. nonsense, frameshift and essential splice site mutations) and missense variants shared between father and daughter, and validated all LoF variants, and missense variants with a Combined Annotation Dependent Depletion (CADD) scaled score of ≥10. Genome-wide copy number analysis was performed on tumour tissue from both individuals to identify regions of LOH. RESULTS: Fifteen novel variants in 15 genes were shared by the father and daughter, including a nonsense mutation in REEP5. None of these germline variants were located in tumour regions of LOH shared by the father and daughter. Four genes (EXOG, RANBP2, RANBP6 and TNFRSF1B) harboured missense variants that fell in a region of LOH in the tumour from the father only, but none showed somatic loss of the wild type allele in the tumour. The REEP5 gene was sequenced in 23 individuals with presumed sporadic AMTs or PMP; no LoF or rare missense germline variants were identified. CONCLUSION: Germline exome sequencing of a father and daughter with AMTs identified novel candidate predisposing genes. Further studies are required to clarify the role of these genes in familial AMTs.

Therapeutic options for mucinous ovarian carcinoma.

OBJECTIVE: Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. METHODS: We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117-166). RESULTS: Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). CONCLUSIONS: Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

Atypical ductal hyperplasia is a multipotent precursor of breast carcinoma.

The current model for breast cancer progression proposes independent 'low grade (LG)-like' and 'high grade (HG)-like' pathways but lacks a known precursor to HG cancer. We applied low-coverage whole-genome sequencing to atypical ductal hyperplasia (ADH) with and without carcinoma to shed light on breast cancer progression. Fourteen out of twenty isolated ADH cases harboured at least one copy number alteration (CNA), but had fewer aberrations than LG or HG ductal carcinoma in situ (DCIS). ADH carried more HG-like CNA than LG DCIS (e.g. 8q gain). Correspondingly, 64% (7/11) of ADH cases with synchronous HG carcinoma were clonally related, similar to LG carcinoma (67%, 6/9). This study represents a significant shift in our understanding of breast cancer progression, with ADH as a common precursor lesion to the independent 'low grade-like' and 'high grade-like' pathways. These data suggest that ADH can be a precursor of HG breast cancer and that LG and HG carcinomas can evolve from a similar ancestor lesion. We propose that although LG DCIS may be committed to a LG molecular pathway, ADH may remain multipotent, progressing to either LG or HG carcinoma. This multipotent nature suggests that some ADH cases could be more clinically significant than LG DCIS, requiring biomarkers for personalising management. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular comparison of interval and screen-detected breast cancers.

Breast cancer (BC) diagnosed after a negative mammogram but prior to the next screening episode is termed an 'interval BC' (IBC). Understanding the molecular differences between IBC and screen-detected BCs (SDBC) could improve mammographic screening and management options. Therefore, we assessed both germline and somatic genomic aberrations in a prospective cohort. Utilising the Lifepool cohort of >54 000 women attending mammographic screening programs, 930 BC cases with screening status were identified (726 SDBC and 204 IBC). Clinico-pathological and family history information were recorded. Germline and tumour DNA were collected where available and sequenced for BC predisposition and driver gene mutations. Compared to SDBC, IBCs were significantly associated with a younger age at diagnosis and tumour characteristics associated with worse prognosis. Germline DNA assessment of BC cases that developed post-enrolment (276 SDBCs and 77 IBCs) for pathogenic mutations in 12 hereditary BC predisposition genes identified 8 carriers (2.27%). The germline mutation frequency was higher in IBC versus SDBC, although not statistically significant (3.90% versus 1.81%, p = 0.174). Comparing somatic genetic features of IBC and SDBC matched for grade, histological subtype and hormone receptor revealed no significant differences, with the exception of higher homologous recombination deficiency scores in IBC, and copy number changes on chromosome Xq in triple negative SDBCs. Our data demonstrates that while IBCs are clinically more aggressive than SDBC, when matched for confounding clinico-pathological features they do not represent a unique molecular class of invasive BC, but could be a consequence of timing of tumour initiation and mammographic screening. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The clinical and biological significance of HER2 over-expression in breast ductal carcinoma in situ: a large study from a single institution.

BACKGROUND: Previous studies have reported up to 50% of ductal carcinoma in situ (DCIS), is HER2 positive, but the frequency of HER2-positive invasive breast cancer (IBC) is lower. The aim of this study is to characterise HER2 status in DCIS and assess its prognostic value. METHODS: HER2 status was evaluated in a large series of DCIS (n = 868), including pure DCIS and DCIS associated with IBC, prepared as tissue microarrays (TMAs). HER2 status was assessed using immunohistochemistry (IHC) and chromogenic in situ hybridisation (CISH). RESULTS: In pure DCIS, HER2 protein was over-expressed in 9% of DCIS (3+), whereas 15% were HER2 equivocal (2+). Using CISH, the final HER2 status was positive in 20%. In mixed DCIS, HER2 amplification of the DCIS component was detected in 15% with amplification in the invasive component of only 12%. HER2-positive DCIS was associated with features of aggressiveness (p < 0.0001) and more frequent local recurrence (p = 0.03). On multivariate analysis, combined HER2+/Ki67+ profile was an independent predictor of local recurrence (p = 0.006). CONCLUSIONS: The frequency of HER2 positivity in DCIS is comparable to IBC- and HER2-positive DCIS is associated with features of poor prognosis. The majority of HER2 over-expression in DCIS is driven by gene amplification.

Legumain is an independent predictor for invasive recurrence in breast ductal carcinoma in situ.

Legumain is a proteolytic enzyme that plays a role in the regulation of cell proliferation in invasive breast cancer. Studies evaluating its role in ductal carcinoma in situ (DCIS) are lacking. Here, we aimed to characterize legumain protein expression in DCIS and evaluate its prognostic significance. Legumain was assessed immunohistochemically in a tissue microarray of a well-characterized cohort of DCIS (n = 776 pure DCIS and n = 239 DCIS associated with invasive breast cancer (DCIS-mixed)). Legumain immunoreactivity was scored in tumor cells and surrounding stroma and related to clinicopathological parameters and patient outcome. High legumain expression was observed in 23% of pure DCIS and was associated with features of high-risk DCIS including higher nuclear grade, comedo necrosis, hormone receptor negativity, HER2 positivity, and higher proliferation index. Legumain expression was higher in DCIS associated with invasive breast cancer than in pure DCIS (p < 0.0001). In the DCIS-mixed cohort, the invasive component showed higher legumain expression than the DCIS component (p < 0.0001). Legumain was an independent predictor of shorter local recurrencefree interval for all recurrences (p = 0.0003) and for invasive recurrences (p = 0.002). When incorporated with other risk factors, legumain provided better patient risk stratification. High legumain expression is associated with poor prognosis in DCIS and could be a potential marker to predict DCIS progression to invasive disease.

Geometric characteristics of collagen have independent prognostic significance in breast ductal carcinoma in situ: an image analysis study.

Collagen plays a key role in normal and malignant tissue homeostasis. While the prognostic significance of collagen fiber remodeling in invasive breast cancer has been studied, its role in ductal carcinoma in situ (DCIS) remains poorly defined. Using image analysis, we aimed to evaluate the prognostic significance of the geometric characteristics of collagen surrounding DCIS. A large well-characterized cohort of DCIS comprising pure DCIS (n = 610) and DCIS coexisting with invasive carcinoma (n = 180) were histochemically stained for collagen using picrosirius red. ImageJ software was used to assess collagen density, degree of collagen fiber dispersion and directionality in relation to DCIS ducts' boundary. We developed a collagen prognostic index and evaluated its prognostic significance. A poor index was observed in 24% of the pure DCIS and was associated with determinants of high-risk DCIS including higher nuclear grade, comedo type necrosis, hormonal receptor negativity, HER2 positivity and high proliferation index. High collagen prognostic index was associated with the collagen remodeling protein prolyl-4-hydroxlase alpha subunit 2 and the hypoxia-related protein hypoxia inducible factor 1α. DCIS coexisting with invasive breast cancer had a higher collagen prognostic index than pure DCIS ( p < 0.0001). High index was an independent poor prognostic factor for DCIS recurrence for all recurrences (HR = 2.3, p = 0.005) and just invasive recurrences (HR = 3.4, p = 0.003). Interaction between collagen prognostic index and radiotherapy showed that the index was associated with poor outcome even with adjuvant radiotherapy ( p = 0.0001). Collagen reorganization around DCIS is associated with poor outcome and provides a potential predictor for disease progression and resistance to radiotherapy. Mechanistic studies are warranted to decipher the underlying mechanisms.