New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation.

BACKGROUND: Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function. METHODS: NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing. RESULTS: In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins. CONCLUSIONS: NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.

Prognostic significance of PD-1 and PD-L1 positive tumor-infiltrating immune cells in ovarian carcinoma.

INTRODUCTION: Ovarian carcinoma is associated with the highest mortality of all gynecologic malignancies. Even after optimal treatment, prognosis remains poor. There is no established biomarker to predict individual patient outcome. OBJECTIVE: To evaluate the prognostic significance of PD-1 and PD-L1 expression in tumor tissues from patients with ovarian cancer. METHODS: Tissue micro-arrays were prepared from routinely formalin-fixed, paraffin-embedded tumor tissues and examined immunohistochemically for the expression of programed cell death protein 1 (PD-1) and one of its ligands (PD-L1) on epithelial tumor cells, as well as on tumor- and stroma-infiltrating immune cells. RESULTS: The presence of PD-1 positive tumor-infiltrating immune cells was significantly associated with prolonged overall survival. PD-1 and PD-L1 positive tumor-infiltrating immune cells were associated with the presence of lymph node metastases and higher tumor grade. Interestingly, the amount of PD-1/PD-L1 positive tumor- and stroma-infiltrating immune cells independent of PD-1 or PD-L1 expression did not show any significant correlation with prognostic variables. CONCLUSION: Our results highlight the prognostic value of PD-1 and PD-L1 positive tumor-infiltrating immune cells in ovarian carcinoma. Their association with favorable prognosis supports the hypothesis that the expression of PD-1 and PD-L1 on tumor-infiltrating immune cells represents a strong immune response.

Intranuclear inclusions in hepatocellular carcinoma contain autophagy-associated proteins and correlate with prolonged survival.

For decades, intranuclear inclusions in many normal and neoplastic cells have been considered to be mere invaginations of cytoplasm into the nucleus without any notable function or influence on disease. We investigated such inclusions in 75 specimens of hepatocellular carcinoma (HCC). In this context we demonstrate that these inclusions are true inclusions, completely closed and delimited by the nuclear membrane, containing degenerate cell organelles and lysosomal proteins. Moreover, their occurrence was positively associated with patient survival but not with tumour grade or stage. In a standardised area a mean of 124 inclusions per specimen was present in the tumorous liver tissue in contrast to 5 inclusions in the non-tumorous adjacent section and 89% of all scrutinised HCC showed at least one membrane-bound nuclear inclusion. Ultrastructural characterisation by transmission electron microscopy revealed degenerative materials such as residues of lysosomes, endoplasmic reticulum and Golgi apparatus within the inclusions. Due to the fact that the content of the inclusions appears to be more condensed than cytoplasm and contains fewer intact cell organelles, we assume that they are not mere invaginations of cytoplasm. Three dimensional (3D) reconstruction of isolated and immunofluorescence stained nuclei showed that the inclusions are completely located within the nucleus without any connection to the cytoplasm. The limiting membrane of the inclusions contained lamin B suggesting nuclear membrane origin. The content of the inclusions stained for the autophagy-associated proteins p62, ubiquitin, LC3B, cathepsin B and cathepsin D. Triple immunofluorescence staining followed by 3D reconstruction revealed co-localisation of p62, ubiquitin and LC3B in the same inclusion. Our observations uncover that these inclusions are real inclusions completely surrounded by the nucleus. We propose that the presence of autophagy-associated proteins and proteases within the inclusions contribute to beneficial survival.

MET Expression in Advanced Non-Small-Cell Lung Cancer: Effect on Clinical Outcomes of Chemotherapy, Targeted Therapy, and Immunotherapy.

BACKGROUND: The receptor tyrosine kinase MET is implicated in malignant transformation, tumor progression, metastasis, and acquired treatment resistance. We conducted an analysis of the effect of MET expression and MET genomic aberrations on the outcome of patients with advanced or metastatic pulmonary adenocarcinomas prospectively enrolled in an institutional precision oncology program. PATIENTS AND METHODS: Standardized immunohistochemistry (IHC) analyses of MET and markers of pathway activation were available in 384 patients, and next-generation sequencing-based MET hotspot mutation analyses were available from 892 patients. Clinical data were retrieved with a median follow-up from initial diagnosis of 37 months. RESULTS: High MET expression, defined as MET IHC 3+ or MET H-Score in the upper quartile, was observed in 102 of 384 patients (26.6%). MET exon 14 mutations were only detected in 7 of 892 patients (0.78%). High MET expression correlated with activation markers of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathways only in cases without Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR), v-Raf murine sarcoma viral oncogene homolog B (BRAF), anaplastic lymphoma kinase (ALK) and proto-oncogene tyrosine-protein kinase ROS (ROS1) aberrations. There was no association of MET expression with outcome during chemotherapy. High MET expression negatively affected the outcome during EGFR-targeting therapy but was associated with more favorable results with programmed death 1/programmed death ligand 1 (PD-L1)-directed therapy, independent of smoking history, PD-L1 expression or KRAS mutation. Two patients with MET exon 14 mutation and high PD-L1 expression failed to respond to pembrolizumab. CONCLUSION: MET expression affects the outcomes of targeted therapies in non-small-cell lung cancer, thus supporting the development of biomarker-informed combination strategies. The interaction of MET expression and MET mutation with immune checkpoint inhibitor therapy is novel and merits further investigation.

Comprehensive Biomarker Analyses in Patients with Advanced or Metastatic Non-Small Cell Lung Cancer Prospectively Treated with the Polo-Like Kinase 1 Inhibitor BI2536.

BACKGROUND: Polo like kinase 1 (PLK1) is frequently upregulated in tumors and is thus viewed as a promising therapeutic target in various cancers. Several PLK1 inhibitors have recently been developed and clinically tested in solid cancers, albeit with limited success. So far, no predictive biomarkers for PLK1 inhibitors have been established. To this end, we conducted a post-hoc biomarker analysis of tumor samples from non-small cell lung cancer (NSCLC) patients treated with the PLK1 inhibitor BI2536 in a phase II study. METHODS: We analyzed formalin-fixed paraffin-embedded surplus tumor tissue from 47 study patients using immunohistochemistry (IHC) and DNA sequencing of KRAS, EGFR, BRAF, and PIK3CA. RESULTS: KRAS-mutated patients showed numerically prolonged progression-free survival, but statistical significance was not established. Interestingly, when pathways rather than single genes were analyzed, a positive correlation between IHC staining of activated ERK (p-ERK) and mutated KRAS was detected, whereas KRAS mutation status was found to be negatively correlated with activated AKT (p-AKT). CONCLUSION: With this hypothesis-generating study in BI2531-treated patients, we could not establish a correlation between KRAS mutations and relevant clinical endpoints. Future clinical trials with concomitant systematic biosampling and comprehensive molecular analyses are required to identify biomarkers predictive for response to PLK1 inhibitors.

Annexin A10 optimally differentiates between intrahepatic cholangiocarcinoma and hepatic metastases of pancreatic ductal adenocarcinoma: a comparative study of immunohistochemical markers and panels.

Discriminating intrahepatic cholangiocarcinoma (ICC) from hepatic metastases of pancreatic ductal adenocarcinoma (mPDAC) can be challenging. While pathologists might depend on clinical information regarding a primary tumor, their diagnosis will lead the patient either to potentially curative surgery (for ICC) or to palliation (for mPDAC). Beyond the validation of recently published potential biomarkers for PDAC (primary or metastatic) in a large cohort, we assessed diagnostic performance of the most promising candidates in the challenging task of discriminating metastatic PDAC (mPDAC) from ICC. In a training set of 87 ICC and 88 pPDAC, our previously identified biomarkers Annexin A1 (ANXA1), ANXA10, and ANXA13 were tested and compared with 11 published biomarkers or panels (MUCIN 1, Agrin, S100P, MUC5 AC, Laminin, VHL, CK 17, N-Cadherin, ELAC2, PODXL and HSPG2). Biomarkers with best results were further tested in an independent series of biopsies of 27 ICC and 36 mPDAC. Highest AUC values (between 0.72 and 0.84) for the discrimination between ICC and pPDAC were found in the training set for Annexin A1, Annexin A10, MUC5 AC, CK17, and N-Cadherin. These markers were further tested on an independent series of liver biopsies containing ICC or mPDAC. Diagnostic characteristics were evaluated for individual markers as well as for 3× panels. ANXA 10 showed the highest diagnostic potential of all single markers, correctly classifying 75% of mPDAC and 85% of ICC. Our results suggest that ANXA10 may be useful to differentiate between ICC and mPDAC, when only a tissue specimen is available.

NOTCH1, HIF1A and other cancer-related proteins in lung tissue from uranium miners--variation by occupational exposure and subtype of lung cancer.

BACKGROUND: Radon and arsenic are established pulmonary carcinogens. We investigated the association of cumulative exposure to these carcinogens with NOTCH1, HIF1A and other cancer-specific proteins in lung tissue from uranium miners. METHODOLOGY/PRINCIPAL FINDINGS: Paraffin-embedded tissue of 147 miners was randomly selected from an autopsy repository by type of lung tissue, comprising adenocarcinoma (AdCa), squamous cell carcinoma (SqCC), small cell lung cancer (SCLC), and cancer-free tissue. Within each stratum, we additionally stratified by low or high level of exposure to radon or arsenic. Lifetime exposure to radon and arsenic was estimated using a quantitative job-exposure matrix developed for uranium mining. For 22 cancer-related proteins, immunohistochemical scores were calculated from the intensity and percentage of stained cells. We explored the associations of these scores with cumulative exposure to radon and arsenic with Spearman rank correlation coefficients (r(s)). Occupational exposure was associated with an up-regulation of NOTCH1 (radon r(s) = 0.18, 95% CI 0.02-0.33; arsenic: r(s) = 0.23, 95% CI 0.07-0.38). Moreover, we investigated whether these cancer-related proteins can classify lung cancer using supervised and unsupervised classification. MUC1 classified lung cancer from cancer-free tissue with a failure rate of 2.1%. A two-protein signature discriminated SCLC (HIF1A low), AdCa (NKX2-1 high), and SqCC (NKX2-1 low) with a failure rate of 8.4%. CONCLUSIONS/SIGNIFICANCE: These results suggest that the radiation-sensitive protein NOTCH1 can be up-regulated in lung tissue from uranium miners by level of exposure to pulmonary carcinogens. We evaluated a three-protein signature consisting of a physiological protein (MUC1), a cancer-specific protein (HIF1A), and a lineage-specific protein (NKX2-1) that could discriminate lung cancer and its major subtypes with a low failure rate.