BRCA gene amplification in primary peritoneal high-grade serous carcinoma patient with intrinsic resistance to platinum treatment: a case report.

Platinum-based chemotherapy is the standard chemotherapy for high grade serous ovarian cancer and primary peritoneal high-grade serous carcinoma. PARP inhibitors have changed the paradigm of the treatment in platinum-sensitive ovarian cancers and primary peritoneal high-grade serous carcinoma with BRCA1/2 mutation or homologous recombination deficiency (HRD). Platinum-resistant ovarian and primary peritoneal high-grade serous carcinoma have a lower chance to treat and have worse outcomes. We described a case of patient with a platinum resistant primary peritoneal high-grade serous carcinoma with a rare somatic BRCA2 amplification. There are no guidelines for the treatment of ovarian cancer or primary peritoneal high-grade serous carcinoma with BRCA2 amplification. BRCA2 amplification could result in extreme homologous recombination repair (HRR) pathway efficiency and in less platinum sensitivity, which could be a molecular signature for platinum resistance. Free platinum chemotherapy regimens could be more effective in cases with BRCA2 amplification. Further studies are necessary to establish better approaches and strategies for oncological management and treatment in BRCA2 amplification high grade ovarian cancer and primary peritoneal high-grade serous carcinoma.

POLE exonuclease domain mutations in endometrial carcinoma: a case report.

Endometrial carcinoma (EC) harboring POLE exonuclease domain mutations occurs in 5-15% of ECs and frequently affects young women with low body mass index (BMI). It presents at early stage as high grade endometrioid histotype with intense tumor infiltrating lymphocytes and has good clinical outcomes and favorable prognosis. In this article we report the case of a 32-year-old woman with endometriod EC (EEC) exhibiting a "ultramutated" molecular profile and an excellent prognosis despite tumor size and grading. Herein, to highlight the importance of defining POLE status in ECs for both clinical and therapeutic implications for patients.

MDM4 (MDMX) localizes at the mitochondria and facilitates the p53-mediated intrinsic-apoptotic pathway.

MDM4 is a key regulator of p53, whose biological activities depend on both transcriptional activity and transcription-independent mitochondrial functions. MDM4 binds to p53 and blocks its transcriptional activity; however, the main cytoplasmic localization of MDM4 might also imply a regulation of p53-mitochondrial function. Here, we show that MDM4 stably localizes at the mitochondria, in which it (i) binds BCL2, (ii) facilitates mitochondrial localization of p53 phosphorylated at Ser46 (p53Ser46(P)) and (iii) promotes binding between p53Ser46(P) and BCL2, release of cytochrome C and apoptosis. In agreement with these observations, MDM4 reduction by RNA interference increases resistance to DNA-damage-induced apoptosis in a p53-dependent manner and independently of transcription. Consistent with these findings, a significant downregulation of MDM4 expression associates with cisplatin resistance in human ovarian cancers, and MDM4 modulation affects cisplatin sensitivity of ovarian cancer cells. These data define a new localization and function of MDM4 that, by acting as a docking site for p53Ser46(P) to BCL2, facilitates the p53-mediated intrinsic-apoptotic pathway. Overall, our results point to MDM4 as a double-faced regulator of p53.

Ovarian high-grade serous carcinoma with transitional-like (SET) morphology: a homologous recombination-deficient tumor.

Thirteen years ago, we pointed out that ovarian transitional cell carcinomas (TCCs) and conventional high-grade serous carcinomas (HGSCs) had similar genetic alterations and clinical behavior. Consequently, ovarian TCC is now classified as a morphologic variant of HGSC. Defective homologous recombination, resulting from genetic or epigenetic inactivation of DNA damage repair genes, such as BRCA1/2, occurs in approximately 50% of the HGSCs. Although BRCA mutations have been associated with HGSCs with solid, pseudoendometrioid or transitional (SET) features, little is known about the role of non-BRCA homologous recombinationrepair (HRR) genes and the HRR status in these tumors. Using two commercially available assays (Myriad Genetics MyChoice CDx Plus test and SOPHiA Dx Homologous Recombination Deficiency Solution), we study mutations of BRCA1/2 and non-BRCA HRR genes (ATM, BARD1, BRIP1, CDK12, CHEK1/2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), and the HRR status in 19 HGSCs with SET features and 20 HGSCs with classic morphology. We also studied, as control cases, 5 endometrioid carcinomas, 1 clear cell carcinoma, 2 low-grade serous carcinomas, and 1 malignant Brenner tumor. Seven HGSCs with SET features (7/19; 37%) showed BRCA mutations (4 BRCA1, 2 BRCA2, and 1 BRCA1/2). Mutations in non-BRCA HRR genes were found in ATM (1/15; 7%), BARD1 (1/15; 7%), and BRIP1 (1/19; 5%). Most HGSCs with SET features (17/19; 90%) were considered to be homologous recombination-deficient tumors. Three HGSCs with classic morphology (3/20; 15%) showed BRCA2 mutations. Mutations in non-BRCA HRR genes were found in CDK12 (2/14; 14%), FANCL (1/14; 7%), RAD51B (1/14; 7%), and RAD54L (1/14; 7%). Eleven HGSCs with classical morphology (11/20; 55%) were considered to be homologous recombination deficient. In contrast, all ovarian carcinoma control cases (5 endometrioid carcinomas, 1 clear cell carcinoma, 2 low-grade serous carcinomas, and 1 malignant Brenner tumor) were homologous recombination proficient and did not have BRCA mutations. Our results show that the majority of HGSCs with SET features are homologous recombination-deficient tumors independently of the BRCA status and highlight the importance of the HRR tumor testing, especially in BRCA wild-type tumors. Recognition of transitional cell variant of HGSCs may help to identify patients most likely to benefit from PARP inhibitors.

A unique microRNA signature associated with plaque instability in humans.

BACKGROUND AND PURPOSE: Atherosclerotic plaque rupture is considered the most important mechanism that underlies the onset of stroke, myocardial infarction, and sudden death. Several evidences demonstrated the pivotal role of inflammatory processes in plaque destabilization. MicroRNAs (miRNAs) are small endogenous RNAs and represent a new important class of gene regulators. Nevertheless, no data exist about the expression profile of miRNAs in atherosclerotic plaques. Thus, the aim of this study was to investigate the expression level of miRNAs in human plaques and to correlate it with clinical features of plaque destabilization. METHODS: Two separate groups of plaques were collected from patients who underwent carotid endarterectomy in Chieti (n=15) and Ancona (n=38) Hospitals. All the plaques were subdivided in symptomatic (n=22) and asymptomatic (n=31) according to the presence/absence of stroke. RESULTS: First, on the plaques collected at Chieti Hospital, we performed large-scale analysis of miRNA expression. Between the 41 miRNAs examined, we discovered profound differences in the expression of 5 miRNAs (miRNA-100, miRNA-127, miRNA-145, miRNA-133a, and miRNA-133b) in symptomatic versus asymptomatic plaques. Remarkably, when we repeated the analysis on the Ancona plaque subset, all these 5 miRNAs confirmed to be significantly more expressed in the symptomatic plaques. Finally, in vitro experiments on endothelial cells transfected with miRNA-145 and miRNA-133a confirmed the importance of these miRNAs in the modulation of stroke-related proteins. CONCLUSIONS: These results are the first to report alterations in the expression of specific miRNAs in human atherosclerotic plaques and suggest that miRNAs may have an important role in regulating the evolution of atherosclerotic plaque toward instability and rupture. Furthermore, by identifying the specific miRNA signature for stroke now, we are able to use computer algorithms to identify previously unrecognized molecular targets.

Reliability and reproducibility among different platforms for tumour BRCA testing in ovarian cancer: a study of the Italian NGS Network.

INTRODUCTION: BRCA tumour testing is a crucial tool for personalised therapy of patients with ovarian cancer. Since different next-generation sequencing (NGS) platforms and BRCA panels are available, the NGS Italian Network proposed to assess the robustness of different technologies. METHODS: Six centres, using four different technologies, provided raw data of 284 cases, including 75 cases with pathogenic/likely pathogenic variants, for a revision blindly performed by an external bioinformatic platform. RESULTS: The third-party revision assessed that all the 284 raw data reached good quality parameters. The variant calling analysis confirmed all the 75 pathogenic/likely pathogenic variants, including challenging variants, achieving a concordance rate of 100% regardless of the panel, instrument and bioinformatic pipeline adopted. No additional variants were identified in the reanalysis of a subset of 41 cases. CONCLUSIONS: BRCA tumour testing performed with different technologies in different centres, may achieve the realibility and reproducibility required for clinical diagnostic procedures.

Survival prediction of stage I lung adenocarcinomas by expression of 10 genes.

Adenocarcinoma is the predominant histological subtype of lung cancer, the leading cause of cancer deaths in the world. At stage I, the tumor is cured by surgery alone in about 60% of cases. Markers are needed to stratify patients by prognostic outcomes and may help in devising more effective therapies for poor prognosis patients. To achieve this goal, we used an integrated strategy combining meta-analysis of published lung cancer microarray data with expression profiling from an experimental model. The resulting 80-gene model was tested on an independent cohort of patients using RT-PCR, resulting in a 10-gene predictive model that exhibited a prognostic accuracy of approximately 75% in stage I lung adenocarcinoma when tested on 2 additional independent cohorts. Thus, we have identified a predictive signature of limited size that can be analyzed by RT-PCR, a technology that is easy to implement in clinical laboratories.

Early prediction of resistance to tyrosine kinase inhibitors by plasma monitoring of EGFR mutations in NSCLC: a new algorithm for patient selection and personalized treatment.

In Non-Small-Cell Lung Cancer (NSCLC) patients treated with Tyrosine Kinase-Inhibitors (TKIs) therapy, the emergence of acquired resistance can be investigated by plasma monitoring of circulating tumor DNA (ctDNA). A series of 116 patients with EGFR-positive lung adenocarcinomas were treated with first/second generation EGFR TKIs. At clinical progression, 64 (55%) EGFR T790M plasma positive patients were subjected to second line-treatment with osimertinib and strictly monitored during the first month of therapy. Plasma analysis by the EGFR Cobas test showed in 57 (89%) cases a substantial decrease in the levels of the sensitizing EGFR mutant allele (sEGFRma), down to a not detectable value. These patients were defined as plasmatic good responders (PGR). In 7 (11%) patients, the sEGFRma did not drop to zero (plasmatic poor responders, PPR). In these latter cases, Massive Parallel Sequencing (MPS) analysis at the end of the first month and at clinical progression showed the presence of resistant-inducing mutations, including MET and HER2 gene amplification, KRAS and PIK3CA gene mutations. PPR showed disease progression in 5 (71%) cases, stable disease in 2 (29%) cases, and a shorter median Progression-free survival (PFS) (4.3 ± 1.1 months) than that observed in PGR (13.3 ± 1.2 months) (P < 0.0001). Our data indicate that plasma monitoring by a simple RT-PCR-based EGFR mutation test in the first month of treatment may be useful for a rapid identification of patients to be subjected to further characterization by MPS. A diagnostic algorithm for an early detection of resistance-inducing mutations and patient management is reported.

Molecular profiling of the "plexinome" in melanoma and pancreatic cancer.

Plexins are transmembrane high-affinity receptors for semaphorins, regulating cell guidance, motility, and invasion. Functional evidences implicate semaphorin signals in cancer progression and metastasis. Yet, it is largely unknown whether plexin genes are genetically altered in human tumors. We performed a comprehensive gene copy analysis and mutational profiling of all nine members of the plexin gene family (plexinome), in melanomas and pancreatic ductal adenocarcinomas (PDACs), which are characterized by high metastatic potential and poor prognosis. Gene copy analysis detected amplification of PLXNA4 in melanomas, whereas copy number losses of multiple plexin genes were seen in PDACs. Somatic mutations were detected in PLXNA4, PLXNB3, and PLXNC1; providing the first evidence that these plexins are mutated in human cancer. Functional assays in cellular models revealed that some of these missense mutations result in loss of plexin function. For instance, c.1613G>A, p.R538H mutation in the extracellular domain of PLXNB3 prevented binding of the ligand Sema5A. Moreover, although PLXNA4 signaling can inhibit tumor cell migration, the mutated c.5206C>T, p.H1736Y allele had lost this activity. Our study is the first systematic analysis of the "plexinome" in human tumors, and indicates that multiple mutated plexins may be involved in cancer progression.

IDH1 mutations at residue p.R132 (IDH1(R132)) occur frequently in high-grade gliomas but not in other solid tumors.

Systematic sequence profiling of the Glioblastoma Multiforme (GBM) genome has recently led to the identification of somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Interestingly, only the evolutionarily conserved residue R132 located in the substrate binding site of IDH1 was found mutated in GBM. At present, the occurrence and the relevance of p.R132 (IDH1(R132)) variants in tumors other than GBMs is largely unknown. We searched for mutations at position R132 of the IDH1 gene in a panel of 672 tumor samples. These included high-grade glioma, gastrointestinal stromal tumors (GIST), melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, prostate, and thyroid carcinoma specimens. In addition, we assessed a panel of 84 cell lines from different tumor lineages. Somatic mutations affecting the IDH1(R132) residue were detected in 20% (23 of 113) high-grade glioma samples. In addition to the previously reported p.R132H and p.R132S alleles, we identified three novel somatic mutations (p.R132C, p.R132G, and p.R132L) affecting residue IDH1(R132) in GBM. Strikingly, no IDH1 mutations were detected in the other tumor types. These data indicate that cancer mutations affecting IDH1(R132) are tissue-specific, and suggest that it plays a unique role in the development of high-grade gliomas.

An innovative diagnostic strategy for the detection of rare molecular targets to select cancer patients for tumor-agnostic treatments.

Targeted therapies are playing an increasing role in oncology. Among them, particular attention is nowadays reserved to histology-agnostic treatments. Rare molecular alterations affecting different neoplastic forms, such as Microsatellite Instability (MSI), Neurotropic Tyrosine Receptor Kinase (NTRK) gene fusions, etc., can allow efficient treatments, irrespective of the histologic type. Developing an effective testing strategy for the detection of rare molecular alterations is challenging. We report an innovative diagnostic strategy for a rapid and economically affordable detection of this uncommon targets. Malignant tumor samples are selected at the time of histopathological diagnosis and further processed for simultaneous analysis of multiple samples on Tissue Micro Arrays (TMAs) and Tissue Slice Arrays (TSAs). The TSA approach was specifically designed for large scale screening of small biopsies. TMA sections and TSA were first screened by immunohistochemistry (IHC) for the expression of mismatch repair and TRK proteins. Positive cases were subjected to confirmation tests (fragment analysis/FISH/NGS). In a series of 1865 malignant tumors, 48 (2.6%) MSI cases and 6 (0.3%) NTRK fusion cases were detected in 9 and 4 different tumor forms, respectively. On average, the TMA/TSA screening approach enabled IHC analysis of about 20 patients simultaneously with significant saving of time and costs. In addition, we have shown that multiplex IHC can further increment the throughput. A detailed procedure for application of this diagnostic approach in clinical practice is reported. The strategy described may allow an efficient and sustainable selection of tumors carrying rare molecular targets, not to leave behind patients for effective agnostic treatments.

Weekly alternate intensive regimen FIrB/FOx in metastatic colorectal cancer patients: an update from clinical practice.

BACKGROUND: Several trials evaluated the role of intensive regimens, made of triplet chemotherapies plus bevacizumab, as first-line treatment for patients with metastatic colorectal cancer (mCRC). We previously reported, in a Phase II prospective study, the efficacy and the tolerability of FIrB/FOx regimen, reporting interesting results in terms of received dose intensities (rDIs) and safety. METHODS: We reported a retrospective update of 85 patients treated with FIrB/FOx, an intensive regimen of 5-fluorouracil, bevacizumab, and weekly alternate irinotecan and oxaliplatin, to confirm its feasibility in "real life". Subgroup analyses were performed, particularly among patients treated with standard and modified FIrB/FOx (based on age, performance status, and/or comorbidities). RESULTS: Overall, 3-month objective response rate (ORR) and 6-month ORR were 75.9% and 55.3%, respectively. Median progression-free survival (PFS) and median overall survival (OS) were 14.4 and 34.9 months, respectively. Among the patients treated with standard and modified regimens, 3-month ORR, PFS, and OS were 75.8% and 76% (P=1.0000), 14.4 and 14.4 months (P=0.8589), and 37.8 and 26.6 months (P=0.7746), respectively. Among the K/NRAS wild-type and K/NRAS mutant patients, 3-month ORR, PFS, and OS were 95.2% and 74.5% (P=0.0526), 15.3 and 14.4 months (P=0.8753), and 37.8 and 51.4 months (P=0.8527), respectively. The rDIs were ≥80% of full doses both in the standard and in the modified regimens subgroups. Cumulative G3/4 toxicities were neutropenia (14.1%), diarrhea (17.6%), asthenia (9.4%), vomiting (5.6%), and hypertension (16.5%). CONCLUSION: This update shows that intensive regimens such as FIrB/FOx are also feasible options for first-line treatment of mCRC patients in the "real-life" setting.

Frequent mutations in the neurotrophic tyrosine receptor kinase gene family in large cell neuroendocrine carcinoma of the lung.

The neurotrophic tyrosine receptor kinase (NTRK) family is potentially implicated in tumorigenesis and progression of several neoplastic diseases, including lung cancer. We investigated a large number of pulmonary neuroendocrine tumors (PNETs) and non-small cell lung carcinomas (NSCLCs) without morphological evidence of neuroendocrine differentiation for mutations in the NTRK gene family. A total of 538 primary lung carcinomas, including 17 typical carcinoids (TCs), 10 atypical carcinoids (ACs), 39 small cell lung carcinomas (SCLCs), 29 large cell neuroendocrine carcinomas (LCNECs), and 443 NSCLCs were evaluated by single-strand conformation polymorphism (SSCP) and sequencing of the tyrosine kinase domain (TKD) of NTRK1, NTRK2, and NTRK3. The NTRK1 gene was never found to be mutated. A total of 10 somatic mutations were detected in NTRK2 and NTRK3, mostly located in the activating and catalytic loops. NTRK mutations were seen in 9 (10%) out of 95 PNETs but in 0 out of 443 NSCLCs investigated. No mutations were observed in TCs, ACs, and SCLCs. Interestingly, all the mutations were restricted to the LCNEC histotype, in which they accounted for 31% of cases. A mutational analysis, performed after microdissection of LCNECs combined with adenocarcinoma (ADC), showed that only neuroendocrine areas were positive, suggesting that NTRK mutations are involved in the genesis of the neuroendocrine component of combined LCNECs. Our data indicate that somatic mutations in the TKD of NTRK genes are frequent in LCNECs. Such mutational events could represent an important step in the cancerogenesis of these tumors and may have potential implications for the selection of patients for targeted therapy.

Different prognostic roles of mutations in the helical and kinase domains of the PIK3CA gene in breast carcinomas.

PURPOSE: In breast cancer, the PIK3CA gene is frequently mutated at "hotspots" in exons 9 and 20, corresponding to the helical and kinase domains, respectively. We decided to investigate the association of PIK3CA mutations with pathologic features and clinical outcome in a large series of patients with breast cancer. EXPERIMENTAL DESIGN: Frozen samples from 163 consecutive patients were analyzed for PIK3CA mutations using PCR single-strand conformation polymorphism and sequence analyses. RESULTS: We identified 46 missense mutations, 24 (53%) in exon 9, and 21 (47%) in exon 20. Twelve (50%) of the 24 mutations in exon 9 were of the E542K type and 11 (46%) were of the E545K type. Twenty (95%) of the 21 mutations in exon 20 were H1047R substitutions. Mutations in exon 9 were more frequent in lobular carcinomas (42% of cases) than in ductal carcinoma (11% of cases; P = 0.002). At univariate survival analysis, PIK3CA exon 20 mutations were associated with prolonged overall and disease-free survival, whereas mutations in exon 9 were associated with significantly worse prognosis. At multivariate analysis, exon 9 PIK3CA mutations were the strongest independent factor to predict poor prognosis for disease-free survival (P = 0.0003) and overall survival (P = 0.001). CONCLUSION: Our data show that exon 9 PIK3CA mutations are typical of infiltrating lobular carcinomas. In addition, they indicate that PIK3CA mutations in different exons are of different prognostic value: exon 9 mutations are independently associated with early recurrence and death, whereas exon 20 PIK3CA mutations are associated with optimal prognosis.

Identification of an aberrantly spliced form of HDMX in human tumors: a new mechanism for HDM2 stabilization.

The HDMX protein is closely related to HDM2 with which it shares different structural domains, particularly the p53 binding domain and the ring finger domain, where the two HDM proteins interact. Several oncogenic forms derived from splicing of HDM2 have been described in cancer. This work aimed at investigating whether analogous forms of HDMX exist in human tumors. Here, we report the characterization of an aberrantly spliced form of HDMX, HDMX211, isolated from the thyroid tumor cell line, ARO. HDMX211 binds and stabilizes the HDM2 protein. Although it lacks the p53 binding domain, HDMX211 also stabilizes p53 by counteracting its degradation by HDM2. However, the resulting p53 is transcriptionally inactive and increasingly associated to its inhibitor HDM2. Expression of HDMX211 strongly enhances the colony-forming ability of human cells in the presence or absence of wild-type p53. Conversely, depletion of HDMX211 by small interfering RNA significantly reduces the growth of ARO cells and increases their sensitivity to chemotherapy. Screening of lung cancer biopsies shows the presence of HDMX211 in samples that overexpress HDM2 protein, supporting a pathologic role for this new protein. This is the first evidence of a variant form of HDMX that has oncogenic potential independently of p53. HDMX211 reveals a new mechanism for overexpression of the oncoprotein HDM2. Most interestingly, it outlines a possible molecular explanation for a yet unclarified tumor phenotype, characterized by simultaneous overexpression of HDM2 and wild-type p53.

ROS1 Gene Fusion in Advanced Lung Cancer in Women: A Systematic Analysis, Review of the Literature, and Diagnostic Algorithm.

PURPOSE: Crizotinib, a mesenchymal-epithelial transition/anaplastic lymphoma kinase/c-ros oncogene 1 (ROS1) inhibitor, has recently been approved by the US Food and Drug Administration for the treatment of patients with advanced ROS1-positive non-small-cell lung cancer (NSCLC). Therefore, interest in ROS1 testing is growing. ROS1 gene fusions affect approximately 0.5% to 2% of unselected NSCLCs. Limited data are available on the prevalence and distribution of ROS1 fusions in patients with advanced-stage NSCLC. MATERIAL AND METHODS: A series of 727 lung adenocarcinomas from patients with stage IV disease, negative for epidermal growth factor receptor and anaplastic lymphoma kinase alterations, were tested for ROS1 fusions by fluorescent in situ hybridization analysis, with confirmation by immunohistochemistry. Results were correlated with clinicopathologic parameters and compared with data from the literature. RESULTS: ROS1 fusions were detected in 29 patients (4%), including 27 of 266 females (10.2%) and two of 461 males (0.4%; P = 1.2E-10). The mean age of patients with ROS1-positive disease was lower than that of patients with ROS1-negative disease (49.21 v 62.96 years, respectively; P = 1.1E-10). Eleven of 583 smokers (1.9%) and 18 of 144 nonsmokers (12.5%) showed ROS1 rearrangement (P = 4.05E-7). By logistic regression analysis, ROS1 fusions were independently associated with female sex, younger age at diagnosis, and absence of smoking history, (odds ratios, 12.4, 7.9, and 3.6, respectively). These data, integrated with those reported in the literature, indicate that the prevalence of ROS1 fusions in females and in nonsmokers was higher in patients with advanced disease than in patients with operable disease (11.2% v 3.1%, P < .001; 11.6% v 2.8%, P < .001, respectively). The mean age at diagnosis was significantly lower in patients with advanced disease (49.8 years) than in patients with operable disease (55.6 years; P < .001). CONCLUSION: Our data indicate that ROS1 fusions in patients with advanced-stage lung adenocarcinoma are more frequent in females, particularly if young and nonsmokers. A diagnostic algorithm for an accurate screening of ROS1 alterations was elaborated.

EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment.

PURPOSE: It has been reported that EGFR mutations in lung carcinomas make the disease more responsive to treatment with tyrosine kinase inhibitors. We decided to evaluate the prevalence of EGFR mutations in a large series of non-small-cell lung carcinomas (NSCLCs) and to develop a rapid and sensitive screening method. PATIENTS AND METHODS We examined 860 consecutive NSCLC patients for EGFR mutations in exons 18, 19, and 21 using a dual technical approach--direct sequencing of polymerase chain reaction (PCR) products and PCR single-strand conformation polymorphism (SSCP) analysis. Moreover, all lung adenocarcinomas were analyzed for K-ras mutations at codon 12 by allele-specific oligoprobe hybriditations. RESULTS: There were no EGFR mutations in 454 squamous carcinomas and 31 large cell carcinomas investigated. Thirty-nine mutations were found in the series of 375 adenocarcinomas (10%). Mutations were present in 26% of 86 bronchioloalveolar carcinomas (BACs) and in 6% of 289 conventional lung adenocarcinomas; P = .000002. EGFR mutations and K-ras mutations were mutually exclusive. A multivariable analysis revealed that BAC histotype, being a never smoker, and female sex were independently associated with EGFR mutations (odds ratios: 4.542, 3.632, and 2.895, respectively). The SSCP analysis was accurate and sensitive, allowing identification of mutations that were undetectable (21% of cases) by direct sequencing. CONCLUSION: Mutations in the EGFR tyrosine kinase domain define a new molecular type of lung carcinoma, more frequent in particular subsets of patients. The SSCP assay is a rapid and reliable method for the detection of EGFR kinase domain mutations in lung cancer.

Int6 expression can predict survival in early-stage non-small cell lung cancer patients.

PURPOSE: The Int6 gene was originally identified as a common insertion site for the mouse mammary tumor virus in virally induced mouse mammary tumors. Recent studies indicate that Int6 is a multifaceted protein involved in the regulation of protein translation and degradation through binding with three complexes: the eukaryotic translation initiation factor 3, the proteasome regulatory lid, and the constitutive photomorphogenesis 9 signalosome. This study aimed to investigate the prognostic role of Int6 in a large series of stage I non-small cell lung cancers (NSCLC) patients with long-term follow-up. EXPERIMENTAL DESIGN: We determined the methylation status of Int6 DNA by methylation-specific PCR and the steady-state levels of Int6 RNA by quantitative real-time reverse transcription-PCR in 101 NSCLCs and matched normal lung tissues. RESULTS: In 27% of the tumors, Int6 RNA levels were reduced relative to normal tissue. In 85% of the tumors with reduced Int6 expression, the transcription promoter and first exon were hypermethylated, whereas only 4% of the tumors with elevated Int6 RNA levels were hypermethylated (P <0.000001). Low levels of Int6 RNA were found a significant predictor of overall and disease-free survival (P=0.0004 and P=0.0020, respectively). A multivariate analysis confirmed that low Int6 expression was the only independent factor to predict poor prognosis, for both overall (P=0.0006) and disease-free (P=0.024) survival. CONCLUSIONS: Our results suggest that Int6 expression, evaluated by quantitative real-time PCR, may represent a new prognostic factor in patients with stage I NSCLC.

Validation of a new algorithm for a quick and easy RT-PCR-based ALK test in a large series of lung adenocarcinomas: Comparison with FISH, immunohistochemistry and next generation sequencing assays.

OBJECTIVES: Anaplastic Lymphoma Kinase (ALK) gene rearrangements have been described in 3-5% of lung adenocarcinomas (ADC) and their identification is essential to select patients for treatment with ALK tyrosine kinase inhibitors. For several years, fluorescent in situ hybridization (FISH) has been considered as the only validated diagnostic assay. Currently, alternative methods are commercially available as diagnostic tests. MATERIAL AND METHODS: A series of 217 ADC comprising 196 consecutive resected tumors and 21 ALK FISH-positive cases from an independent series of 702 ADC were investigated. All specimens were screened by IHC (ALK-D5F3-CDx-Ventana), FISH (Vysis ALK Break-Apart-Abbott) and RT-PCR (ALK RGQ RT-PCR-Qiagen). Results were compared and discordant cases subjected to Next Generation Sequencing. RESULTS: Thirty-nine of 217 samples were positive by the ALK RGQ RT-PCR assay, using a threshold cycle (Ct) cut-off ≤35.9, as recommended. Of these positive samples, 14 were negative by IHC and 12 by FISH. ALK RGQ RT-PCR/FISH discordant cases were analyzed by the NGS assay with results concordant with FISH data. In order to obtain the maximum level of agreement between FISH and ALK RGQ RT-PCR data, we introduced a new scoring algorithm based on the ΔCt value. A ΔCt cut-off level ≤3.5 was used in a pilot series. Then the algorithm was tested on a completely independent validation series. By using the new scoring algorithm and FISH as reference standard, the sensitivity and the specificity of the ALK RGQ RT-PCR(ΔCt) assay were 100% and 100%, respectively. CONCLUSIONS: Our results suggest that the ALK RGQ RT-PCR test could be useful in clinical practice as a complementary assay in multi-test diagnostic algorithms or even, if our data will be confirmed in independent studies, as a standalone or screening test for the selection of patients to be treated with ALK inhibitors.

ALK Protein Analysis by IHC Staining after Recent Regulatory Changes: A Comparison of Two Widely Used Approaches, Revision of the Literature, and a New Testing Algorithm.

INTRODUCTION: Recent regulatory changes have allowed the diagnostic use of immunohistochemical (IHC) analysis for the identification of patients with non-small cell lung cancer who are eligible for treatment with anaplastic lymphoma receptor tyrosine kinase (ALK) inhibitors. The U.S. Food and Drug Administration has approved the VENTANA ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ) as companion diagnostics, and the Italian Medicines Agency has recognized IHC analysis as a diagnostic test indicating an algorithm for patient selection. METHODS: On the basis of the new regulations, we compared two commonly used IHC assays on 1031 lung adenocarcinomas: the VENTANA ALK (D5F3) CDx Assay with the OptiView Amplification Kit (Ventana Medical Systems) and a standard IHC test with the clone 5A4 (Novocastra, Leica Biosystems, Newcastle Upon Tyne, United Kingdom) along with their interpretative algorithms. Fluorescence in situ hybridization (FISH) was performed in all cases. Next-generation sequencing was performed in FISH/IHC analysis-discordant samples. RESULTS: FISH gave positive results in 33 (3.2%) cases. When FISH was used as a reference, the VENTANA ALK (D5F3) CDx assay had a sensitivity of 90.9% ± 2.6%, a specificity of 99.8% ± 0.6%, and positive and negative predictive values of 93.8% ± 2.1% and 99.7% ± 0.6%, respectively. The clone 5A4-based IHC test showed a sensitivity of 90.9% ± 2.6%, a specificity of 98.3% ± 1.3%, and positive and negative predictive values of 63.8% ± 4.2% and 99.7% ± 0.6%, respectively. Five cases with IHC analysis/FISH-discordant results in our series were analyzed together with those previously reported in the literature. Overall, data from 35 patients indicate a response rate to ALK inhibitors in 100% of FISH-negative/IHC analysis-positive cases (seven of seven) and 46% of FISH-positive/IHC analysis-negative cases (13 of 28), respectively. CONCLUSIONS: Our results confirm the difficulty in managing an IHC test without amplification in the absence of confirmatory FISH analysis, as well as the possibility of performing a direct diagnosis in approximately 90% of patients by the VENTANA ALK (D5F3) CDx Assay. On the basis of the recent regulatory changes, the data that have emerged from the literature, and the results of the present study, a new algorithm for ALK assessment in non-small cell lung cancer has been devised.