Artificial intelligence-based digital scores of stromal tumour-infiltrating lymphocytes and tumour-associated stroma predict disease-specific survival in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is known to have a relatively poor outcome with variable prognoses, raising the need for more informative risk stratification. We investigated a set of digital, artificial intelligence (AI)-based spatial tumour microenvironment (sTME) features and explored their prognostic value in TNBC. After performing tissue classification on digitised haematoxylin and eosin (H&E) slides of TNBC cases, we employed a deep learning-based algorithm to segment tissue regions into tumour, stroma, and lymphocytes in order to compute quantitative features concerning the spatial relationship of tumour with lymphocytes and stroma. The prognostic value of the digital features was explored using survival analysis with Cox proportional hazard models in a cross-validation setting on two independent international multi-centric TNBC cohorts: The Australian Breast Cancer Tissue Bank (AUBC) cohort (n = 318) and The Cancer Genome Atlas Breast Cancer (TCGA) cohort (n = 111). The proposed digital stromal tumour-infiltrating lymphocytes (Digi-sTILs) score and the digital tumour-associated stroma (Digi-TAS) score were found to carry strong prognostic value for disease-specific survival, with the Digi-sTILs and Digi-TAS scores giving C-index values of 0.65 (p = 0.0189) and 0.60 (p = 0.0437), respectively, on the TCGA cohort as a validation set. Combining the Digi-sTILs feature with the patient's positivity status for axillary lymph nodes yielded a C-index of 0.76 on unseen validation cohorts. We surmise that the proposed digital features could potentially be used for better risk stratification and management 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.

Complete pathological response following levonorgestrel intrauterine device in clinically stage 1 endometrial adenocarcinoma: Results of a randomized clinical trial.

PURPOSE: Intrauterine levonorgestrel (LNG-IUD) is used to treat patients with endometrial adenocarcinoma (EAC) and endometrial hyperplasia with atypia (EHA) but limited evidence is available on its effectiveness. The study determined the extent to which LNG-IUD with or without metformin (M) or weight loss (WL) achieves a pathological complete response (pCR) in patients with EAC or EHA. PATIENTS AND METHODS: This phase II randomized controlled clinical trial enrolled patients with histologically confirmed, clinically stage 1 FIGO grade 1 EAC or EHA; a body mass index > 30 kg/m2; a depth of myometrial invasion of less than 50% on MRI; a serum CA125 ≤ 30 U/mL. All patients received LNG-IUD and were randomized to observation (OBS), M (500 mg orally twice daily), or WL (pooled analysis). The primary outcome measure was the proportion of patients developing a pCR (defined as absence of any evidence of EAC or EHA) after 6 months. RESULTS: From December 2012 to October 2019, 165 patients were enrolled and 154 completed the 6-months follow up. Women had a mean age of 53 years, and a mean BMI of 48 kg/m2. Ninety-six patients were diagnosed with EAC (58%) and 69 patients with EHA (42%). Thirty-five participants were randomized to OBS, 36 to WL and 47 to M (10 patients were withdrawn). After 6 months the rate of pCR was 61% (95% CI 42% to 77%) for OBS, 67% (95% CI 48% to 82%) for WL and 57% (95% CI 41% to 72%) for M. Across the three treatment groups, the pCR was 82% and 43% for EHA and EAC, respectively. CONCLUSION: Complete response rates at 6 months were encouraging for patients with EAC and EHA across the three groups. TRIAL REGISTRATION: U.S. National Library of Medicine, NCT01686126.

Improved relapse-free survival on aromatase inhibitors in breast cancer is associated with interaction between oestrogen receptor-α and progesterone receptor-b.

BACKGROUND: Recent pre-clinical studies indicate that activated progesterone receptor (PR) (particularly the PR-B isoform) binds to oestrogen receptor-α (ER) and reprogrammes transcription toward better breast cancer outcomes. We investigated whether ER and PR-B interactions were present in breast tumours and associated with clinical parameters including response to aromatase inhibitors. METHODS: We developed a proximity ligation assay to detect ER and PR-B (ER:PR-B) interactions in formalin-fixed paraffin-embedded tissues. The assay was validated in a cell line and patient-derived breast cancer explants and applied to a cohort of 229 patients with ER-positive and HER2-negative breast cancer with axillary nodal disease. RESULTS: Higher frequency of ER:PR-B interaction correlated with increasing patient age, lower tumour grade and mitotic index. A low frequency of ER:PR-B interaction was associated with higher risk of relapse. In multivariate analysis, ER:PR-B interaction frequency was an independent predictive factor for relapse, whereas PR expression was not. In subset analysis, low frequency of ER:PR-B interaction was predictive of relapse on adjuvant aromatase inhibitor (HR 4.831, p = 0.001), but not on tamoxifen (HR 1.043, p = 0.939). CONCLUSIONS: This study demonstrates that ER:PR-B interactions have utility in predicting patient response to adjuvant AI therapy.

Ectopic Intralaryngo-Tracheal Thyroid Tissue Causing Neonatal Death.

INTRODUCTION: Ectopic thyroid tissue can be found anywhere along the embryologic path of thyroid descent. Intralaryngo-tracheal thyroid tissue is the least common site of ectopia and can present with upper airways obstruction. Its presentation in the neonate is exceptional. CASE REPORT: We describe a term female neonate with subglottic thyroid tissue causing near-total occlusion of the larynx, which led to upper airways obstruction and neonatal death. CONCLUSION: This emphasizes the importance of considering intralaryngo-tracheal tumors as a cause of acute and otherwise unexplainable respiratory distress immediately after birth. The cause of this neonatal death would not have been elucidated without careful autopsy examination.

Cell line and patient-derived xenograft models reveal elevated CDCP1 as a target in high-grade serous ovarian cancer.

BACKGROUND: Development of targeted therapies for high-grade serous ovarian cancer (HGSC) remains challenging, as contributing molecular pathways are poorly defined or expressed heterogeneously. CUB-domain containing protein 1 (CDCP1) is a cell-surface protein elevated in lung, colorectal, pancreas, renal and clear cell ovarian cancer. METHODS: CUB-domain containing protein 1 was examined by immunohistochemistry in HGSC and fallopian tube. The impact of targeting CDCP1 on cell growth and migration in vitro, and intraperitoneal xenograft growth in mice was examined. Three patient-derived xenograft (PDX) mouse models were developed and characterised for CDCP1 expression. The effect of a monoclonal anti-CDCP1 antibody on PDX growth was examined. Src activation was assessed by western blot analysis. RESULTS: Elevated CDCP1 was observed in 77% of HGSC cases. Silencing of CDCP1 reduced migration and non-adherent cell growth in vitro and tumour burden in vivo. Expression of CDCP1 in patient samples was maintained in PDX models. Antibody blockade of CDCP1 significantly reduced growth of an HGSC PDX. The CDCP1-mediated activation of Src was observed in cultured cells and mouse xenografts. CONCLUSIONS: CUB-domain containing protein 1 is over-expressed by the majority of HGSCs. In vitro and mouse model data indicate that CDCP1 has a role in HGSC and that it can be targeted to inhibit progression of this cancer.

Ovarian Microcystic Stromal Tumor: A Rare Clinical Manifestation of Familial Adenomatous Polyposis.

Microcystic stromal tumor (MST) is a rare tumor of presumed sex-cord stromal differentiation. We present a case of MST arising within a patient with constitutional 5q deletion syndrome, whose deletion encompassed the APC gene. Genomic analysis of the MST revealed a point mutation in the remaining APC allele, predicted to result in abnormal splicing of Exon 7. Subsequent clinical investigation revealed multiple gastrointestinal polyps qualifying for a diagnosis of familial adenomatous polyposis. This case emphasizes the importance of an aberrant Wnt/ß-catenin pathway in the development of MST and adds credence to the inclusion of MST as a rare phenotype of familial adenomatous polyposis. In a search for additional genetic aberrations which may contribute to the development of this rare tumor, genomic analysis revealed a frameshift mutation in FANCD2, a protein which plays a key role in DNA repair. This protein is expressed in human ovarian stromal cells and FANCD2-knockout mice are known to develop sex cord-stromal tumors, factors which further support a possible role of aberrant FANCD2 in the development of MST.

Interstitial lung disease in infancy: A general approach.

Childhood Interstitial lung disease (chILD) is an umbrella term used to define a broad range of rare, diffuse pulmonary disorders with altered interstitial structure that leads to abnormal gas exchange. Presentation of chILD in infancy can be difficult to differentiate from other common causes of diffuse lung disease. This article aimed at paediatricians provides an overview of interstitial lung disease presenting in infancy and includes key clinical features, a suggested approach to investigation and a summary of management. An overview of three clinical cases has been included to demonstrate the diagnostic approach, characteristic investigation findings and varied clinical outcomes.

Genetic segregation of inflammatory lung disease and autoimmune disease severity in SHIP-1-/- mice.

Alternatively activated M2 macrophages are implicated as both regulators and agents of lung disease, but their control is poorly understood. SHIP-1 is a 5' inositol phosphatase that negatively regulates the PI3K signaling pathway implicated in inflammation. SHIP-1-deficient mice have defects in hematopoiesis and B cell development, and die prematurely due to consolidation of lungs with M2-skewed macrophages. SHIP-1 is thought to restrain M2 macrophage polarization, with deregulated M2 skewing coinciding with severe lung disease in SHIP-1-deficient mice. To determine the influence of genetic background on the lung phenotype in SHIP-1(-/-) mice, we backcrossed the SHIP-1 null mutation onto C57BL/6 (Th2-resistant) and BALB/c (Th2-prone) backgrounds. Remarkably, we found that inflammatory lung disease was severe in C57.SHIP-1(-/-) mice, but absent in BALB.SHIP-1(-/-) mice. C57.SHIP-1(-/-), but not BALB.SHIP-1(-/-) mice had greatly increased myeloid progenitors, myeloid hyperplasia, markedly enhanced numbers of activated alveolar macrophages, and elevated amounts of Th2 and proinflammatory cytokines in bronchoalveolar lavage fluid and serum, suggesting that deregulated cytokine production induced disease. C57.SHIP-1(-/-) mice also developed severe B cell-dependent autoimmune disease, which was markedly attenuated on the BALB/c background. These data demonstrate that, contrary to current concepts, loss of SHIP-1 alone is not sufficient to cause lung inflammation, with disease only manifest on a permissive genetic background. This finding questions the nature of the lung disease in SHIP-1(-/-) mice, suggesting that its M2 classification is not strictly correct. Future identification of disease-promoting loci might reveal determinants of comorbid lung disease and autoimmunity and uncover potentially useful therapeutic targets.

Assessing HER2 amplification in breast cancer: findings from the Australian In Situ Hybridization Program.

In August 2006, the Australian government approved subsidized trastuzumab therapy for human epidermal growth factor receptor 2 (HER2)-positive early breast cancer, and it was mandated that HER2 testing should be performed using in situ hybridization (ISH) rather than immunohistochemistry (IHC). Here we review results of the first regulated, nationwide program to provide HER2 ISH testing for all newly diagnosed breast cancer patients, with a particular emphasis on cases where IHC and ISH results were discordant. Data from all laboratories participating in the program were collated. Cases with an equivocal ISH test result [by chromogenic ISH (CISH) or silver ISH (SISH)] were tested centrally by fluorescence ISH. Most laboratories also performed HER2 IHC, and 200 cases with discordant IHC and ISH results were selected for further analysis in a central laboratory. A total of 26 laboratories were involved and 53,402 tests were reported. Over a 4-year period the HER2 positivity rate decreased for primary cancers from 23.8 to 14.6 %, but remained relatively constant for samples from metastases. Average ISH reporting times were <5 days for all yearly reporting periods. Test-repeat rates decreased for CISH (8.9-3.6 %) and SISH (13.7-8.4 %). Only 12 of 196 cases remained discordant after retesting in a central laboratory. These findings demonstrate the successful implementation of a regulated, national program that continues to collect data on HER2 status. The results also highlight the differences in IHC interpretation between local laboratories and a central, more experienced, laboratory. This model could be used to establish future biomarker-testing programs in other countries.

Bodyweight and other correlates of symptom-detected breast cancers in a population offered screening.

OBJECTIVE: To determine the factors associated with symptom-detected breast cancers in a population offered screening. METHODS: We interviewed 1,459 Australian women aged 40-69, 946 with symptom-detected and 513 with mammogram-detected invasive breast cancers ≥ 1.1 cm in diameter about their personal, mammogram, and breast histories before diagnosis and reviewed medical records for tumor characteristics and mammogram dates, calculating ORs and 95% confidence intervals (CIs) for symptom- versus mammogram-detected cancers in logistic regression models. RESULTS: Lack of regular mammograms (<2 mammograms in the 4.5 years before diagnosis) was the strongest correlate of symptom-detected breast cancer (OR = 3.04 for irregular or no mammograms). In women who had regular mammograms (≥ 2 mammograms in the 4.5 years before diagnosis), the independent correlates of symptom-detected cancers were low BMI (OR < 25 kg/m(2) vs. ≥ 30 kg/m(2) = 2.18, 95% CI 1.23-3.84; p = 0.008), increased breast density (available in 498 women) (OR highest quarter vs. lowest = 3.50, 95% CI 1.76-6.97; p (trend) = 0.004), high-grade cancer, and a larger cancer (each p < 0.01). In women who did not have regular mammograms, the independent correlates were age <50 years, a first cancer, and a ≥ 2-cm cancer. Smoking appeared to modify the association of symptom-detected cancer with low BMI (higher ORs for low BMI in current smokers) and estrogen receptor (ER) status (higher ORs for low BMI in ER cancers). CONCLUSION: Women with low BMI may benefit from a tailored approach to breast cancer detection, particularly if they smoke.

Increased plasma concentrations of anterior gradient 2 protein are positively associated with ovarian cancer.

Ovarian cancer is often asymptomatic and is diagnosed at an advanced stage with poor survival rates, thus there is an urgent need to develop biomarkers for earlier detection of ovarian cancer. In the present study, we demonstrate for the first time that the previously reported metastasis-inducing protein AGR2 (anterior gradient protein 2) can be detected in the blood of ovarian cancer patients. Using a newly developed ELISA, we show significantly increased concentrations of AGR2 protein in plasma from cancer patients relative to normal controls. Plasma AGR2 concentrations were highest in stages II and III ovarian cancer patients and were similarly elevated in patients with both serous and non-serous tumours. The identification of elevated plasma concentrations of AGR2 may provide a useful biomarker to aid in the discrimination of normal and ovarian cancer patients particularly when used in combination with CA125.

Disruption of Glycogen Utilization Markedly Improves the Efficacy of Carboplatin against Preclinical Models of Clear Cell Ovarian Carcinoma.

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses-of more than 10-fold lower than previously reported for other cancers-significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

Effects of life event stress and social support on the odds of a >or= 2 cm breast cancer.

OBJECTIVE: To examine the contribution of life event and social support factors to diagnosis with a >or= 2 cm breast cancer. METHODS: We studied 1,459 Australian women aged 40-69 diagnosed in 2002-2003 with a first primary invasive breast cancer 1.1 cm or larger. We measured stressful life events, perceived stress levels, and social support in the year before diagnosis and collected information on other potential risk factors and confounders. RESULTS: The odds of a >or= 2 cm breast cancer relative to a 1.1-1.9 cm breast cancer were reduced in women who reported tension or change in an intimate relationship in the year before diagnosis (OR=0.71 95% CI 0.54-0.92; p=0.009); the reduction was greatest in women living with a partner (OR=0.64 95% CI 0.47-0.88; p=0.006) and was largely unaffected by adjustment for other variables independently associated with a >or= 2 cm breast cancer in our study. There was no evidence that the total number or severity of all studied life events influenced cancer size. Low partner support increased the odds of a >or= 2 cm cancer but only in women not living with a partner. CONCLUSION: Intimate relationship stress may reduce risk of a >or= 2 cm breast cancer. Suppression by stress of estrogen synthesis and metabolism is a possible mechanism.

DNA extraction from placental, fetal and neonatal tissue at autopsy: what organ to sample for DNA in the genomic era?

Incorporation of genome and exome sequencing into fetal and neonatal autopsy investigations has been shown to improve diagnostic yield. This requires deoxyribonucleic acid (DNA) to be extracted from either the placenta or autopsy tissue for molecular testing. However, the sources and quality of DNA obtained are highly variable and there are no adequate published data on what tissue is most ideal to sample for DNA extraction in this setting. Here we compare the quality of DNA extracted from sampling the placenta and various solid organs at fetal and neonatal autopsy, thereby determining the optimal tissue from which to source DNA for ancillary testing as part of the modern perinatal autopsy. A total of 898 tissue samples were obtained at autopsy from 176 fetuses (gestational ages 17-40 weeks) and 44 neonates (age range 0-28 days) at our tertiary institution. Fetal tissue was processed using the QIAsymphony DSP DNA Mini kit and placental tissue was extracted using the New iGENatal Kit. DNA concentration was quantified using the Qubit dsDNA BR Assay Kit. DNA integrity, as stratified by gel electrophoresis was classified as high (≥5 kb) or low quality (<5 kb). Genome sequencing was performed on the extracted DNA, together with respective parental DNA from blood samples, and confirmed absence of maternal contamination in all cases. Analyses used logistic mixed models to test for associations between tissue types, intrauterine retention times, delivery to autopsy and death to autopsy intervals with DNA quality. In the fetal cohort, the placenta had the highest proportion of high quality DNA samples (93.1%), and liver had the lowest proportion (35.3%). Among the neonates, all tissue samples with the exception of liver had over 88% high DNA quality with the placenta also yielding the highest quality (100%). There was statistically significant deterioration in DNA quality with prolonged time interval between demise and autopsy (≥5 days). In the 726 fetal samples, the odds of obtaining higher quality DNA from the placenta, thymus, and spleen were 70.4 [95% confidence interval (CI) 29.2-169.6], 3.6 (95% CI 2.0-6.6) and 3.3 (95% CI 1.8-6.1) times, respectively, more likely than samples from the liver (p values <0.001). DNA yield from other fetal solid organs investigated was not significantly superior to that from the liver. This study shows that, when available, refrigerated unfixed placenta is the most suitable source of high quality DNA during perinatal investigations. Of the solid fetal organs sampled at autopsy, lymphocyte-rich, lytic enzymes-poor organs such as thymus and spleen were significantly more likely to yield good quality DNA than the liver.

Application of Whole Genome Sequencing Technology in the Investigation of Genetic Causes of Fetal, Perinatal, and Early Infant Death.

Death in the fetal, perinatal, and early infant age-group has a multitude of causes, a proportion of which is presumed to be genetic. Defining a specific genetic aberration leading to the death is problematic at this young age, due to limited phenotype-genotype correlation inherent in the underdeveloped phenotype, the inability to assess certain phenotypic traits after death, and the problems of dealing with rare disorders. In this study, our aim was to increase the yield of identification of a defined genetic cause of an early death. Therefore, we employed whole genome sequencing and bioinformatic filtering techniques as a comprehensive, unbiased genetic investigation into 16 fetal, perinatal, and early infant deaths, which had undergone a full autopsy. A likely genetic cause was identified in two cases (in genes; COL2A1 and RYR1) and a speculative genetic cause in a further six cases (in genes: ARHGAP35, BBS7, CASZ1, CRIM1, DHCR7, HADHB, HAPLN3, HSPG2, MYO18B, and SRGAP2). This investigation indicates that whole genome sequencing is a significantly enabling technology when determining genetic causes of early death.

L1 Retrotransposon Heterogeneity in Ovarian Tumor Cell Evolution.

LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity.

The outcome of papillary lesions of the breast diagnosed by standard core needle biopsy within a BreastScreen Australia service.

Papillary lesions of the breast are most commonly diagnosed via mammographic screening. The standard practice has been to excise these lesions, since a subset of papillary lesions are neoplastic. However, this approach leads to a high proportion of negative excisions. In order to identify papillary lesions which could be managed by surveillance alone, we assessed the outcome of 103 papillary lesions diagnosed on core needle biopsy in a public screening program. Subsequent excision biopsy led to an upgrade to malignancy in 30% of cases. Segregation via presence or absence of atypia stratified the outcome into 72% upgrade, compared with 7% upgrade, respectively. Further, in the latter group (i.e., no atypia on core needle biopsy with 7% upgrade to malignancy), the neoplasia found in the targeted excision area was low to intermediate grade ductal carcinoma in situ only, with no invasive neoplasia (4 cases). Of the lesions identified due to microcalcification, the microcalcification was present within an adjacent benign lesion in 35% of cases and hence the papillary lesion was detected incidentally. Overall therefore, we have identified a cohort of papillary lesions in which conservative management, rather than excision, could be considered, i.e., those without atypia, including those without atypia in which the papillary lesion was found incidental to microcalcification in an adjacent benign lesion.

Extracorporeal Life Support in Multisystem Smooth Muscle Dysfunction Syndrome.

We describe an infant with congenital mydriasis, patent ductus arteriosus (PDA), pulmonary hypertension, and cystic lung disease. She had all the major components of multisystemic smooth muscle dysfunction syndrome. Due to progressive respiratory deterioration, she required surgical PDA interruption, extracorporeal life support, and subsequent prolonged respiratory support. Genetic testing revealed ACTA2 R179H mutation and cystic lung disease on biopsy.

Histologic Evaluation of Explanted Tissue-Engineered Bovine Pericardium (CardioCel).

CardioCel is a bovine pericardium that is subjected to a novel anticalcification tissue-engineering process. We present the histopathologic findings of human explants of CardioCel that were used in operations for congenital heart disease in children. Six explants were identified from 140 patients undergoing CardioCel implants from October 2012 to March 2015. CardioCel explants were evaluated histologically using hematoxylin and eosin, Masson trichrome, and immunohistochemical staining. A variable inflammatory response was seen in the surrounding native tissue, but not within the CardioCel graft in any of the explants. A neointimal layer of varying thickness developed on the visceral surface of 5 CardioCel explants with endothelialization of the longest duration explant. A granulation tissue layer developed on the parietal surface of the graft (consistently thicker than the neointima). Maintained collagen fiber architecture (laminated) and variable fibroblastic invasion (which increased with the age of the implant) were identified in all 6 cases. Scattered capillary vessels were noted in the majority of the explants with new collagen fibers in one, suggesting early remodeling. Calcium was seen in 1 explant at the interface of the graft and inflammatory response on its parietal surface. Evidence of graft remodeling was noted in the majority of the explants without inflammatory cells or calcification within the explanted graft material. A noticeable feature was the differential thickness of the host reaction to the parietal compared with the visceral surface of the graft. We will continue to evaluate CardioCel as a cardiovascular substitute for extracardiac and intracardiac reconstructions.