Does subtyping of high-grade pulmonary neuroendocrine carcinomas have an impact on therapy selection?

Background: Small cell lung cancer (SCLC) and large cell neuroendocrine carcinomas (LCNEC) are characterized by a rapid progressive course. Therapy for SCLC has not much changed for decades, and in LCNEC controversies exist, favoring either SCLC-like or non-small cell lung cancer (NSCLC)-like therapy. Three subtypes of SCLC identified in cell cultures, namely ASCL1, NeuroD1, and POU2F3 have been confirmed by immunohistochemistry. The fourth type based on the expression of YAP1 was questioned, and another type, inflamed SCLC, was proposed. Methods: SCLC and LCNEC samples were investigated by immunohistochemistry for different subtypes. Additionally, immunohistochemical markers as potential tools to identify patients who might respond to targeted treatment were investigated. For validation a biopsy set was added. Results: ASCL1, NeuroD1, and POU2F3 were expressed in different percentages in SCLC and LCNEC. Similar percentages of expression were found in biopsies. ATOH was expressed in combination with one of the subtypes. YAP1 and TAZ were expressed in some SCLC and LCNEC cases. HES1 expression was seen in few cases. Predominantly stroma cells expressed programmed cell death ligand 1 (PD-L1). The dominant MYC protein was N-MYC. Aurora kinase A (AURKA) was expressed in the majority of both carcinomas, whereas fibroblast growth factor receptor 2 (FGFR2) in few. Conclusions: SCLC and LCNEC can be subtyped into ASCL1-, NeuroD1-, and POU2F3-positive types. AURKA expression and positivity for N-MYC protein was not associated with subtypes. AURKA and FGFR2 are both possible targets for inhibition in SCLC and LCNEC, but patients' selection should be based on expression of the enzyme. Combined chemo- and immunotherapy might be decided by PD-L1 staining of stroma cells.

Senescence and autophagy in usual interstitial pneumonia of different etiology.

Idiopathic pulmonary fibrosis (IPF) is a disease with a dismal prognosis. Currently, the causing agent(s) are poorly understood. Recent data suggest that senescence and autophagy might play a role in its development, as well as changes in metabolism due to hypoxic conditions. In this study, the expression of senescence markers in 23 cases of usual interstitial pneumonia (UIP)/IPF and UIP/chronic autoimmune diseases (UIP/AuD) was investigated. The status of autophagy was evaluated with respect to either antiinflammatory or antihypoxia function. Formalin-fixed paraffin-embedded tissues of UIP were selected for immunohistochemistry with antibodies for p21, p16, and ß-galactosidase (senescence); for LC3, SIRT1, MAP1S, and pAMKα (autophagy); and for LDH and GLUT1 (metabolism). Epithelial cells in cystic remodeled areas of UIP stained for p16 and p21, p16 being more specific compared with p21. Myofibroblasts were negative in all cases. An upregulation of all four autophagy markers was seen not only in epithelia within remodeled areas and proliferating myofibroblasts, but also in bronchial epithelia and pneumocytes. Upregulated autophagy points to a compensatory mechanism for hypoxia; therefore, LDH and GLUT1 were investigated. Their expression was present in epithelia within cystic remodeling and in myofibroblasts. The cells within the remodeled areas stained for cytokeratin 5, but coexpressed TTF1, confirming their origin from basal cells of bronchioles. Within this population, senescent cells arise. Our results indicated that autophagy in UIP very likely helps cells to survive in hypoxic condition. By phagocytosis of cellular debris, they supplement their need for nutrition, and by upregulating LDH and GLUT1, they compensate for local hypoxia.

Pleuropulmonary blastoma type I might arise in congenital pulmonary airway malformation type 4 by acquiring a Dicer 1 mutation.

Congenital pulmonary airway malformation (CPAM) occurs most commonly in infants. It is divided into 5 types. The most common types 1 and 2 are cystic, type 0 presents as bronchial buds without alveolar tissue, most likely corresponding to alveolar dysgenesis, while type 3 is composed of branching bronchioles and appears as a solid lesion. A defect in the epithelial-mesenchymal crosstalk might be the underlying mechanism for all. Type 4 is a peripheral cystic lesion with a thin cyst wall covered by pneumocytes. CPAM 4 has been mixed up with pleuropulmonary blastoma (PPB) type I and some authors question its existence. We investigated five cases of CPAM type 4 for the presence or absence of rhabdomyoblasts, and for markers associated with CPAM development. In addition, all cases were evaluated for mutations within the Dicer gene and for mutations of the RAS family of oncogenes. All five cases showed smooth muscle actin and desmin-positive cells; however, only one case showed a few cells positive for MyoD. The same case showed a mutation of Dicer 1. All cases were negative for mutations of the RAS family of genes. Fibroblast growth factor 10 was similarly expressed in all cases, and thus cannot be used to differentiate CPAM4 from PPB-I. Low expression of the proliferation marker Ki67 was seen in our CPAM 4 cases and the probable PPB-I case. YingYang-1 protein seems to play an active role in the development of PPB-I. CPAM 4 can be separated from PPB-I based on the presence of rhabdomyoblasts and mutations in Dicer 1 gene. These cells might not be numerous; therefore, all available tissue has to be evaluated. As CPAM 4 morphologically looks very similar to PPB-I, it might be speculated, that there exists a potential for progression from CPAM 4 to PPB-I, by acquiring somatic mutations in Dicer 1.

Atypical goblet cell hyperplasia occurs in CPAM 1, 2, and 3, and is a probable precursor lesion for childhood adenocarcinoma.

Congenital pulmonary airway malformation (CPAM) is a developmental disorder. Types 1-2-3 are the more common ones. Atypical goblet cell hyperplasia (AGCH) in CPAM might be a precursor lesion for pulmonary adenocarcinomas. In nine out of 33 CPAM cases, types 1-3 showed foci of goblet cell proliferations. As these cells completely replace normal epithelium, we prefer to name these proliferations AGCH. In 5 cases, adenocarcinomas were seen (AC). All cases were analyzed for proteins possibly being associated with CPAM development: fibroblast growth factor 10 (FGF10) and receptor 2 (FGFR2), forkhead box A1 (FOXA1) and A2 (FOXA2), MUC protein 5AC (MUC5AC), human epidermal growth factor receptor 2 (erbB2, HER2/neu), hepatocyte nuclear factor 4α (HNF4α), SOX2, and Ying Yang protein 1 (YY1). By next generation sequencing, AGCH and adenocarcinomas were evaluated for driver mutations. Expression for FGF10, FGFR2, FOXA1, and FOXA2 was seen in CPAM epithelium and stroma, but not differently in AGCH and AC. SOX2 was positive in CPAM epithelium and AGCH, however weakly in AC. YY1 and MUC5AC showed more intense staining in AGCH and AC than in CPAM epithelium. HER2 was intensely expressed in AC and less intensely in AGCH, but not in CPAM epithelium. KRAS mutation in exon 2 was detected in all AGCH and AC, but was absent in CPAM epithelia. AGCH can arise in CPAM types 1-3. Oncogenic KRAS mutation seems to be the oncogenic driver already in AGCH, proving its role as a precursor lesion for adenocarcinoma. It might upregulate HER2 at the protein level. YY1 seems to be involved in carcinogenesis.

Comparison of four DLL3 antibodies performance in high grade neuroendocrine lung tumor samples and cell cultures.

BACKGROUND: Small cell lung cancer (SCLC) is usually diagnosed in the advanced stage. It has a very poor prognosis, with no advancements in therapy in the last few decades. A recent phase 1 clinical study, using an antibody-drug conjugate directed against DLL3, showed promising results. A prerequisite for this therapy is an immunohistochemical test for DLL3 expression. The antibody used in the clinical trial was bound to a specific platform, which is not available in all pathology laboratories. In this study, the expression of DLL3 was analyzed using different DLL3 antibodies in high-grade neuroendocrine tumors of the lung and cell cultures. Additionally, correlation of DLL3 expression with Rb1 loss and TP53 mutation was evaluated. METHODS: The study cohort consisted of surgically resected cases, 24 SCLC and 29 large cell neuroendocrine carcinoma (LCNEC), from which tissue microarrays (TMAs) were constructed. The validation cohort included 46 SCLC samples, mostly small biopsies. Additionally, well-characterized SCLC cell lines were used. Immunohistochemical analysis was performed using four different DLL3 antibodies, as well as TP53 and Rb1 antibodies. Expression was evaluated microscopically and manually scored. RESULTS: The comparison of all DLL3 antibodies showed poor results for the overall agreement, as well as positive and negative agreement. Differences were observed regardless of the applied cut-off values and the tumor type. The antibody used in the clinical trial was the only which always positively stained the tumor cells obtained from cell cultures with known DLL3 expression and was negative on cells that did not express DLL3. There was no correlation between p53 and DLL3 expression in SCLC and LCNEC. RB1 loss in SCLC showed statistical significant correlation with the DLL3 positivity (p = 0.037), while no correlation was found in LCNEC. CONCLUSION: The DLL3 antibody used in the clinical trial demonstrated superiority in the detection of DLL3 expression. Cell cultures, which can be used for DLL3 antibodies as positive and negative probes, were established. Evidence of DLL3 expression in high proportions of patients with LCNEC might provide basis for studies of new therapy options in this group of patients.

Bulk tumour cell migration in lung carcinomas might be more common than epithelial-mesenchymal transition and be differently regulated.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is one mechanism of carcinoma migration, while complex tumour migration or bulk migration is another - best demontrated by tumour cells invading blood vessels. METHODS: Thirty cases of non-small cell lung carcinomas were used for identifying genes responsible for bulk cell migration, 232 squamous cell and adenocarcinomas to identify bulk migration rates. Genes expressed differently in the primary tumour and in the invasion front were regarded as relevant in migration and further validated in 528 NSCLC cases represented on tissue microarrays (TMAs) and metastasis TMAs. RESULTS: Markers relevant for bulk cancer cell migration were regulated differently when compared with EMT: Twist expressed in primary tumour, invasion front, and metastasis was not associated with TGFß1 and canonical Wnt, as Slug, Snail, and Smads were negative and ß-Catenin expressed membraneously. In the majority of tumours, E-Cadherin was downregulated at the invasive front, but not absent, but, coexpressed with N-Cadherin. Vimentin was coexpressed with cytokeratins at the invasion site in few cases, whereas fascin expression was seen in a majority. Expression of ERK1/2 was downregulated, PLCγ was only expressed at the invasive front and in metastasis. Brk and Mad, genes identified in Drosophila border cell migration, might be important for bulk migration and metastasis, together with invadipodia proteins Tks5 and Rab40B, which were only upregulated at the invasive front and in metastasis. CXCR1 was expressed equally in all carcinomas, as opposed to CXCR2 and 4, which were only expressed in few tumours. CONCLUSION: Bulk cancer cell migration seems predominant in AC and SCC. Twist, vimentin, fascin, Mad, Brk, Tsk5, Rab40B, ERK1/2 and PLCγ are associated with bulk cancer cell migration. This type of migration requires an orchestrated activation of proteins to keep the cells bound to each other and to coordinate movement. This hypothesis needs to be proven experimentally.

Critical evaluation of KCNJ3 gene product detection in human breast cancer: mRNA in situ hybridisation is superior to immunohistochemistry.

Increased expression levels of KCNJ3 have been correlated with lymph node metastases and poor prognosis in patients with breast cancer, suggesting a prognostic role of KCNJ3 We aimed to establish protocols for the detection of KCNJ3 in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue. Several antibodies were tested for sensitivity and specificity by western blot, followed by optimisation of the immunohistochemistry (IHC) procedure and establishment of KCNJ3 mRNA in situ hybridisation (ISH). Methods were validated by processing 15 FFPE breast cancer samples for which microarray data were available. Spearman's rank correlation analysis resulted in borderline significant correlation for IHC versus ISH (rS: 0.625; p<0.05) and IHC versus microarray (rS: 0.668; p<0.01), but in significant correlation for ISH versus microarray (rS: 0.861; p<0.001). The ISH method was superior to IHC, regarding robustness, sensitivity and specificity and will aid to further study expression levels of KCNJ3 in both malignant and physiological conditions.

Mutation Profiling of Usual Ductal Hyperplasia of the Breast Reveals Activating Mutations Predominantly at Different Levels of the PI3K/AKT/mTOR Pathway.

Usual ductal hyperplasia (UDH) of the breast is generally regarded as a nonneoplastic proliferation, albeit loss of heterozygosity has long been reported in a part of these lesions. To gain deeper insights into the molecular drivers of these lesions, an extended mutation profiling was performed. The coding regions of 409 cancer-related genes were investigated by next-generation sequencing in 16 cases of UDH, nine unassociated with neoplasia (classic) and seven arising within papillomas. Phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) activation was investigated by phosphorylated AKT, mTOR, and S6 immunohistochemistry. Of 16 lesions, 10 (63%) were mutated; 56% of classic lesions were unassociated with neoplasia, and 71% of lesions arose in papillomas. Fourteen missense mutations were detected: PIK3CA [6 (43%) of 14], AKT1 [2 (14%) of 14], as well as GNAS, MTOR, PIK3R1, LPHN3, LRP1B, and IGF2R [each 1 (7%) of 14]. Phosphorylated mTOR was seen in 83% and phosphorylated S6 in 86% of evaluable lesions (phospho-AKT staining was technically uninterpretable). In conclusion, UDH displays mutations of the phosphatidylinositol 3-kinase/AKT/mTOR axis at different levels, with PIK3R1, MTOR, and GNAS mutations not previously described. Specifically, oncogenic G-protein activation represents a yet unrecognized route to proliferation in UDH. On the basis of evidence of activating mutations, loss of heterozygosity, and a mass forming proliferation, we propose that UDH is most appropriately viewed as an early neoplastic intraductal proliferation.