Valentina Monica

Desmocollin-3: a new marker of squamous differentiation in undifferentiated large-cell carcinoma of the lung

Lung cancer classification in small-cell and non-small-cell types was recently challenged by data on the differential efficacy of new cytotoxic agents in specific histotypes. An accurate histotype definition has therefore gained interest in both preoperative and surgical materials, but is a hard task especially in undifferentiated large-cell tumors lacking morphological signs of squamous or glandular differentiation. The responsiveness of these latter subtypes to new drugs apparently more selective for adenocarcinomas or squamous carcinomas is not fully understood, also due to the heterogeneity of diagnostic criteria for this tumor entity. Current immunohistochemical markers are not fully specific and new molecules are to be explored. On the basis of gene expression profiling data, reporting a remarkable differential expression of desmocollin-3 (a protein localized in desmosomal junctions of stratified epithelial) between adeno- and squamous cancers, we immunostained 62 cases of resected undifferentiated large-cell lung carcinomas for desmocollin-3 (and for TTF-1, p63 and mucin stain), to test its ability to identify a (residual) squamous phenotype, if present. Desmocollin-3 was expressed in almost half of the undifferentiated large-cell cancers and was mutually exclusive with TTF-1 (positive in 39%; the remaining 18 % of cases was double negative). Special large-cell carcinoma variants expressed desmocollin-3 in 6 of 6 basaloid, 7 of 12 clear-cell types, again mutually exclusive with TTF-1 expression. None of seven sarcomatoid carcinomas reacted for either marker. In 31 cytological samples diagnosed as ‘non-small-cell lung carcinoma’, desmocollin-3 was again mutually exclusive with TTF-1 and stained all squamous carcinomas, 1 of 19 adenocarcinoma only, and 50% of large-cell carcinoma (all histologically confirmed). This combined morphophenotypic approach may represent a valid adjunct (for both surgical and cytological samples) in the selection of patients with lung cancer to medical treatments tailored according to different efficacy in different lung carcinomas of the squamous, adeno- and large-cell types.

Targeted Next-Generation Sequencing of Cancer Genes in Advanced Stage Malignant Pleural Mesothelioma: A Retrospective Study

Introduction: Malignant pleural mesothelioma (MPM) is a rare malignant disease, and the understanding of molecular pathogenesis has lagged behind other malignancies. Methods: A series of 123 formalin-fixed, paraffin-embedded tissue samples with clinical annotations were retrospectively tested with a commercial library kit (Ion AmpliSeq Cancer Hotspot Panel v.2, Life Technologies, Grand Island, NY) to investigate 50 genes plus other two, BRCA1-associated protein-1 (BAP-1) and neurofibromatosis-2 (NF2), frequently altered in MPM. DNA was obtained from tissues after manual microdissection and enriched for at least 50% cancer cells. Variations affecting protein stability or previously correlated to cancer, more frequently identified (≥25 patients with at least 10% of allelic frequency), were subsequently evaluated by Sanger sequencing. Immunohistochemistry staining for BAP1 and NF2 proteins was also performed. Results: The commonest genetic variations were clustered in two main pathways: the p53/DNA repair (TP53, SMACB1, and BAP1) and phosphatidylinositol 3-kinase–AKT pathways (PDGFRA, KIT, KDR, HRAS, PIK3CA, STK11, and NF2). PIK3CA:c.1173A>G mutation, STK11:rs2075606 (T>C), or TP53:rs1042522 (Pro/Pro) was significantly associated with time to progressive disease (TTPD; all p values < 0.01). Furthermore, the accumulation of genetic alterations correlated with shorter TTPD and reduced overall survival (TTPD p value = 0.02, overall survival p value = 0.04). BAP1 genetic variations identified were mainly located in exons 13 and 17, and BAP1 nonsynonymous variations were significantly correlated with BAP1 protein nuclear localization. Conclusion: Next-generation sequencing was applied to a relatively large retrospective series of MPM using formalin-fixed, paraffin-embedded archival material. Our results indicate a complex mutational landscape with a higher number of genetic variations in the p53/DNA repair and phosphatidylinositol 3-kinase pathways, some of them with prognostic value.

Polymerase eta mRNA expression predicts survival of non-small cell lung cancer patients treated with platinum-based chemotherapy.

Purpose: The effect of translesion DNA synthesis system in conferring cellular tolerance to DNA-damaging agents has been recently described. DNA polymerase η (Pol η) is part of this machinery and in vitro models showed that it can overcome DNA damages caused by cisplatin and UV rays. The aim of the present study was to investigate the role of Pol η mRNA expression levels in non–small cell lung cancer (NSCLC). Experimental Design: Pol η mRNA expression levels were evaluated by real-time PCR in (a) formalin-fixed paraffin-embedded biopsies of 72 NSCLC patients treated with platinum-based chemotherapy, (b) fresh snap-frozen surgical specimens of tumor and corresponding normal lung tissue from 50 consecutive patients not treated with perioperative or postoperative chemotherapy, and (c) five NSCLC cell lines. Results: High Pol η expression levels were strongly associated with shorter survival at both univariate (6.9 versus 21.1 months; P = 0.003) and multivariate (hazard ratio, 3.18; 95% confidence interval, 1.73-5.84; P = 0.008) analysis in the group of platinum-treated patients. By contrast, Pol η expression was not significantly correlated with the prognosis in surgically resected patients (P = 0.54) and mRNA levels did not significantly differ in tumor versus normal lung (P = 0.82). Moreover, endogenous Pol η mRNA expression was found to be inducible by cisplatin in three of five cell lines and significantly associated with in vitro sensitivity (P = 0.01). Conclusions: Taken together, these data indicate Pol η as a predictive rather than prognostic marker worth of further investigation in NSCLC patients candidate to platinum-based chemotherapy.

Differential Thymidylate Synthase Expression in Different Variants of Large-Cell Carcinoma of the Lung

Purpose: In non–small cell lung cancer, higher thymidylate synthase (TS) levels have been reported in squamous cell carcinoma (SCC) compared with adenocarcinoma (ADC). Data on TS expression in large-cell carcinoma (LCC) are scanty. Experimental Design: TS mRNA and protein levels were analyzed in 42 surgical cases of pulmonary LCC, including 8 large-cell neuroendocrine carcinomas, and were compared with controls represented by ADC (n = 41), SCC (n = 30), and small-cell lung carcinoma (SCLC; n = 33). TS levels were also correlated with the expression of Ki67 and E2F1. Moreover, the reliability of TS expression analysis was assessed in 22 matched cytologic and surgical specimens of non–small cell lung cancer. Results: TS mRNA levels of LCC were comparable with those of control SCC, but significantly higher than those of ADC (P < 0.001) and lower than SCLC (P < 0.001). A correlation between TS mRNA and protein levels was observed in control ADC and SCC, but not in LCC. Large-cell neuroendocrine carcinomas had the highest TS expression, whereas in non-neuroendocrine LCCs, TS protein levels were significantly higher (P = 0.02) in LCC immunoreactive for p63 and desmocollin3 (markers of squamous differentiation) than those expressing TTF-1 (a marker of ADC). Both E2F1 and Ki67 levels were not correlated with TS in LCCs. Finally, a linear correlation in TS protein levels was observed between matched cytologic and surgical specimens. Conclusion: The pulmonary LCC immunoprofile may resemble that of SCCs or ADCs. This immunoprofile is associated with differential TS expression levels, which may support a more appropriate therapeutic strategy decision. (Clin Cancer Res 2009;15(24):7547–52)

Effects of Src kinase inhibition induced by dasatinib in non–small cell lung cancer cell lines treated with cisplatin

c-Src is a tyrosine kinase involved in tumor proliferation, migration, and angiogenesis and has been shown to modulate the cytotoxicity following cisplatin-induced DNA damages. c-Src is frequently activated in non–small cell lung cancer (NSCLC) tissues and cell lines, but no preclinical data regarding the effects of the novel potent Src inhibitor, dasatinib (BMS-354825), in the modulation of cisplatin resistance are currently available. The present study reports that treatment with dasatinib completely abrogated Src phosphorylation in the majority of the NSCLC cell lines tested (n = 7), with modest effects on cell proliferation and survival. In five cell lines, a higher cytotoxicity was observed delivering cisplatin in combination with dasatinib: the most evident effects were found in the squamous H520 cells due to the effective block of cisplatin-induced Src phosphorylation. Moreover, dasatinib treatment significantly blocked cisplatin-induced transcription of a panel of DNA repair and synthesis genes. In addition, a real-time PCR analysis done on tumor and matched normal lung specimens from 44 surgically resected NSCLC patients showed that Src transcripts are significantly upregulated in 23% of cases. In conclusion, Src-directed therapeutic strategies could interfere with cisplatin resistance, possibly allowing to reduce cisplatin doses, thus improving its efficacy. The data of this study support further clinical studies aimed to evaluate the efficacy of Src-inhibiting agents in combination with cisplatin in the treatment of NSCLC. [Mol Cancer Ther 2009;8(11):3066–74]

Heterogeneity of large cell carcinoma of the lung: an immunophenotypic and miRNA-based analysis.

Large cell carcinomas (LCCs) of the lung are heterogeneous and may be of different cell lineages. We analyzed 56 surgically resected lung tumors classified as LCC on the basis of pure morphologic grounds, using a panel of immunophenotypic markers (adenocarcinoma [ADC]-specific, thyroid transcription factor-1, cytokeratin 7, and napsin A; squamous cell carcinoma [SQCC]-specific, p63, cytokeratin 5, desmocollin 3, and Δnp63) and the quantitative analysis of microRNA-205 (microRNA sample score [mRSS]). Based on immunoprofiles 19 (34%) of the cases were reclassified as ADC and 14 (25%) as SQCC; 23 (41%) of the cases were unclassifiable. Of these 23 cases, 18 were classified as ADC and 5 as SQCC according to the mRSS. Our data show that an extended panel of immunohistochemical markers can reclassify around 60% of LCCs as ADC or SQCC. However, a relevant percentage of LCCs may escape convincing immunohistochemical classification, and mRSS could be used for further typing, but its clinical relevance needs further confirmation.

Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed, a multi-target folate antagonist, has demonstrated efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS) expression. Among many other potential targets, histone deacetylase inhibitors (HDACi) modulate TS expression, potentially sensitizing to the cytotoxic action of anti-cancer drugs that target the folate pathway, such as pemetrexed. Since high levels of TS have been linked to clinical resistance to pemetrexed in NSCLC, herein we investigated the molecular and functional effects of combined pemetrexed and ITF2357, a pan-HDACi currently in clinical trials as an anti-cancer agent.ResultsIn NSCLC cell lines, HDAC inhibition by ITF2357 induced histone and tubulin acetylation and downregulated TS expression at the mRNA and protein level. In combination experiments in vitro ITF2357 and pemetrexed demonstrated sequence-dependent synergistic growth-inhibitory effects, with the sequence pemetrexed followed by ITF2357 inducing a strikingly synergistic reduction in cell viability and induction of both apoptosis and autophagy in all cell line models tested, encompassing both adenocarcinoma and squamous cell carcinoma. Conversely, simultaneous administration of both drugs achieved frankly antagonistic effects, while the sequence of ITF2357 followed by pemetrexed had additive to slightly synergistic growth-inhibitory effects only in certain cell lines. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells (LCSC) exposed to pemetrexed followed by ITF2357. In terms of molecular mechanisms of interaction, the synergistic growth-inhibitory effects observed were only partially related to TS modulation by ITF2357, as genetic silencing of TS expression potentiated growth inhibition by either pemetrexed or ITF2357 and, to a lesser extent, by their sequential combination. Genetic and pharmacological approaches provided an interesting link between the autophagic and apoptotic pathways, and showed that sequential pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival.ConclusionsOverall, these data provide a strong rationale for the clinical development of sequential schedules employing pemetrexed followed by HDACi in NSCLC.

p63 and p73 Isoform Expression in Non-small Cell Lung Cancer and Corresponding Morphological Normal Lung Tissue

Background: The TP73 and TP63 genes are members of the p53 tumor suppressor family and are expressed in different N-terminal isoforms either with proapoptotic (transactivation domain, TA) and antiapoptotic (N-terminally truncated, &Dgr;N) function. Unlike p53, the role of p73 and p63 in tumor is controversial. It has been recently hypothesized that altered &Dgr;N:TA expression ratio, rather than single isoform overexpression, plays a role in the pathogenesis of many diseases, including lung cancer. Methods: Isoform-specific, real-time polymerase chain reaction and immunohistochemistry analysis on matched cancer and corresponding normal tissues from surgically resected non-small cell lung cancers (NSCLCs) have been performed aiming to explore the expression levels of each p63 and p73 N-terminal isoforms and their &Dgr;N:TA expression ratio. Results: For both p63 and p73, a N-terminal isoform-specific modulation that alter &Dgr;N:TA isoform balance was identified. In particular, &Dgr;Np63 isoform was significantly up-modulated, whereas TAp63 was slightly down-modulated in NSCLC specimens. Likewise, &Dgr;2p73 and &Dgr;2/3p73 were up-modulated, whereas &Dgr;Np73 and &Dgr;N′p73 isoforms were down-modulated. Moreover, a higher TAp63 and &Dgr;N′p73 transcripts expression, detected in the normal tissue surrounding the tumors, correlates with poor patient outcome, representing independent prognostic factors for overall survival (&Dgr;N′p73: p = 0.049, hazard ratio = 3.091, 95% confidence interval = 1.005–9.524 and TAp63: p = 0.001, hazard ratio = 8.091, 95% confidence interval = 2.254–29.05). Conclusion: Our findings suggest that p63 and p73 altered &Dgr;N:TA expression ratio occurs in NSCLC likely contributing to the molecular pathogenesis of this tumor.

ATF2 contributes to cisplatin resistance in non-small cell lung cancer and celastrol induces cisplatin resensitization through inhibition of JNK/ATF2 pathway.

ATF2 is a transcription factor involved in stress and DNA damage. A correlation between ATF2 JNK‐mediated activation and resistance to damaging agents has already been reported. The purpose of the present study was to investigate whether ATF2 may have a role in acquired resistance to cisplatin in non‐small cell lung cancer (NSCLC). mRNA and protein analysis on matched cancer and corresponding normal tissues from surgically resected NSCLC have been performed. Furthermore, in NSCLC cell lines, ATF2 expression levels were evaluated and correlated to platinum (CDDP) resistance. Celastrol‐mediated ATF2/cJUN activity was measured. High expression levels of both ATF2 transcript and proteins were observed in lung cancer specimens (p << 0.01, Log2(FC) = +4.7). CDDP‐resistant NSCLC cell lines expressed high levels of ATF2 protein. By contrast, Celastrol‐mediated ATF2/cJUN functional inhibition restored the response to CDDP. Moreover, ATF2 protein activation correlates with worse outcome in advanced CDDP‐treated patients. For the first time, it has been shown NSCLC ATF2 upregulation at both mRNA/protein levels in NSCLC. In addition, we reported that in NSCLC cell lines a correlation between ATF2 protein expression and CDDP resistance occurs. Altogether, our results indicate a potential increase in CDDP sensitivity, on Celastrol‐mediated ATF2/cJUN inhibition. These data suggest a possible involvement of ATF2 in NSCLC CDDP‐resistance.

Role of Hormone Receptor Expression in Patients With Advanced-Stage Lung Cancer Treated With Chemotherapy.

BACKGROUND Evidence that supports a role for hormonal status in lung cancer has been inconsistently reported and is still unclear. We retrospectively assessed the potential correlation between sex-linked hormone receptor expression and the clinical outcome of patients with advanced-stage lung cancer treated with chemotherapy. PATIENTS AND METHODS Based on tissue availability, 130 consecutive patients diagnosed at San Luigi Hospital from January 2008 to June 2010 were collected, including 24 small-cell lung cancer, 57 adenocarcinomas, 34 squamous cell carcinomas, 5 large-cell carcinomas, and 10 non-small-cell lung cancer-not otherwise specified. The immunohistochemical expression of estrogen receptors (ER-α and ER-β) and progesterone receptor, aromatase, epidermal growth factor receptor (EGFR), and excision repair cross-complementing 1 (ERCC1) was assessed. RESULTS ER-β nuclear expression was higher than ER-α and progesterone receptor, whose expression was null or weak (mainly in women). ER-β expression was significantly higher in patients with metastatic disease compared with all other disease stages (P = .02). EGFR expression was strongly correlated with non-small-cell lung cancer histology, being higher in squamous types and stage related. In men, aromatase positive cases had a worse outcome (P = .03) as well as in men with non-small-cell lung cancer and high ER-β expression. In the latter group, the combined aromatase negative and/or low ER-β expression and low ERCC1 and/or low ER-β expression showed a better outcome (P = .026; P = .03, respectively). CONCLUSION In patients with advanced-stage lung cancer treated with chemotherapy, the prognostic and predictive role of sex-linked hormone receptor expression, if any, is of borderline significance and is restricted to selected subgroups of patients.