Differential Gene Expression of SARS-CoV-2 Positive Bronchoalveolar Lavages: A Case Series.

BACKGROUND: Transcriptomic data on bronchoalveolar lavage (BAL) from COVID-19 patients are currently scarce. OBJECTIVES: This case series seeks to characterize the intra-alveolar immunopathology of COVID-19. METHOD: BALs were performed on 14 patients (5 COVID-19, of which 3 mild and 2 largely asymptomatic, 9 controls). Controls included asthma (n = 1), unremarkable BALs (n = 3), infections with respiratory syncytial virus (n = 1), influenza B (n = 1), and infections with other coronaviruses (n = 3). SARS-CoV-2 RNA load was measured by quantitative nucleic acid testing, while the detection of other pathogens was performed by immunofluorescence or multiplex NAT. RESULTS: Gene expression profiling showed 71 significantly downregulated and 5 upregulated transcripts in SARS-CoV-2-positive lavages versus controls. Downregulated transcripts included genes involved in macrophage development, polarization, and crosstalk (LGALS3, MARCO, ERG2, BTK, RAC1, CD83), and genes involved in chemokine signaling and immunometabolism (NUPR1, CEBPB, CEBPA, PECAM1, CCL18, PPARG, ALOX5, ALOX5AP). Upregulated transcripts featured genes involved in NK-T cell signaling (GZMA, GZMH, GNLY, PRF1, CD3G). Patients with mild COVID-19 showed a significant upregulation of genes involved in blood mononuclear cell/leukocyte function (G0S2, ANXA6, FCGR2B, ADORA3), coagulation (von Willebrand factor [VWF]), interferon response (IFRD1, IL12RB2), and a zinc metalloprotease elevated in asthma (CPA3) compared to asymptomatic cases. In-silico comparison of the 5 COVID-19 BAL cases to a published cohort of lethal COVID-19 showed a significant upregulation of "antigen processing and presentation" and "lysosome" pathways in lethal cases. CONCLUSIONS: These data underscore the heterogeneity of immune response in COVID-19. Further studies with a larger dataset are required to gain a better understanding of the hallmarks of SARS-CoV-2 immunological response.

A multicenter phase II trial of anti-EGFR-immunoliposomes loaded with doxorubicin in patients with advanced triple negative breast cancer.

Advanced triple negative breast cancer (TNBC) is an aggressive, but initially chemo-sensitive disease. The prognosis is poor and more than three quarters of patients experience progression 12 months after the initiation of conventional first-line chemotherapy. Approximately two thirds of TNBC express epidermal growth factor receptor 1 (EGFR). We have developed an anti-EGFR targeted nanocontainer drug by inserting anti-EGFR antibody fragments into the membrane of pegylated liposomes (anti-EGFR-ILs-dox). The payload consists of doxorubicin, a standard drug for TNBC. In a first-in-human phase I trial in 26 patients with various advanced solid malignancies, anti-EGFR-ILs-dox has shown little toxicity and encouraging efficacy. In this single-arm phase II trial, we assessed the efficacy of anti-EGFR-ILs-dox as first-line therapy in patients with advanced, EGFR + TNBC. The primary endpoint was progression-free survival at 12 months (PFS12m). Secondary endpoints included overall response rate (ORR), duration of response (DOR), time to progression (TTP), overall survival (OS) and adverse events (AEs). 48 patients received anti-EGFR-ILs-dox 50 mg/m2 iv, on day one of a 28 days-cycle until progression. The Kaplan-Meier estimate for PFS12m was 13% (one-sided 90% CI 7%, 95% CI [5%, 25%]), median PFS was 3.5 months (95% CI 1.9, 5.4). The trial has not reached its primary endpoint. There were no new toxicity signals. Based on these results, anti-EGFR-ILs-dox should not be further developed for TNBC. It remains an open question whether anti-EGFR-ILs-dox would offer more opportunities in other EGFR-expressing malignancies, where targeting this receptor has already shown anticancer effects.Trial registration: This trial was registered at clinicaltrials.gov: NCT02833766. Registered 14/07/2016.

Multiple infections in an immunocompromised patient-How cytology can help.

Opportunistic infections in immunocompromised patients can present a diagnostic challenge. This case report describes multiple pulmonary infections-Pneumocystic jirovecii, Histoplasma capsulatum, and cytomegalovirus-in an HIV-positive patient. The morphological findings are described, and the importance of cytology for the rapid diagnosis of these infections is highlighted.

Inter- and intraobserver agreement in whole-slide digital ThinPrep samples of low-grade squamous lesions of the cervix uteri with known high-risk HPV status: A multicentric international study.

BACKGROUND: High-risk human papilloma virus (HR HPV) testing and liquid-based cytology are used for primary cervical screening. Digital cytology, based on whole-slide scanned samples, is a promising technique for teaching and diagnostic purposes. The aim of our study was to evaluate the interobserver and intraobserver variation in low-grade squamous lesions, HR HPV status bias, and the use of whole-slide scanned digital cervical cytology slides. METHODS: Fifteen expert cytopathologists evaluated 71 digitalized ThinPrep slides (31 atypical squamous cells of undetermined significance [ASC-US], 21 negative for intraepithelial lesion or malignancy, and 19 low-grade squamous intraepithelial lesion cases). HR HPV data were accessible only in the second round. RESULTS: In interobserver analysis, Kendall's coefficient of concordance was 0.52 in the first round and 0.58 in the second round. Fleiss' kappa values were 0.29 in the first round and 0.31 in the second round. In the ASC-US category, Fleiss kappa increased from 0.19 to 0.22 in the second round and the increase was even higher expressed by Kendall's coefficient: from 0.42 to 0.52. In intraobserver analysis, personal scores were higher in the second round. CONCLUSIONS: The interobserver and intraobserver variability in low-grade squamous lesions was within fair agreement values in the present study, in line with previous works. The comparison of two rounds showed that expert cytopathologists are generally unbiased by the knowledge of HR HPV data, but that being informed of the HR HPV status leads to a better agreement. Stain quality and back discomfort were highlighted as factors affecting digital cytopathology use.

An Integrated Epigenomic and Genomic View on Phyllodes and Phyllodes-like Breast Tumors.

Fibroepithelial lesions (FL) of the breast, in particular, phyllodes tumors (PT) and fibroadenomas, pose a significant diagnostic challenge. There are no generally accepted criteria that distinguish benign, borderline, malignant PT and fibroadenomas. Combined genome-wide DNA methylation and copy number variant (CNV) profiling is an emerging strategy to classify tumors. We compiled a series of patient-derived archival biopsy specimens reflecting the FL spectrum and histological mimickers including clinical follow-up data. DNA methylation and CNVs were determined by well-established microarrays. Comparison of the patterns with a pan-cancer dataset assembled from public resources including "The Cancer Genome Atlas" (TCGA) and "Gene Expression Omnibus" (GEO) suggests that FLs form a methylation class distinct from both control breast tissue as well as common breast cancers. Complex CNVs were enriched in clinically aggressive FLs. Subsequent fluorescence in situ hybridization (FISH) analysis detected respective aberrations in the neoplastic mesenchymal component of FLs only, confirming that the epithelial component is non-neoplastic. Of note, our approach could lead to the elimination of the diagnostically problematic category of borderline PT and allow for optimized prognostic patient stratification. Furthermore, the identified recurrent genomic aberrations such as 1q gains (including MDM4), CDKN2a/b deletions, and EGFR amplifications may inform therapeutic decision-making.

Bronchoalveolar lavage-detected SARS-CoV-2 infection in presence of serial negative nasopharyngeal swabs: a case report.

We describe a case of a SARS coronavirus 2 (SARS-CoV-2) infection in a Swiss 54-years-old immunocompromised patient (lymphoma, therapy with the anti-CD20 antibody Rituximab® ), with initial scarce respiratory symptoms but typical coronavirus disease 2019 (COVID-19) radiological presentation, and symptoms onset during a holiday trip to Texas (USA). Three nasopharyngeal swabs in the 96 hours following hospital admission were negative, despite a CT thorax suggestive for an early stage of infection. COVID-infection was finally confirmed in the bronchoalveolar lavage (BAL) fluid, performed for exclusion of an alternative diagnosis in immunocompromised. In the BAL an increased cellularity with marked lymphocytosis of 35%, a reduced CD4/CD8 ratio of 0.1 and borderline neutrophilia of 3% were found. This finding might be due to the concomitant therapy with anti-CD20 antibodies, but the presence of lymphocytosis in the BAL despite peripheral lymphopenia with decreased CD4/CD8 T-cells ratio are described here for the first time in a SARS-CoV-2 infection. Persistent gastrointestinal symptoms (diarrhea), fever and initially headache were the predominant symptoms. The respiratory symptoms were scarce (variable mild dyspnea mMRC1). The respiratory conditions worsened during the hospital stay, with tachypnea up to 35/min, increased need for supplemental oxygen up to 8 L/min and worsening lung infiltrates on CT thorax on day 5. A therapy with hydroxychloroquine (HCQ) and an immunoglobulin-supplementation were given, with clinical and respiratory improvement, without need for intensive care or any ventilator support, and hospital discharge on day 16. Our case highlights some diagnostic and therapeutical challenges occurring in patients with COVID-19 infection. As take-home message, in the presence of clinical and radiological findings compatible with SARS-CoV-2 infection we outline the importance of treating patients accordingly, also in presence of repeated negative nasopharyngeal swabs. In selected patients as in our case a bronchoscopic BAL should be considered to exclude other infections, but in our opinion not primarily to confirming COVID-19 infection. Our unique finding of a lymphocytosis in the BAL during a COVID-19 infection needs further investigations.

Length Scale Matters: Real-Time Elastography versus Nanomechanical Profiling by Atomic Force Microscopy for the Diagnosis of Breast Lesions.

Real-time elastography (RTE) is a noninvasive imaging modality where tumor-associated changes in tissue architecture are recognized as increased stiffness of the lesion compared to surrounding normal tissue. In contrast to this macroscopic appraisal, quantifying stiffness properties at the subcellular level by atomic force microscopy (AFM) reveals aggressive cancer cells to be soft. We compared RTE and AFM profiling of the same breast lesion to explore the diagnostic potential of tissue elasticity at different length scales. Patients were recruited from women who were scheduled for a biopsy in the outpatient breast clinic of the University Hospital Basel, Switzerland. RTE was performed as part of a standard breast work-up. Individual elastograms were characterized based on the Tsukuba elasticity score. Additionally, lesion elasticity was semiquantitatively assessed by the strain ratio. Core biopsies were obtained for histologic diagnosis and nanomechanical profiling by AFM under near-physiological conditions. Bulk stiffness evaluation by RTE does not always allow for a clear distinction between benign and malignant lesions and may result in the false assessment of breast lesions. AFM on the other hand enables quantitative stiffness measurements at higher spatial, i.e., subcellular, and force resolution. Consequently, lesions that were false positive or false negative by RTE were correctly identified by their nanomechanical AFM profiles as confirmed by histological diagnosis. Nanomechanical measurements can be used as unique markers of benign and cancerous breast lesions by providing relevant information at the molecular level. This is of particular significance considering the heterogeneity of tumors and may improve diagnostic accuracy compared to RTE.

Human Papillomavirus (HPV) Detection in Cytologic Specimens: Similarities and Differences of Available Methodology.

Accumulating evidence regarding the causative role of human papillomavirus (HPV) in a wide range of malignant and nonmalignant diseases highlights the importance of HPV testing. This study describes and discusses the efficacy and characteristics of 4 well-established and commercially available tests. Here, 181 cytologic specimens from cervical smears were analyzed using the HPV SIGN PQ (Diatech) and the Linear Array (Roche) method. Discrepant results were further studied with the Real Time High-Risk HPV (Abbott) method and the INNO-LiPA (Fujirebio) method. Of 181 cytologic specimens, 61 (34%) showed discrepant results. High-risk HPV was not detected in 9 cases by HPV SIGN PQ, in 16 cases by Linear Array, in 10 cases by Real Time High-Risk HPV, and in 6 cases by INNO-LiPA, respectively. Lack of DNA detection or problems in interpreting the result were seen in 9 cases with HPV SIGN PQ, 8 cases with Linear Array, 3 cases with Real Time High-Risk HPV, and 3 cases with INNO-LiPA, respectively. This study indicates that the choice of HPV detection method has a substantial influence on the HPV risk classification of tested PAP smears and clinical follow-up decisions.

Detection of CCNE1/URI (19q12) amplification by in situ hybridisation is common in high grade and type II endometrial cancer.

One TCGA subgroup of endometrial cancer (EC) is characterised by extensive genomic DNA copy number alterations. CCNE1 located at 19q12 is frequently amplified in EC and a target for anti-cancer therapy. The relevance of URI, also located at 19q12, is unknown. To evaluate the prevalence of 19q12 (CCNE1/URI) in EC, we investigated different histologic types by in situ hybridisation (ISH) and copy number assay. We applied a previously established 19q12 ISH for the detection of CCNE1/URI copy numbers in EC (n = 270) using conventional bright field microscopy. In a subset (n = 21), 19q12 amplification status was validated by OncoScan assay. Manual ISH was controlled by a recently developed computational ISHProfiler algorithm. Associations of 19q12 status with Cyclin E1, URI and p53 expression, and clinico-pathological parameters were tested.Amplification of 19q12 (CCNE1/URI) was found in 10.4% (28/270) and was significantly associated with type II EC (high grade and non-endometrioid; p < 0.0001), advanced FIGO stage (p = 0.001), high Cyclin E1 expression (p = 0.008) and aberrant p53 expression (p = 0.04). 19q12 ISH data were confirmed by OncoScan and computational ISHProfiler techniques. The 19q12 in situ hybridisation is a feasible and robust biomarker assay in molecular pathology. Amplification of CCNE1/URI predominantly occurred in type II endometrial cancer. Prospective clinical trials are warranted to assess the utility of combined 19q12 amplification and Cyclin E1/URI protein expression analysis for the prediction of therapeutic response to chemotherapy and/or cyclin-dependent kinase inhibitors in patients with endometrial cancer.

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine.

INTRODUCTION: The use of patients' own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs. METHODS: MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists. RESULTS: We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs. CONCLUSIONS: We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.

Renal Cell Carcinoma in a Young Adult - Do We Need Further Investigations?

Renal cell carcinomas (RCC), mostly occurring in adults aged 60-70 years, can result from well-known factors like cigarette smoking, obesity and hypertension. However, they have been associated with genetic alterations in children and young adults. A 28 year-old male patient with a confirmed RCC underwent biomolecular and immunohistochemical analyses due to his young age. A point mutation of the von Hippel-Lindau tumor suppressor gene was identified. Young patients under 40 years with diagnosed RCC should undergo additional diagnostic investigation, hence the discovery of an underlying cause. This could be important for further treatment and counseling of these young patients.

Targeting the ROR1 and ROR2 receptors in epithelial ovarian cancer inhibits cell migration and invasion.

AIM: In recent years, the Wnt signalling pathway has been implicated in epithelial ovarian cancer and its members have potential as diagnostic, prognostic and therapeutic targets. Here we investigated the role of two Wnt receptor tyrosine kinases (RTKs), ROR1 and ROR2, and their putative ligand, Wnt5a, in ovarian cancer. METHODS: Immunohistochemistry for ROR2 was performed in a large patient cohort, including benign controls, borderline tumours and epithelial ovarian cancer. In addition, siRNA was used to silence ROR1, ROR2 and Wnt5a individually, and together, in two ovarian cancer cell lines, and the effects on cell proliferation, adhesion, migration and invasion were measured. RESULTS: ROR2 expression is significantly increased in ovarian cancer patients compared to patients with benign disease. In vitro assays showed that silencing either receptor inhibits ovarian cancer cell migration and invasion, and concurrently silencing both receptors has an even stronger inhibitory effect on proliferation, migration and invasion. CONCLUSIONS: ROR2 expression is increased in epithelial ovarian cancer, and silencing ROR2 and its sister receptor ROR1 has a strong inhibitory effect on the ability of ovarian cancer cells to proliferate, migrate and invade through an extracellular matrix.

KPNA2 is overexpressed in human and mouse endometrial cancers and promotes cellular proliferation.

Endometrial cancer is the most frequently occurring malignancy of the female genital tract in Western countries. Although in many cases surgically curable, about 30% of the tumours represent an aggressive and untreatable disease. In an attempt to establish a reliable prognostic marker for endometrial carcinomas disregarding their histological diversity, we investigated the expression of KPNA2, a mediator of nucleocytoplasmic transport, and other cell proliferation-associated proteins and their correlation with cancer progression. We analysed patient tissue microarrays (TMAs) assembled from 527 endometrial cancer tissue specimens and uterus samples from a Trp53 knockout mouse model of endometrial cancer. Our data show that KPNA2 expression was significantly up-regulated in human endometrial carcinomas and associated with higher tumour grade (p = 0.026), higher FIGO stage (p = 0.027), p53 overexpression (p < 0.001), activation of the PI3K/AKT pathway, and epithelial-mesenchymal transition. Increased nuclear KPNA2 immunoreactivity was identified as a novel predictor of overall survival, independent of well-established prognostic factors in Cox regression analyses (hazard ratio 1.7, 95% CI 1.13-2.56, p = 0.01). No significant association between KPNA2 expression and endometrial cancer subtype was detected. In the mouse model, KPNA2 showed increased expression levels from precancerous (EmgD, EIC) to far-advanced invasive lesions. We further investigated the cell proliferation capacity after siRNA-mediated KPNA2 knockdown in the human endometrial cancer cell line MFE-296. KPNA2 silencing led to decreased proliferation of the cancer cells, suggesting interplay of the protein with the cell cycle. Taken together, increased expression of KPNA2 is an independent prognostic marker for poor survival. The mechanism of enhanced nucleocytoplasmic transport by KPNA2 overexpression seems a common event in aggressive cancers since we have shown a significant correlation of KPNA2 expression and tumour aggressiveness in a large variety of other solid tumour entities. Introducing KPNA2 immunohistochemistry in routine diagnostics may allow for the identification of patients who need more aggressive treatment regimens.

Targeted multiprobe fluorescence in situ hybridization analysis for elucidation of inconclusive pancreatobiliary cytology.

BACKGROUND: Endoscopic fine-needle aspiration (FNA) and brush cytology are standard methods for the diagnosis of pancreatobiliary malignancies. Although the majority of cytological diagnoses are straightforward, there remains a difficult category of inconclusive cytology. This study explored the utility of fluorescence in situ hybridization (FISH) to improve the diagnostic stratification between reactive and malignant cells in cases of inconclusive cytology. METHODS: The multiprobe FISH assay UroVysion was used for copy number assessment of chromosomes 3, 7, 17, and the 9p21 locus on Papanicolaou-stained specimens with a diagnosis of inconclusive cytology (n = 50), adenocarcinoma (n = 31) and no evidence of malignancy (n = 9). The target cells were photographed and their coordinates saved on an automated stage prior to hybridization. A positive test was defined as increased copy number (> 2) of at least 2 chromosomes (3, 7, or 17) in at least 4 atypical cells, or loss of 9p21 in at least 12 cells. RESULTS: FISH confirmed all 31 cytological diagnoses of pancreatobiliary adenocarcinomas, and was negative in the 9 patients with negative cytology. Among the 50 cases with inconclusive cytology, FISH detected 19 of 31 cases with a final diagnosis of adenocarcinoma, and was negative in all 19 cases with no final evidence of malignancy (sensitivity of 61.3%, specificity of 100%, positive predictive value of 100%, negative predictive value of 61.3%). Loss of 9p21 was found in 43 (86%) of all 50 FISH-positive cases. CONCLUSIONS: Multiprobe FISH combined with automated relocation of atypical cells is a powerful technique to clarify inconclusive cytology of the pancreatobiliary tract, allowing for a better distinction between reactive atypia and malignancy.

Putative stem cell markers in non-small-cell lung cancer: a clinicopathologic characterization.

INTRODUCTION: The cancer stem cell (CSC) theory postulates the existence of a distinct population of undifferentiated cells responsible for tumor initiation and maintenance. CSCs may be naturally resistant to the cytotoxic effect of radio-chemotherapy because of slow cell cycling, lower proliferation, and increased expression of DNA repair and antiapoptosis genes. To date, a universal marker for CSCs has not been identified. Proposed CSC markers are expressed both by cancer cells as well as by benign stem cells. Although many putative CSC markers exist, a precise characterization for non-small-cell lung cancer (NSCLC) is lacking. METHODS: We explored the expression of multiple alleged stemness associated markers in 371 surgically resected NSCLCs. Extensive clinical data and a postoperative follow-up period of up to 15 years enabled detailed clinicopathological correlations. ABCG5, ALDH1, CD24, CD44, CD133, CD166, epithelial cell adhesion molecule epitopes (ESA, MOC-31, Ber-EP4), nestin, OCT4, and sex-determining region Y-box 2 were analyzed immunohistochemically by using a standardized tissue microarray platform. RESULTS: Sex-determining region Y-box 2, CD44, ABCG5, ALDH1, and nestin were associated with poorer tumor differentiation and/or an increased proliferation index. ALDH1, CD44, and SOX2 were frequently found in squamous cell carcinoma, whereas CD24, CD166, and epithelial cell adhesion molecule markers were encountered in adenocarcinomas. CD44 expression was an independent marker associated with better overall survival in squamous cell carcinoma and Ber-EP4 was associated with tumor recurrences. CONCLUSION: The expression and prognostic significance of CSC markers obviously varies depending on histologic NSCLC subtype. Importantly, our findings suggest that CD44 and Ber-EP4 may be promising for ongoing targeted therapies in specific NSCLC subgroups.

Heterogeneity of ERG expression in core needle biopsies of patients with early prostate cancer.

Prostate cancer is a heterogeneous, frequently multifocal disease with a broad spectrum of clinical, pathologic, and molecular characteristics. The TMPRSS2-ERG gene rearrangement is highly specific for prostate cancer. We used immunohistochemistry as a surrogate marker of the TMPRSS2-ERG fusion to study the heterogeneity of ERG expression in 280 prostate core needle biopsy series from 256 patients with early prostate cancer defined as 3 or less positive cores with no more than 50% of cancer per biopsy and a Gleason score of 7 or lower (3 + 4). Among the 163 patients with 2 or 3 cancer-positive biopsies, we found a subset of 19 patients (11.7%) with heterogeneous ERG expression. Thirteen (68.4%) of these patients showed biopsies with distinct positive and negative ERG staining in separate cores. The remaining 6 patients showed a mixture of both positive and negative staining within 1 biopsy core. This was either caused by different cancer foci (n = 3) or by one single, ERG-heterogeneous cancer focus (n = 3) in 1 core. Furthermore, we observed a heterogeneous ERG staining pattern over time in 6 (2.3%) of the 256 patients, in biopsies taken at various time points. An interobserver study of 21 cases with 2 separate cancer foci revealed that heterogeneity of ERG status in different cancer foci can be suspected based on morphologic differences (κ = 0.44). We conclude that heterogeneity of ERG expression is detectable in 10% to 15% of core biopsies of early prostate cancer. Further studies are needed to explore the clinical impact of heterogeneous ERG status in this patient group.

Negligible nuclear FOXP3 expression in breast cancer epithelial cells compared with FOXP3-positive T cells.

BACKGROUND: The presence of forkhead box protein 3 (FOXP3) in breast cancer cells is a matter of debate. We systematically analyzed expression of FOXP3 in 1574 breast carcinomas. MATERIALS AND METHODS: For nuclear FOXP3 detection in epithelial breast cancer cells, tissue microarray technology was used. In addition, cultured breast cancer cell lines (ZR75-1, MCF7-WH, BT20, T47D, and SKBR3) were tested for FOXP3 expression using real-time polymerase chain reaction and flow cytometry for messenger RNA (mRNA) and protein quantification. RESULTS: We could neither detect any significant levels of mRNA or protein expression of FOXP3 in human breast cancer cell lines, nor in breast cancer specimens. Weak nuclear staining for FOXP3 was detectable in 16 of 1574 breast cancer cases (1%) and was only seen in a small proportion of malignant cells. There was no difference in survival between patients with FOXP3-positive or -negative tumors. CONCLUSION: FOXP3 does most likely not play a significant role in breast cancer biology, because it is only scarcely expressed in a very small proportion of breast cancer samples.

Src homology phosphotyrosyl phosphatase-2 expression is an independent negative prognostic factor in human breast cancer.

AIMS: Src homology phosphotyrosyl phosphatase-2 (SHP2) is a ubiquitously expressed phosphatase that plays an essential role in the downstream signalling pathways of multiple growth factor receptors, thus representing a potential target for cancer therapy. Recent studies suggest that SHP2 contributes to tumour initiation, progression and metastasis in breast cancer, yet the impact of SHP2 expression on prognosis in human breast cancer has not been evaluated. METHODS AND RESULTS: To explore further the role of SHP2 in breast cancer, we conducted an immunohistochemical study using a tissue microarray encompassing 1401 formalin-fixed breast cancer specimens with detailed clinical annotation and outcome data. Of 1401 evaluable breast cancers, 651 (46%) were positive for SHP2. SHP2 expression was associated positively with tumour grade, lymph node status and tumour stage. In univariate survival analysis, cases with SHP2 expression had a significantly worse overall survival (OS). In multivariate analysis, SHP2 remained an independent negative prognostic factor for OS. SHP2 expression was a negative prognostic factor for OS in the luminal A and the luminal B HER2(-) intrinsic subtypes. CONCLUSIONS: Our data demonstrate for the first time that SHP2 is an independent predictor of survival in breast cancer, suggesting that SHP2 may be a potential target for therapy.

Detection of ALK-positive non-small-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone.

INTRODUCTION: Lung cancer is often diagnosed by cytology, necessitating predictive molecular marker analyses on cytological specimens. The gold standard for detection of predictive anaplastic lymphoma kinase (ALK)-rearrangements is fluorescence in situ hybridization (FISH), but FISH is both expensive and often challenging to interpret. The aim of our study was to investigate the accuracy of ALK immunocytochemistry (ICC) on cytological specimens of non-small-cell lung cancers (NSCLCs). METHODS: Forty-one cytological specimens with available ALK FISH results were retrospectively analyzed with the 5A4 monoclonal antibody (Novocastra; Leica Biosystems) on a fully automated slide stainer. The specimens were enriched for ALK FISH-positive NSCLCs (14 of 41; 34.1%). Evaluation of the ICC staining was performed blinded to the FISH results. The staining intensity and the percentage of stained cancer cells were recorded. Any ICC staining was regarded as a positive result. The ALK ICC results were compared with the FISH results. In case of a discrepancy the ICC-stained slide and the FISH signals were reviewed. RESULTS: ICC was evaluable on 40 of 41 specimens. Fifteen of 40 NSCLCs (37.5%) were ALK ICC-positive, with staining of the majority of cancer cells (median 100%; mean 82.3%). Twelve of the ICC-positive NSCLCs (80.0%) showed an intense staining (3+). Compared with the ALK FISH results, only one NSCLC was false-negative, and one false-positive by ICC, respectively. The sensitivity, specificity, and positive and negative predictive values for ALK ICC compared with ALK FISH were 93.3%, 96.0%, 93.3%, and 96%, respectively. CONCLUSION: ALK ICC is highly accurate for detecting ALK-rearranged NSCLCs.

HPV L1 detection discriminates cervical precancer from transient HPV infection: a prospective international multicenter study.

The benefits of cytology-based cervical cancer screening programs in reducing morbidity and mortality are well recognized. Especially, overtreatment of human papillomavirus (HPV) high-risk positive early dysplastic lesions may have a negative impact on reproductive outcomes for fertile women. To optimize the clinical management an objective standard is needed to distinguish precancer that requires treatment, from spontaneously resolving HPV infections. In the current study, we examined the prognostic relevance of HPV-L1 capsid protein analysis with Cytoactiv in an international prospective multicenter study including 908 HPV high-risk positive early dysplastic lesions (LSIL/HSIL) during a follow-up period of 54 months. The clinical end points of the study were histologically confirmed CIN3+ as progression, CIN1/2 for stable disease and repeated negative Pap smears as spontaneous clinical remission. The difference of the clinical outcome of HPV-L1-negative and HPV-L1-positive cases was statistically highly significant (P-value<0.0001) independent of the classification as mild dysplasia (LSIL) and moderate dysplasia (HSIL). Of the HPV-L1-negative HPV high-risk positive mild/moderate dysplasias 84% progressed to CIN3, as compared with only 20% of the HPV-L1-positive cases. The data from our study show that HPV-L1 detection allows to identify transient HPV infections and precancerous lesions within the group of HPV high-risk positive early dysplastic lesions. The high progression rate of HPV-L1-negative mild and moderate dysplasia emphasizes the precancerous nature of these lesions. A close follow-up with colposcopy and histological evaluation is advisable and removal of these lesions should be considered. The low malignant potential of HPV-L1-positive cases, however, indicates transient HPV infection, justifying a watch and wait strategy with cytological follow-up, thus preventing overtreatment especially for women in their reproductive age.