Osteosarcoma microenvironment: whole-slide imaging and optimized antigen detection overcome major limitations in immunohistochemical quantification.

BACKGROUND: In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment. METHODS: Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis. RESULTS: More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35-65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method. CONCLUSION: Standard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations

Variability of predictive markers (hormone receptors, Her2, Ki67) and intrinsic subtypes of breast cancer in four consecutive years 2015-2018

PURPOSE Accurate monitoring of predictive markers is of utmost importance as oncological treatment decisions almost entirely depend on these factors. In this study, we conducted a quality control assessment on hormone receptors, Her2 status, Ki67 Labelling Index (LI) and histological grading in breast cancer over 4 years (2015-2018). METHODS Altogether 2214 consecutive breast cancer cases were included. Data on estrogen (ER) and progesterone receptors (PR), Her2 and Ki67, were available in all cases and were tested mostly on preoperative biopsies, in selected cases on postoperative surgical specimens. ER, PR, and Ki67 were assessed with immunohistochemistry (IHC), Her2 status with IHC and fluorescence in situ hybridization. RESULTS ER/PR were positive in 74-79% cases, ER/PR/Her2 negative in 6.16-10.70% and Her2 positive in 11.49-13.88%/year. Ki67 had median values as 15-17.5% in ER/PR-positive cases, 55-60% in triple-negative cases and 30-32.50% in Her2-positive cases. Histological grading distribution for well (G1), moderately (G2) and poorly (G3) differentiated carcinomas was 15.8-19.1% for G1, 54.2-54.8% for G2 and 21.7-23.7% for G3 cases. Variation in yearly distributions was not significant in any of these markers. CONCLUSIONS Predictive markers displayed a yearly similar distribution in breast cancer cases independently of grading or of intrinsic subtypes. These results point to a qualitative high performance of predictive marker assessment in breast cancer, corresponding to expected on average positivity rate per marker and per year. It is recommended to monitor positivity rate of ER, PR, Ki67 and Her2 yearly or periodically to comply with quality assurance requirements

Juvenile papillomatosis of the breast (Swiss cheese disease) has frequent associations with PIK3CA and/or AKT1 mutations

Juvenile papillomatosis (JP), the so-called Swiss cheese disease is a rare benign breast disease of young adults. An association (up to 28 %) with breast-cancer within the family of affected patients has been reported. A multinodular cystic breast-mass lesion and calcifications characterizes JP in imaging studies. The histological picture is diverse and comprises multiple intraductal-papillomas, usual ductal hyperplasia (UDH), ductectasias, perifocal sclerosing adenosis and calcification. Patients with complete excision of JP lesions have an excellent follow-up; breast cancer develops only on a very low subset of patients. Molecular background of JP has not been investigated until now. In this study, we addressed mutational analysis of JP cases and correlated these results with follow-up and family-history in context with a comprehensive review of JP literature. We identified 13 cases fulfilling the criteria of JP. All patients were female with a median-age of 38 years (26 to 50 years). Follow-up information was available in 11 of 13 patients. Sufficient paraffin embedded tissue and good DNA quality for next generation sequencing (NGS) was available in 10 patients. Paraffin blocks were microdissected in the area of intraductal proliferative disease, the tissue cores underwent NGS analysis using Oncomine Comprehensive Panel. In 5 of 10 patients, we found PIK3CA mutations, in 2 of 10 patients AKT1 mutations in known hotspot regions. Further mutations in MET, FGFR3, PTEN, ATM, NF1 and GNAS genes were detected in individual patients. Some of these mutations were present at high allelic frequencies suggesting germ line mutations. 2 of 3 patients with positive family history had PIK3CA mutation; one patient with positive family history had an AKT1 mutation. One patient who subsequently developed invasive ductal carcinoma in the contralateral breast possibly had a germ line ATM mutation. Our results confirm hotspot mutations in PIK3CA and AKT1 genes in JP associated with positive family history for breast cancer, although these mutations are not specific for JP. The genetic link between JP, positive family history and subsequent risk of breast cancer, needs further studies

Inter-observer reproducibility of classical lobular neoplasia (B3 lesions) in preoperative breast biopsies: a study of the Swiss Working Group of breast and gynecopathologists

PURPOSE Classical type of lobular neoplasia (LN) spans a spectrum of disease, including atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS), classical lobular neoplasia (LN), and the three-tiered classification of lobular intraepithelial neoplasia (LIN-1, -2, -3). This study addressed inter-observer variability of classical lobular neoplasias (LN) (B3 lesions) in preoperative breast biopsies among breast and gynecopathologists METHODS: A retrospective, observational, cross-sectional study was conducted. 40 preoperative digital images of breast core/vacuum biopsies were analyzed by eight experienced breast- and gynecopathologists. Evaluation criteria were ALH, LCIS, LN classic, LIN-1, LIN-2, LIN-3, focal B3 (one focus), extensive B3 (> one focus). Kappa-index and Chi-square tests were used for statistics. Digital scanned slides were provided to each participant. Agreement between the categories was defined as at least six of eight (cut-off 75%) concordant diagnoses. RESULTS The highest agreement between eight pathologists was reached using the category lobular neoplasia (LN, classical), 26/40 (65%) cases were diagnosed as such. Agreements in other categories was low or poor: 12/40 (30%) (ALH), 9/40 (22%) (LCIS), 8/40 (20%) (LIN-1), 8/40 (20%) (focal B3), 3/40 (7.5%) (LIN-2), and 2/40 (5%) (extensive B3). Chi-square-test (classical LN versus the other nomenclatures) was significant (p = 0.001137). CONCLUSION Our data suggest that among Swiss breast pathologists, the most reproducible diagnosis for B3 lobular lesions is the category of classical LN. These data further support lack of consistent data in retrospective studies using different terminologies. Validation of reproducible nomenclature is warranted in further studies. This information is useful especially in view of retro- and prospective data analysis with different diagnostic categories

Differential prognostic value of positive HER2 status determined by immunohistochemistry or fluorescence in situ hybridization in breast cancer

Purpose Human epidermal growth factor-receptor-2 (HER2) is a membrane-tyrosine-kinase that is amplified/overexpressed up to 20% in breast cancer. HER2 positive status is associated with faster disease progression, higher metastatic potential, and shorter disease-free/overall survival and also has emerged as an important therapeutic target in breast cancer. HER2 status can be determined by in-situ-hybridization (ISH) or immunohistochemistry (IHC). Although the concordance rate between ISH and IHC is well-known, the prognostic power of both technologies if tested in parallel on the same tumor has not been studied extensively. Methods In this study we retrospectively analyzed a large HER2 positive breast cancer cohort tested both with fluorescence labeled ISH (FISH) and IHC in parallel on each case. We stratified HER2 positive results by FISH and IHC with long-term overall survival, 5-year survival and metastases/recurrence rates. Positive HER2 status both FISH and IHC was available in 364 patients. Results The number of HER2 FISH-positive and FISH-negative patients was 342 and 22, respectively. The number of HER2 IHC 0/1 + , IHC 2 + , and IHC 3 + patients was 12, 42, and 310, respectively. Among the patients with IHC 3 + status, 288 were FISH-positive and 22 FISH-negative. HER2 status determined by FISH correlated with clinical outcomes (overall survival and with metastases/recurrence, p = 0.036, p = 0.039), whereas HER2 status determined by IHC did not

Preferential expression of NY-BR-1 and GATA-3 in male breast cancer

BACKGROUND: Male breast cancer is an uncommon disease often discovered in advanced stage; thus, in the setting of metastatic adenocarcinoma, breast origin must be taken to account. Breast markers as NY-BR-1, GATA-3, mammaglobin, and BRST-2 are established tools for labelling primary and metastatic female breast cancer; however, none of them has been sufficiently studied in male breast cancer. The aim of this study was to analyze the expression of these markers in male breast cancer. MATERIALS AND METHODS: Thirty consecutive cases of male breast cancer and eight loco-regional metastases were re-revaluated, assembled in tissue micro array (TMA), and stained with immunohistochemistry (IHC) for NY-BR-1, GATA-3, mammaglobin, and BRST-2. The IHC stains were scored either positive or negative. In addition, concordant expression patterns of primary tumors and matched metastasis were noted. RESULTS: 30 of 30 (100%) primary tumors and 8 of 8 (100%) metastases were positive for NY-BR-1. 30 of 30 (100%) primary tumors and 6 of 8 (75%) metastases were positive for GATA-3. 22 of 30 (73.3%) primary tumors and 6 of 8 (75%) metastases were positive for Mammaglobin. 18 of 30 (60%) primary tumors and 5 of 8 (62.5%) metastases were positive for BRST-2. Differences in staining percentage were not significant with Fisher's exact test. CONCLUSION: We found a high sensitivity for all the markers analyzed. Moreover, the expression of NY-BR-1 and GATA-3 seemed the most effective for labelling male breast cancer in primary and metastatic setting

Discrepancies between radiological and histological findings in preoperative core needle (CNB) and vacuum-assisted (VAB) breast biopsies

BACKGROUND: Ultrasound (US)-guided breast biopsy is a routine diagnostic method used to correlate imaging finding to a histological diagnosis which is still the gold standard in preoperative diagnostics. The accuracy of US-guided breast biopsies relies on a precise radiologic-histopathologic correlation, which is discussed amongst an interdisciplinary team of gynecologists, radiologists and pathologists. However, false-negative or non-diagnostic biopsy results occur. Hence, a thorough and honest discussion to clarify the reason for discrepancies and to decide the next diagnostic step between specialists of the different disciplines is warranted. In this retrospective study, we analyzed discrepant findings between imaging and pathology results on preoperative breast biopsies. METHODS: Core and vacuum-assisted breast biopsies from 232 patients were included in this study. Inclusion criteria were (1) non-diagnostic (B1) category on histology independent from imaging category and (2) histological benign (B2) category with a BIRADS 5 (Breast Imaging Reporting and Data System) rating on imaging. Histological diagnoses were retrieved from all cases. Follow-up data were available in most cases. RESULTS: 138 biopsies were classified as B1, 94 biopsies as B2 category. 51 of 138 B1 cases (37%) underwent re-biopsy. Re-biopsy found malignancy (B5) in 19 of 51 cases, and B3/4 (premalignant) lesions in 3 of 51 cases. All B2 cases underwent second-look imaging-diagnosis, in 57 of 94 cases (66%) consecutive direct surgery or re-biopsy. Of these, malignancy was diagnosed histologically in 26 of 57 cases (45.6%). CONCLUSION: Determining imaging-pathology concordance after US-guided breast biopsy is essential. Discrepant cases and further diagnostic steps need to be discussed with an interdisciplinary approach

Primary urethral squamous cell carcinoma: a unique manifestation of a penile tumor

This case report describes a unique manifestation of a primary urethral squamous cell carcinoma (SCC) as the underlying pathology in an 80-year-old male patient who underwent partial penectomy due to an enlarging penile mass. Persistent pain in the right knee was discovered to be a pathologic fracture using magnetic resonance imaging. Computed tomography-guided biopsy confirmed metastatic SCC. Whole-body positron emission tomography revealed systemic dissemination to multiple sites. Orthopedic knee replacement was performed in combination with local radiotherapy. Palliative chemotherapy was rejected due to poor performance status. Primary urethral SCC is rare and an uncommon cause of advanced penile cancer. These findings could be of great interest to clinicians for two reasons. First, a tumor's appearance can be misleading. Consequently, histological work-up in accordance with clinical guidelines is necessary for accurate diagnosis. Second, a more comprehensive investigation is required when clinical symptoms persist despite the use of conventional treatment. Our case is an instance in which persistent pain masked the presence of downstream metastasis. We believe that these aforementioned points are of significant clinical importance and present a salient learning opportunity