Up-regulation by overexpression of c-MET in fibroblastic foci of usual interstitial pneumonia.

Background: Usual interstitial pneumonia (UIP) is the radiologic and histologic hallmark of idiopathic pulmonary fibrosis (IPF) and the commonest histologic pattern of other progressive fibrosing interstitial lung diseases (e.g., fibrotic hypersensitivity pneumonia). Analogous to lung cancer, activation of epithelial-to-mesenchymal transition (EMT) is one of the main molecular pathways recently identified by transcriptomic studies in IPF. Fibroblastic foci (FF) are considered the active/trigger component of UIP pattern. The proto-oncogene C-MET is a key gene among molecules promoting EMT against which several inhibitors are currently available or promising in ongoing studies on lung cancer. Methods: Twenty surgical cases of diffuse fibrosing interstitial lung diseases (fILD) with UIP pattern and FF-rich (17 IPF and 3 patients with fibrotic hypersensitivity pneumonia, fHP) were retrospectively selected. FF were manually microdissected and analysed for c-MET gene alterations (FISH amplification and gene hot-spot mutations Sanger sequencing) and tested with a c-MET companion diagnostic antibody (clone SP44 metmab) by immunohistochemistry. Results: FF are characterized by upregulation of c-MET as shown by overexpression of the protein in 80% of cases, while no gene amplification by FISH or mutations were detected. C-MET upregulation of FF was observed either in IPF and fHP, with a tropism for the epithelial cell component only. Conclusion: Upregulation of c-MET in FF of ILD with UIP pattern further confirms the key role of the proto-oncogene c-MET in its pathogenesis, possibly representing an interesting and easily-detectable molecular target for selective therapy using specific inhibitors in future clinical trials, similar to lung cancer. It is reasonable to speculate that molecular alterations in FF can also be detected in FF by transbronchial cryobiopsy.

Loss of CDKN2A Promoter Methylation Coincides With the Epigenetic Transdifferentiation of Uterine Myosarcomatous Cells.

Leiomyosarcoma is the most common type of uterine sarcoma and usually displays typical morphology. Heterologous leiomyosarcoma is the rarest variant, in which the tumor contains liposarcomatous, osteosarcomatous, or rhabdomyosarcomatous components. We have investigated the largest series of uterine leiomyosarcoma with a rhabdomyosarcomatous component and we have disclosed a molecular finding, which coincides to the process of transdifferentiation from smooth muscle into striated muscle phenotype. The surgical specimens of 5 rare cases of uterine leiomyosarcoma with a rhabdomyosarcomatous component were formalin fixed and paraffin embedded. In addition to hematoxylin/eosin stains, phosphotungstic acid hematoxylin staining, immunohistochemistry, and methylation-specific polymerase chain reaction the CDKN2A promoter region were performed. Leiomyosarcomatous cells were found to be strongly immunoreactive for both desmin and α-smooth muscle actin. Rhabdomyosarcomatous cells were immunoreactive for sarcomeric actin, desmin, vimentin, CD10, and p16. The methylation-specific polymerase chain reaction revealed the presence of a methylated allele and an unmethylated allele in the microdissected samples, coming from leiomyosarcomatous cells. On the contrary, 2 unmethylated alleles, molecular expression of a loss of heterozygosity, were detected in all the microdissected samples in the rhabdomyosarcomatous cells. The loss of heterozygosity methylation in the promoter region of the CDKN2A gene, occurred only in the rhabdomyosarcomatous cells with increases in both p16 and p14 expression. This event may result in an inhibition of cdk4/cdk6 activity, stabilizes the tumor suppressor protein p53, and coincides with the transdifferentiation from smooth muscle into striated muscle.

MUC5AC, cytokeratin 20 and HER2 expression and K-RAS mutations within mucinogenic growth in congenital pulmonary airway malformations.

AIMS: To analyse the expression of several mucins (MUC1, MUC2, MUC3, MUC5AC and MUC6), epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukaemia viral oncogene homologue 2 (HER2), thyroid transcription factor-1 (TTF-1), caudal type homeobox 2 (CDX2) and cytokeratin 20 (CK20), and the presence of mutations of EGFR, KRAS and HER2 in congenital pulmonary airway malformations (CPAM). METHODS AND RESULTS: Forty-one cases of CPAM and six pulmonary sequestrations were included. TTF-1 expression was observed in all cases but was not seen in mucinogenic growths in CPAM. CDX2 expression was completely negative. MUC1 expression was noted in 12 (29%) CPAM and in 33% sequestrations. MUC5AC was noted in only five cases (26%) by immunohistochemistry and was found in the mucinogenic proliferations of type 1 CPAM. No immunolabelling was noted for the other mucins. EGFR was expressed variably in almost all cases, while HER2 and CK20 was seen exclusively in the mucinogenic proliferations. All mucinous growths were characterized by KRAS mutations. No EGFR and HER2 gene alterations were identified. CONCLUSIONS: KRAS mutations and MUC5AC, CK20 and HER2 expression was seen in all mucinogenic proliferations, supporting the neoplastic nature of these cytologically bland growths. These findings emphasize the importance of complete surgical resection of such lesions.

EGFR polysomy in squamous cell carcinoma of the thyroid. Report of two cases and review of the literature.

AIMS AND BACKGROUND: Primary squamous cell carcinoma of the thyroid gland (PSCCT) is an uncommon malignancy characterized by a poor prognosis. A radical surgical approach combined with radiotherapy or chemotherapy is the generally accepted treatment for this tumor. The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor modulating the cell proliferation and biological progression of many human epithelial tumors. The EGFR overexpression in PSCCT suggests an additional therapeutic option for the treatment of this tumor. METHODS AND STUDY DESIGN: The clinicopathological features and immunohistochemical profiles of two cases of primary squamous cell carcinoma of the thyroid in a 66-year-old and an 83-year-old woman are presented. EGFR status was valued in both cases. RESULTS: Overexpression of EGFR protein was detected in 50% and 75% of the tumor cell membranes. EGRF gene polysomy was detected in both tumors. CONCLUSIONS: Pharmaceuticals targeting EGFR may help to provide the rationale for an additional, novel therapeutic option for this rare tumor, especially when other therapeutic options have been exhausted.

Infarct-Like Spindle Cell Carcinoma of the Lung: Clinicopathologic, Immunohistochemical, and Molecular Analysis of 4 Cases.

Pulmonary spindle cell carcinoma is a rare and aggressive malignancy that often mimics benign conditions. We report 4 cases that simulate a pulmonary infarction, 2 of which were misdiagnosed. Patients were 3 men and 1 woman, smokers, presenting chest pain. All cases appeared as pleural-based, solitary, and rounded nodules. Patients underwent wedge resections followed by adjuvant chemotherapy (3/4) but died of disease. At histology, lesions consisted of widely necrotic nodules surrounded by organizing fibrosis and pleuritis. Examination and immunostains with pan-cytokeratins and epithelial membrane antigen (EMA) revealed atypical spindle cells encircling necrotic tissue and involving the vascular wall. Positive staining with PD-L1 was noted. Molecular analysis showed KRAS (2/4) and TP53 (1/4) mutations, whereas EGFR, ALK, and ROS1 alterations were not detected. Although in a limited series, these cases further evidence the treacherous appearance of spindle cell carcinomas and the need for careful attention when examining pulmonary infarcted tissue, thus requiring extensive sampling, meticulous examination of vascular structures, and immunostaining with cytokeratins.

Mucoepidermoid carcinoma of the lung, response to EGFR inhibitors, EGFR and K-RAS mutations, and differential diagnosis.

Mucoepidermoid carcinoma (MEC) of the lung needs to be carefully distinguished from other lung tumors with similar features, particularly from adenosquamous carcinoma, this latter tumor frequently showing EGFR mutations. In contrast with the data reported by Han et al in the last July issue of Lung Cancer, neither EGFR nor K-RAS mutations were observed in MEC from caucasian patients.

A subset of lung adenocarcinomas and atypical adenomatous hyperplasia-associated foci are genotypically related: an EGFR, HER2, and K-ras mutational analysis.

Atypical adenomatous hyperplasia (AAH) is considered the preinvasive lesion of pulmonary adenocarcinoma, and mutations of EGFR, HER2, and K-ras are involved in the early stage of lung adenocarcinoma carcinogenesis, also predicting clinical response to anti-EGFR small molecule inhibitors. We analyzed 18 cases of primary lung adenocarcinoma with concomitant AAH foci from 13 patients for mutations of EGFR (exons 18-21), HER2 (exons 19-20), and K-ras (exon 2) by direct sequencing polymerase chain reaction. Among mutated cases, concordant mutations of EGFR or K-ras in adenocarcinoma and related AAH were observed in 5 (63%) of 8 cases. In particular, 3 of 4 adenocarcinomas with EGFR mutations (all L858R point mutations in women, never or former smokers) had a concomitant and identical mutation in AAH, and 2 of 4 adenocarcinomas with K-ras mutations (both at codon 12 in women, a never and a current smoker) showed the same mutation in concomitant AAH. All cases were wild-type for HER2. Mutations of EGFR and K-ras genes represent an early event in lung adenocarcinomagenesis, and AAH convincingly seems to be a precursor lesion in a subset of cases of adenocarcinoma.

Microsatellite and EGFR, HER2 and K-RAS analyses in sclerosing hemangioma of the lung.

Sclerosing hemangioma (SH) is an uncommon pulmonary tumor thought to derive from primitive respiratory epithelium consisting of 2 cell populations (cuboidal surface and polygonal stromal cells) and sharing some clinical characteristics (frequent occurrence in nonsmoking women of Asian ethnicity) with bronchioloalveolar carcinoma with which it has been suggested a possible common origin. We investigated 11 cases of SH by immunohistochemistry, fluorescence in situ hybridization, and polymerase chain reaction-based microsatellite and mutational analyses with particular emphasis on possible alterations of microsatellite loci located at tumor suppressor genes (FHIT, p16, Rb, and p53) involved in lung adenocarcinoma genesis and EGFR, HER2, and K-RAS genes. Although EGFR expression was observed in all tested cases, none showed HER2 immunostaining. Fluorescence in situ hybridization and mutational analysis of EGFR and HER2 and also K-RAS sequencing did not reveal molecular alterations, whereas allelic losses at p16 and Rb loci (4 and 2 out of 9 tested cases, respectively) with an identical microsatellite allelic loss pattern in both cuboidal and polygonal cells were observed. The finding of microsatellite alterations in chromosomal regions related to genes deeply involved in early stage lung adenocarcinoma could suggest a possible link between SH and bronchioloalveolar carcinoma, but tumor pathway promoted by EGFR, HER2, and K-RAS does not represent a common molecular mechanism of tumorigenesis. Microsatellite alterations identified in cuboidal and polygonal cells further confirm the clonal and neoplastic nature of both components of SH.

Mucinous cells in type 1 pulmonary congenital cystic adenomatoid malformation as mucinous bronchioloalveolar carcinoma precursors.

Type 1 congenital cystic adenomatoid malformation (CCAM), the most frequent malformation of the lung, is the only type to present intracystic mucinous cell clusters, which may form beyond the cysts extracystic mucinous proliferation resembling mucinous bronchioloalveolar carcinomas (BACs). As mucinous BACs are increasingly described in the literature in young patients with CCAM, we hypothesized that type 1 CCAM mucinous cells could represent BAC precursors. We reviewed 7 cases of type 1 CCAM including 6 with intracystic mucinous cell clusters, 3 with extracystic mucinous proliferations, and 4 with mucinous BAC or mixed adenocarcinoma with predominant BAC. K-ras mutations at codon 12 were detected in 3/3 intracystic mucinous cell clusters, in 2/3 extracystic mucinous proliferations, and in 3/4 BAC. Loss of heterozygosity (LOH) at p16(INK4) locus, with microsatellite alterations in 3 cases, was observed in 2/3 intracystic mucinous cell clusters, in 2/3 extracystic mucinous proliferations, and in all BAC. Two extracystic mucinous proliferations showed LOH at FHIT and Rb loci, respectively. P16(INK4) expression was lost in 2 intracystic mucinous cell clusters, 1 extracystic mucinous proliferation, and 1 BAC. Neither epidermal growth factor receptor mutation on exons 18, 19, and 21 nor P53 accumulation was observed. All lesions expressed MUC5AC, but were negative for MUC2, CDX2, and TTF-1. In conclusion, type 1 CCAM mucinous cells share the same differentiation profile with corresponding mucinous BAC, consistent with a common bronchial origin. Moreover, the high frequency of K-ras mutation and LOH and/or microsatellite alterations at p16(INK4) locus presented by these mucinous cells justifies their consideration as BAC precursors.

Role of chemotherapy and the receptor tyrosine kinases KIT, PDGFRalpha, PDGFRbeta, and Met in large-cell neuroendocrine carcinoma of the lung.

PURPOSE: Pulmonary large-cell neuroendocrine carcinoma (LCNEC) is a relatively uncommon, high-grade neuroendocrine tumor sharing several features with small-cell lung carcinoma (SCLC) but currently considered as a variant of non-SCLC and accordingly treated with poor results. Little is known about the optimal therapy of LCNEC and the possible therapeutic molecular targets. PATIENTS AND METHODS: We reviewed 83 patients with pure pulmonary LCNEC to investigate their clinicopathologic features, therapeutic strategy, and immunohistochemical expression and the mutational status of the receptor tyrosine kinases (RTKs) KIT, PDGFRalpha, PDGFRbeta, and Met. RESULTS: LCNEC histology predicted a dismal outcome (overall median survival, 17 months) even in stage I patients (5-year survival rate, 33%). LCNEC strongly expressed RTKs (KIT in 62.7% of patients, PDGFRalpha in 60.2%, PDGFRbeta in 81.9%, and Met in 47%), but no mutations were detected in the exons encoding for the relevant juxtamembrane domains. Tumor stage and size (> or = 3 cm) and Met expression were significantly correlated with survival. At univariate and multivariate analysis, SCLC-based chemotherapy (platinum-etoposide) was the most important variable correlating with survival, both in the adjuvant and metastatic settings (P < .0001). CONCLUSION: Pulmonary LCNEC represents an aggressive tumor requiring multimodal treatment even for resectable stage I disease, and LCNEC seems to respond to adjuvant platinum-etoposide-based chemotherapy. Patients who received this therapy had the best survival rate. Despite our failure in finding mutational events in the tested RTKs, the strong expression of KIT, PDGFRalpha, PDGFRbeta, and Met in tumor cells suggests an important role of these RTKs in LCNEC, and these RTKs seem to be attractive therapeutic targets.

Pleural malignant solitary fibrous tumor with sarcomatous overgrowth showing PDGFRbeta mutation.

Pleural malignant solitary fibrous tumors (SFTs) are uncommon, and little is known about their histogenesis and molecular features. We report a case of pleural SFT with sarcomatous overgrowth that showed expression for PDGFRbeta and a missense mutation on exon 18 of the PDGFRbeta gene. The involvement of the PDGFRbeta gene in SFT is compatible with a pericytic derivation, also supporting a possible role of this tyrosine kinase in malignant transformation and in the adoption of novel molecular therapies.

Diamond: immunohistochemistry versus sequencing in EGFR analysis of lung adenocarcinomas.

AIMS: Identification of epidermal growth factor receptor (EGFR) mutations in lung adenocarcinomas is the single most important predictor of clinical response and outcome using EGFR tyrosine kinase inhibitors (TKIs). EGFR E746-A750del and L858R mutations are the most common gene alterations, also predicting the best clinical response to TKIs. We evaluated the accuracy of EGFR mutation-specific antibodies in a large cohort of lung adenocarcinomas, with different molecular settings and types of tissue samples. METHODS: 300 lung adenocarcinomas diagnosed on cytology (48 cell blocks), biopsy (157 cases) and surgical resections (95 cases) were selected. All cases were investigated for EGFR by sequencing and two mutation-specific antibodies (clone 6B6 for E746-A750del; clone 43B2 for L858R) were tested using an automated immunostainer. Discordant results were investigated by next-generation sequencing (NGS). RESULTS: Overall sensitivity and specificity of mutant-specific antibodies were 58.6% and 98.0%, respectively, and they increased up to 84% and 100% if only tumours harbouring E746-A750del were considered. In 13 discordant cases, NGS confirmed immunohistochemistry results in eight samples. CONCLUSIONS: The EGFR mutation-specific antibodies have a fair/good sensitivity and good/high specificity in identifying classic mutations, but they cannot replace molecular tests. The antibodies work equally well on biopsies and cell blocks, possibly permitting a rapid screening in cases with poor material.

Primary mixed adenocarcinoma and small cell carcinoma of the appendix: a clinicopathologic, immunohistochemical, and molecular study of a hitherto unreported tumor.

Appendiceal carcinoids range from well-differentiated endocrine tumor to well-differentiated endocrine carcinoma, while poorly differentiated (small cell) carcinoma has not been described in this site. We report herein a case of mixed intestinal-type adenocarcinoma associated with a small cell carcinoma arisen in a 35-year-old woman and clinically presenting as an appendiceal abscess. The resected tumor histologically appeared as a biphasic lesion composed of a nonmucinous adenocarcinoma closely juxtaposed with a poorly differentiated (small cell) endocrine carcinoma. The subsequent right hemicolectomy was unremarkable, but one pericolic lymph node showed a metastatic deposit consisting of the adenocarcinoma only. The patient thus underwent a chemotherapeutic protocol for colorectal cancer, and she is alive and well at the 65-month follow-up. Immunohistochemically, the adenocarcinoma strongly stained for cytokeratin 20 and carcinoembryonic antigen, while the endocrine component displayed a dot-like positivity for pan-cytokeratins and chromogranin. Of note, both components did not stain with CDX2 and p53. At genotypic analysis by microsatellite instability, both components shared many microsatellite alterations as well as a normal p53 gene setup, although small cell carcinoma harbored additional alterations. Clinical and molecular findings led us to consider this lesion as a clonal tumor in which the endocrine component seems to derive from a progressive differentiation of the adenocarcinoma following a glandular-to-endocrine sequence.

Placental transmogrification of the lung: clinicopathologic, immunohistochemical and molecular study of two cases, with particular emphasis on the interstitial clear cells.

Two cases of placental transmogrification of the lung are reported. The lesions presented in the left lung, in one case as a giant bulla of the upper lobe and in the other as a cystic nodule of the lower lobe. A segmentectomy was performed in both cases, and the patients were alive and well 5 years and 2 months after surgery, respectively. In our opinion, pulmonary placental transmogrification is not a variant of emphysema, as generally considered, but rather probably represents a benign proliferation of immature interstitial clear cells with secondary cystic change. This report presents a histological, immunohistochemical, ultrastructural and molecular study of these peculiar cells, together with a review of the literature.

Alveolar adenoma of the lung: a clinicopathologic, immunohistochemical, and molecular study of an unusual case.

We describe an alveolar adenoma of the lung with 3 previously unreported findings, which expand both the clinical and the morphologic spectrum of this rare tumor: presentation as a cystic nodule, foci of mature adipocytes, and S-100 positivity of the mesenchymal cells. Furthermore, using a laser capture microdissection technique under microscope visualization, we analyzed multiple chromosomal loci in both the epithelial and mesenchymal components of the lesion, showing microsatellite alterations and loss of heterozygosity in the former but not in the latter.

Napsin-A, TTF-1, EGFR, and ALK Status Determination in Lung Primary and Metastatic Mucin-Producing Adenocarcinomas.

Pulmonary mucin-producing adenocarcinomas may be indistinguishable on conventional histology from a metastasis, as thyroid transcription factor-1 (TTF-1) expression often is lacking and KRAS mutations are widely present even in extrapulmonary sites. Few data have been reported on the diagnostic role of napsin-A and epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) gene alterations in this challenging differential diagnosis. Seventy-seven surgically resected cases, including 53 primary and 24 metastatic tumors from different sites, were evaluated for napsin-A, TTF-1, and ALK by immunohistochemistry and for EGFR mutations by direct sequencing. Overall, napsin-A expression in primary lung mucin-producing adenocarcinomas was 36% (8% mucinous, 17% colloid, 87.5% solid, and 100% signet ring cell) and TTF-1 expression reached an overall figure of 42% (12.5% mucinous, 33% colloid, 87.5% solid, and 100% signet ring cell). Metastatic mucinous adenocarcinomas did not react with napsin-A or with TTF-1. All primary and metastatic tumors lacked EGFR mutations, while a single case of signet ring cell lung adenocarcinoma showed ALK expression and rearrangement at fluorescent in situ hybridization analysis. Napsin-A has a lower sensitivity compared with TTF-1 in primary mucin-producing adenocarcinomas of the lung. However, both antibodies have an absolute specificity, being always negative in metastatic mucinous adenocarcinomas. EGFR mutations and ALK translocation or expression are exceedingly rare in mucin-producing adenocarcinomas of the lung, resulting unnecessary as diagnostic tool in this setting.

Gene mutations in small-cell lung cancer (SCLC): results of a panel of 6 genes in a cohort of Italian patients.

BACKGROUND: No target therapies are presently available in the treatment of small-cell lung cancer (SCLC). We investigated the presence of potentially drugable mutations in the EGFR, c-MET, BRAF, KRAS, PDGFRa and c-KIT genes in a retrospective series of SCLC from 2 Italian Institutions. Correlations with immunohistochemical, clinical and outcome features were evaluated. MATERIALS AND METHODS: Genes were studied by direct sequencing of DNA extracted from formalin-fixed paraffin-embedded tissues. Immunohistochemical expression of TTF-1, p63, chromogranin, synaptophysin, CD56 and bcl-2 was assessed. RESULTS: Samples from 113 SCLC patients were analyzed. All cases were wild-type for BRAF, KRAS, PDGFRa and c-KIT (data available for 82 patients). Two (1.8%) patients were EGFR-mutated (exon 19 delE746-A750 and exon 21 L858R); both were females, non-smoker and had limited disease. Overall survival of EGFR-mutated patients was 21 months as compared to 11 months in wild-type. Five (4.4%) patients were c-MET-mutated (4 on exon 14: 2 R988C, 1 D990N, 1 D102Y; 1 on exon 17 R1166Q); all were smokers, 3 were males and 4 had extensive disease. Their OS was comparable to wild-type cases (12 vs. 11 months). EGFR and c-MET mutations were mutually exclusive. Gene mutations did not correlate with immunophenotype. CONCLUSIONS: Targetable mutations are uncommon in SCLC. EGFR-mutated patients tended to be female and non-smoker and experienced a prolonged OS suggesting a possible positive prognostic effect. c-MET mutations did not affect survival. Target therapy might be considered in EGFR and c-MET-mutated patients.

Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing: potential clinical implications.

Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.