FOLFOXIRI plus bevacizumab versus FOLFIRI plus bevacizumab as first-line treatment of patients with metastatic colorectal cancer: updated overall survival and molecular subgroup analyses of the open-label, phase 3 TRIBE study.

BACKGROUND: In the TRIBE study, FOLFOXIRI (fluorouracil, leucovorin, oxaliplatin, and irinotecan) plus bevacizumab significantly improved progression-free survival of patients with metastatic colorectal cancer compared with FOLFIRI (fluorouracil, leucovorin, and irinotecan) plus bevacizumab. In this updated analysis, we aimed to provide mature results for overall survival-a secondary endpoint-and report treatment efficacy in RAS and BRAF molecular subgroups. METHODS: TRIBE was an open-label, multicentre, phase 3 randomised study of patients (aged 18-70 years with Eastern Cooperative Oncology Group [ECOG] performance status of 2 or less and aged 71-75 years with an ECOG performance status of 0) with unresectable metastatic colorectal cancer who were recruited from 34 Italian oncology units. Patients were randomly assigned (1:1) via a web-based procedure to receive FOLFIRI plus bevacizumab or FOLFOXIRI plus bevacizumab. Bevacizumab was given as a 5 mg/kg intravenous dose. FOLFIRI consisted of a 180 mg/m(2) intravenous infusion of irinotecan for 60 min followed by a 200 mg/m(2) intravenous infusion of leucovorin for 120 min, a 400 mg/m(2) intravenous bolus of fluorouracil, and a 2400 mg/m(2) continuous infusion of fluorouracil for 46 h. FOLFOXIRI consisted of a 165 mg/m(2) intravenous infusion of irinotecan for 60 min, followed by an 85 mg/m(2) intravenous infusion of oxaliplatin given concurrently with 200 mg/m(2) leucovorin for 120 min, followed by a 3200 mg/m(2) continuous infusion of fluorouracil for 48 h. Tissue samples for RAS and BRAF mutational status analyses were centrally collected. In this updated analysis, we assessed the secondary endpoint of overall survival in the main cohort and treatment efficacy in RAS and BRAF molecular subgroups. All analyses were by intention to treat. TRIBE was concluded on Nov 30, 2014. The trial is registered with ClinicalTrials.gov, number NCT00719797. FINDINGS: Between July 17, 2008, and May 31, 2011, 508 patients were randomly assigned. At a median follow-up of 48·1 months (IQR 41·7-55·6), median overall survival was 29·8 months (95% CI 26·0-34·3) in the FOLFOXIRI plus bevacizumab group compared with 25·8 months (22·5-29·1) in the FOLFIRI plus bevacizumab group (hazard ratio [HR] 0·80, 95% CI 0·65-0·98; p=0·03). Median overall survival was 37·1 months (95% CI 29·7-42·7) in the RAS and BRAF wild-type subgroup compared with 25·6 months (22·4-28·6) in the RAS-mutation-positive subgroup (HR 1·49, 95% CI 1·11-1·99) and 13·4 months (8·2-24·1) in the BRAF-mutation-positive subgroup (HR 2·79, 95% CI 1·75-4·46; likelihood-ratio test p<0·0001). Treatment effect was not significantly different across molecular subgroups (pinteraction=0·52). INTERPRETATION: FOLFOXIRI plus bevacizumab is a feasible treatment option for those patients who meet the inclusion criteria of the present study, irrespective of baseline clinical characteristics and RAS or BRAF mutational status.

Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer.

NRAS mutations occur in 3-5% of colorectal cancer. Differently from KRAS and BRAF mutations, the role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer (mCRC) has been investigated to a lesser extent. A retrospective series suggested the role of NRAS mutations as predictors of resistance to anti-EGFR monoclonal antibodies (MoAbs) in chemo-refractory patients with mCRC. In our study, KRAS codons 12, 13, 61 and BRAF codon 600 mutational status were evaluated in mCRCs referred to our Institution from 2009 to 2012. NRAS codons 12, 13 and 61 mutational status was analyzed in KRAS/BRAF wt patients. We collected pathological and clinical features in the overall population and outcome data in a subset of NRAS mutated chemo-refractory patients treated with anti-EGFR MoAbs in advanced lines. NRAS was mutated in 47/786 (6%) mCRCs. NRAS and KRAS mutated tumors did not show significant differences in terms of clinical and pathological characteristics, except for a lower prevalence of mucinous histology (p = 0.012) and lung metastases (p = 0.012) among NRAS mutated tumors. In the uni- and multivariate model, NRAS mutations were associated with shorter overall survival (OS) compared to all wt patients (median OS 25.6 vs 42.7 months; univ: HR = 1.91, 95% CI 1.39-3.86, p = 0.0013; multiv: HR = 1.75, 95% CI 1.1.3-2.72, p = 0.013). None of the chemo-refractory NRAS mutated patients evaluable for response to anti-EGFRs achieved response. In conclusion, NRAS mutations have a relevant incidence in patients with mCRC and showed an association with specific clinical and pathological features. NRAS mutations affect mCRC patients' prognosis and predict lack of response to anti-EGFRs.

Molecular and pathological characterization of the EZH2 rs3757441 single nucleotide polymorphism in colorectal cancer.

BACKGROUND: The enhancer of zeste-homolog 2 (EZH2) is involved in cancer development through gene silencing by trimethylation of lysine 27 of histone 3 (H3K27me3). The C/C genotype for the EZH2 rs3757441 single-nucleotide polymorphism (SNP) is linked with poor prognosis in metastatic colorectal cancer (CRC), but molecular and pathological characterization of this SNP is lacking. METHODS: 119 primary CRCs were analyzed. SNP was evaluated by real-time PCR from colonic healthy tissue, while EZH2 and H3K27me3 expression were studied by immunohistochemistry. We primarily looked for correlation between EZH2 rs3757441 genotypes and EZH2/H3K27me3 expression. Potential associations between EZH2/H3K27me3 expression and clinico-pathological features or KRAS exon 2 and BRAF exon 15 mutations were secondary endpoints. Statistical analysis was performed by chi-square test, T-test or ANOVA. RESULTS: The C/C genotype was significantly associated with higher EZH2 (100 vs. 44 %; P = 0.019) and H3K27me3 (100 vs. 38 %; P = 0.009) staining intensity compared with C/T and T/T. EZH2 3+ staining significantly correlated with stronger H3K27me3 expression (P = 0.039). KRAS and BRAF mutations were not associated with EZH2 or H3K27me3 expression. CONCLUSION: EZH2 rs3757441 C/C genotype is associated with stronger EZH2 and H3K27me3 immunoreactivity in primary CRC: this SNP may serve as a promising biomarker for EZH2-targeting agents and may add independent information to KRAS and BRAF testing.

Bevacizumab with FOLFOXIRI (irinotecan, oxaliplatin, fluorouracil, and folinate) as first-line treatment for metastatic colorectal cancer: a phase 2 trial.

BACKGROUND: The FOLFOXIRI (irinotecan, oxaliplatin, fluorouracil, and folinate) regimen has been shown to be better than FOLFIRI (fluorouracil, folinate, and irinotecan) in a phase 3 trial in patients with metastatic colorectal cancer. Results of various studies have shown that the addition of bevacizumab to chemotherapy increases treatment efficacy. We therefore assessed the safety and activity of the combination of FOLFOXIRI plus bevacizumab in patients with colorectal cancer. METHODS: In a phase 2 study, patients (aged 18-75 years) with colorectal cancer, which was judged to be unresectable for metastatic disease, were given the combination of intravenous bevacizumab (5 mg/kg on day 1) and intravenous FOLFOXIRI (irinotecan 165 mg/m(2) on day 1, oxaliplatin 85 mg/m(2) on day 1, folinate 200 mg/m(2) on day 1, and fluorouracil 3200 mg/m(2) for 48 h continuous infusion starting on day 1 and repeated every 2 weeks) as first-line treatment in seven centres in Italy. Induction treatment (FOLFOXIRI and bevacizumab) was administered for a maximum of 6 months, followed by maintenance treatment with bevacizumab (5 mg/kg intravenously on day 1, repeated every 2 weeks). The primary endpoint was progression-free survival (PFS) at 10 months from study entry in the intention-to-treat population. This study has been completed and is registered with ClinicalTrials.gov, number NCT01163396. FINDINGS: From July 2, 2007, to April 1, 2008, 57 patients were enrolled; all patients were assessed for safety and efficacy. Median follow-up time was 28.8 months (95% CI 24.9-32.5). PFS at 10 months was 74% (95% CI 62-85). Main grade 3 or 4 adverse events during induction treatment were neutropenia (n=28 [49%], including one case of febrile neutropenia), diarrhoea (n=8 [14%]), stomatitis (n=2 [4%]), neurotoxicity (n=1 [2%]), deep-vein thrombosis (n=4 [7%]), and hypertension (n=6 [11%]). No treatment-related deaths occurred. Six serious adverse events occurred during the induction treatment: febrile neutropenia (n=1 [2%]), grade 3 diarrhoea with dehydration (n=2 [4%]), grade 4 stomatitis (n=1 [2%]), grade 4 hypertension (n=1 [2%]), and fluorouracil-related cardiac ischaemia (n=1 [2%]). The most common grade 3 or 4 adverse events noted in the 37 patients who received maintenance treatment were hypertension (n=5 [14%]) and neurotoxicity (n=3 [8%]). One case of acute myocardial infarction due to coronary thrombosis was noted during the maintenance treatment. INTERPRETATION: Bevacizumab can be safely used with FOLFOXIRI without causing unforeseen adverse events. Treatment achieved promising results in terms of PFS. A phase 3 study for the comparison of FOLFOXIRI plus bevacizumab with FOLFIRI plus bevacizumab is in progress. FUNDING: Gruppo Oncologico Nord Ovest, ARCO Foundation, and Roche.

Feasibility of BRCA1/2 Testing of Formalin-Fixed and Paraffin-Embedded Pancreatic Tumor Samples: A Consecutive Clinical Series.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer, with most patients diagnosed at advanced stages. First-line treatment based on a combined chemotherapy (FOLFIRINOX or gemcitabine plus nab-paclitaxel) provides limited benefits. Olaparib, a PARP inhibitor, has been approved as maintenance for PDAC patients harboring germline BRCA1/2 pathogenic mutations and previously treated with a platinum-based chemotherapy. BRCA1/2 germline testing is recommended, but also somatic mutations could predict responses to PARP inhibitors. Analysis of tumor tissues can detect both germline and somatic mutations and potential resistance alterations. Few data are available about BRCA1/2 testing on pancreatic tumor tissues, which often include limited biological material. We performed BRCA1/2 testing, by an amplicon-based Next Generation Sequencing (NGS) panel, on 37 consecutive PDAC clinical samples: 86.5% of cases were adequate for NGS analysis, with a success rate of 81.2% (median DNA input: 10 nanograms). Three BRCA2 mutations were detected (11.5%). Failed samples were all from tissue macrosections, which had higher fragmented DNA than standard sections, biopsies and fine-needle aspirations, likely due to fixation procedures. BRCA1/2 testing on pancreatic tumor tissues can also be feasible on small biopsies, but more cases must be analyzed to define its role and value in the PDAC diagnostic algorithm.

Consistency and reproducibility of next-generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens.

BACKGROUND: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 105 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. METHODS: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). RESULTS: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. CONCLUSIONS: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Functional characterization of the novel T599I-VKSRdel BRAF mutation in a follicular variant papillary thyroid carcinoma.

CONTEXT: Mutations in BRAF are rare in the follicular variant of papillary thyroid carcinoma (FV-PTC). OBJECTIVE: We identified and functionally characterized a novel T599I-VKSR(600-603)del BRAF mutation in a FV-PTC patient. We analyzed in vitro the effects of this novel mutation in comparison with other thyroid cancer-associated mutations. DESIGN: Expression vectors for the BRAF mutants were generated and their in vitro kinase activity, signaling along the MAPK pathway, and capability of stimulating transcription from an AP1-responsive reporter evaluated. RESULTS: BRAF kinase and signaling were increased to a similar extent by the T599I-VKSR (600-603)del, V600E, and K601E mutations. Instead, the G474R, a mutation previously found in a FV-PTC, knocked down the BRAF kinase and its intracellular signaling. Some cancer-associated low-activity BRAF mutants stimulate the MAPK cascade via CRAF; however, the G474R protein lacked also this property. CONCLUSION: The T599I-VKSR(600-603)del is a novel gain-of-function mutation that targets BRAF in FV-PTC. Moreover, G474R is the first example of a mutation knocking down enzymatic BRAF activity in a FV-PTC. These findings underscore the importance of functional studies to characterize the role of BRAF mutations associated with thyroid cancer.

BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: a 15-year median follow-up study.

BACKGROUND: The BRAF(V600E) mutation is the most frequent genetic alteration in papillary thyroid carcinoma (PTC). The role of BRAF(V600E) mutation as a poor prognostic factor has been controversially reported in series with short-term follow-ups. In this study we verified the prognostic value of the BRAF(V600E) mutation in PTC patients with a long-term follow-up. METHODS: We studied 102 PTC patients with a median follow-up of 15 yr. The BRAF(V600E) mutation was analyzed by PCR-single-strand conformational polymorphism and sequencing. The correlation between the presence/absence of the BRAF(V600E) mutation, clinicopathological features, and outcome of PTC patients were evaluated. RESULTS: The BRAF(V600E) mutation was found in 38 of 102 (37.3%) PTC patients, and was significantly more frequent in patients older than 60 yr (P = 0.02), in advanced stages (P = 0.03), and in cases with vascular invasion (P = 0.02). At univariate analysis the worst outcome for PTC patients was significantly correlated with clinicopathological features (i.e. age, tumor size, extrathyroid extension, lymph node and distant metastases, advanced stage, vascular endothelial growth factor expression, and vascular invasion) and the BRAF(V600E) mutation (P < 0.002). However, at multivariate analysis only the BRAF(V600E) mutation showed an independent correlation with the worst outcome (P = 0.03). Moreover, the survival curves of PTC patients showed a lower percentage of survivors in the BRAF(V600E)-mutated group (P = 0.015). CONCLUSIONS: In this study the BRAF(V600E) mutation correlated with the worst outcome for PTC patients, who were not only at a higher risk not to be cured but also for death. In particular, the BRAF(V600E) mutation was demonstrated to be a poor prognostic factor independent from other clinicopathological features.

Activity and Safety of Cetuximab Plus Modified FOLFOXIRI Followed by Maintenance With Cetuximab or Bevacizumab for RAS and BRAF Wild-type Metastatic Colorectal Cancer: A Randomized Phase 2 Clinical Trial.

IMPORTANCE: The combination of a triple-drug chemotherapy regimen with an anti-epidermal growth factor receptor (EGFR) agent as a first-line treatment of metastatic colorectal cancer (mCRC) showed promising activity along with safety concerns in single-arm phase 2 trials. The role of maintenance following chemotherapy and anti-EGFR and the optimal regimen to be adopted are not established. OBJECTIVES: To evaluate the activity and safety of cetuximab plus modified FOLFOXIRI (mFOLFOXIRI) and explore the role of maintenance with cetuximab or bevacizumab in RAS and BRAF wild-type mCRC. DESIGN, SETTING, AND PARTICIPANTS: In a prospective, noncomparative, open-label, multicenter, randomized phase 2 trial, patients aged 18 to 75 years with unresectable, previously untreated RAS and BRAF wild-type (before amendment, KRAS wild-type) mCRC were recruited from 21 oncology units in Italy from October 19, 2011, to March 1, 2015 (followed up through May 31, 2017). In total, 323 patients were screened and 143 were randomized to 2 treatment arms to receive as a first-line induction a regimen of mFOLFOXIRI plus cetuximab followed by cetuximab (arm A) or bevacizumab (arm B) until disease progression. Primary analyses were conducted in a modified intention-to-treat population. INTERVENTIONS: mFOLFOXIRI plus cetuximab repeated every 2 weeks for up to 8 cycles, followed by maintenance with cetuximab or bevacizumab until disease progression. MAIN OUTCOMES AND MEASURES: The primary end point was the 10-month progression-free rate (PFR); secondary end points included progression-free and overall survival, response rate, rate of metastases resection, and adverse events. RESULTS: Of 143 patients randomized, 116 (81.1%) (median [interquartile range (IQR)] age, 59.5 [53-67] years; 34 [29.3%] women) had RAS and BRAF wild-type mCRC. At a median (IQR) follow-up of 44.0 (30.5-52.1) months, 10-month PFRs were 50.8% (90% CI, 39.5%-62.2%) in arm A and 40.4% (90% CI, 29.4%-52.1%) in arm B. The overall response rate was 71.6% (95% CI, 62.4%-79.5%). Main grade 3/4 adverse events were neutropenia (occurring in 36 patients [31%]), diarrhea (in 21 patients [18%]), skin toxic effects (in 18 patients [16%]), asthenia (in 11 patients [9%]), stomatitis (in 7 patients [6%]), and febrile neutropenia (in 3 patients [3%]). CONCLUSIONS AND RELEVANCE: Although neither of the 2 arms met the primary end point, the findings indicate that a 4-month induction regimen of mFOLFOXIRI plus cetuximab is feasible and provides relevant activity results, leading to a high surgical resection rate. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02295930.

The heterogeneous distribution of BRAF mutation supports the independent clonal origin of distinct tumor foci in multifocal papillary thyroid carcinoma.

CONTEXT: Papillary thyroid carcinoma (PTC) is frequently multifocal. Independent PTC foci may occur either from intraglandular metastases from a single dominant tumor or as unrelated neoplastic clones. In rare cases, the simultaneous presence of PTC foci of different histopathological subtypes points to independent sites of tumor formation. OBJECTIVES: We examined the pattern of BRAF mutations in noncontiguous tumor foci and node metastases from 69 patients affected by multicentric PTC. These included 19 cases characterized by the simultaneous presence of different PTC histopathological variants. DESIGN: BRAF (exon 15) mutation was examined by PCR-single strand conformational polymorphism followed by DNA sequencing in laser-capture microdissected tissue samples. RESULTS: Discordant patterns of BRAF mutation were found in about 40% of the multifocal PTCs. In node metastases, BRAF mutations were, in most but not all the cases, concordant with the dominant tumor. A discordant pattern of BRAF mutation was also found in about 50% of the cases in which multiple foci of different histopathological variants were present. CONCLUSIONS: The heterogeneous distribution of BRAF mutations suggests that discrete tumor foci in multifocal PTC may occur as independent tumors. This information has to be considered in the design of targeted therapeutic approaches with BRAF pathway inhibitors.

Association of BRAF V600E mutation with poor clinicopathological outcomes in 500 consecutive cases of papillary thyroid carcinoma.

CONTEXT: Because very few studies have examined the correlation between BRAF mutations and clinicopathological features of papillary thyroid carcinoma (PTC), we analyzed here a large and homogeneous cohort of patients with PTC for the presence of the BRAF mutation. OBJECTIVE: We examined BRAF mutations in a consecutive series of 500 PTC patients who underwent surgery in the Department of Surgery of the University of Pisa, and we correlated the presence of the mutation with clinicopathological parameters of the patients: age, gender, tumor size, presence of tumor capsule, extrathyroidal invasion, multicentricity, presence of node metastases, and tumor class. DESIGN: BRAF (exon 15) mutation was examined by PCR-single strand conformational polymorphism followed by DNA sequencing in laser-capture microdissected tissue samples. RESULTS: In this study, BRAF mutation was found in 219 of 500 cases (43.8%). In particular, we found the most common BRAF V600E mutation in 214 cases (42.8%), BRAF K601E mutation in three cases (0.6%), BRAF VK600-1E (0.2%) in one case, whereas in one case we found a new 14-bp deletion with concomitant 2-bp insertion, VKSR600-3del and T599I, respectively. BRAF V600E was associated with extrathyroidal invasion (P < 0.0001), multicentricity (P = 0.0026), presence of nodal metastases (P = 0.0009), class III vs. classes I and II (P < 0.00000006), and absence of tumor capsule (P < 0.0001), in particular in follicular- and micro-PTC variants. By multivariate analysis, the absence of tumor capsule remained the only parameter associated (P = 0.0005) with BRAF V600E mutation. CONCLUSIONS: Our data suggest that BRAF V600E mutation is associated with high-risk PTC and in particular in follicular variant with invasive tumor growth.

Presence of BRAF V600E in very early stages of papillary thyroid carcinoma.

OBJECTIVE: Extremely small (often 0.5-4 mm) incidental thyroid malignancies (incidental microcarcinomas, IM) might be discovered at histological examination after surgery in patients who were operated on for benign thyroid conditions. Morphologically, these malignancies have virtually always features of differentiated papillary thyroid carcinoma (PTC). Although IM are in general considered of little clinical significance their potential malignant behavior cannot be ignored. BRAF(V600E) mutation has emerged as the most prevalent genetic alteration in PTC. DESIGN: The incidence of BRAF(V600E) was studied in 85 microdissected cases of IM detected in two series of 334 and 398 patients operated on for benign thyroid disease in 2005 and in 2006, respectively. IM ranged in size from 0.5 to 4 mm and all featured cytology consistent with the diagnosis of PTC. Microdissected lesions were also evaluated for galectin-3 expression by immunohistochemistry. MAIN OUTCOME: BRAF(V600E) was identified in 15 out of 85 (17.6%) IM compared to 45% clinically evident PTCs (n = 91) and 38.3% preoperatively identified microcarcinomas (n = 47). CONCLUSION: These data suggest that it is possible to find BRAF(V600E) in IM despite their extremely small (<1 mm) size. This is not a formal demonstration that IM can evolve into clinical PTC, but on these bases, patients with BRAF(V600E) IM may need to be managed more carefully.

EGFR and KRAS mutational analysis in a large series of Italian non-small cell lung cancer patients: 2,387 cases from a single center.

Activating EGFR mutations are important genetic alterations that have strong therapeutic implications for non-small cell lung cancer (NSCLC) patients. However, the role of KRAS mutations in this process is still under evaluation. Here, we report on the feasibility of a large­scale EGFR and KRAS mutation analysis in the daily routine of a single center. NSCLCs from 2,387 patients were screened for EGFR and KRAS mutations from January 2010 to September 2015. Mutational analyses were performed in a single laboratory using single strand conformation polymorphism (SSCP)-Sanger sequencing and matrix­assisted laser desorption ionization­time of flight (MALDI­TOF) on Sequenom platform for EGFR and pyrosequencing for KRAS. Activating EGFR mutations were found in 14.1% of all tumors, whereas KRAS mutations were found in 30.5% of all tumors. Direct sequencing showed analyzable cytological, small biopsy and surgical specimen percentages of 90.3, 90.9 and 98.1%, respectively, whereas the MALDI­TOF platform showed analyzable cytological samples, small biopsies and surgical specimens percentages of 94.6, 95.7 and 96.9%, respectively. The mean analytical turnaround times (TAT) were 4 and 3 days for direct sequencing and the MALDI­TOF platform, respectively. Our results confirm that small biopsy or cytological samples can be used for reliable EGFR and KRAS mutation testing and indicate that adopting the MALDI­TOF platform reduces the rate of missed samples among the samples. Moreover, the 3-day analytical TAT of the MALDI-TOF multi-target technique is appropriate for clinical management and reduces the overall treatment decision time.

Response to erlotinib in a patient with lung adenocarcinoma harbouring the EML4-ALK translocation: A case report.

Lung cancer is the leading cause of cancer-associated mortality worldwide, and the mainstay of treatment remains to be personalised therapy. Tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKIs) have been reported to exert a significant impact in the treatment of non-small cell lung cancer (NSCLC), particularly in patients harbouring mutations in the EGFR gene. The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) gene translocation has been described in a subset of patients with NSCLC and possesses potent oncogenic activity. This translocation represents one of the most novel molecular targets in the treatment of NSCLC. Patients who harbour the EML4-ALK rearrangement possess lung tumours that lack EGFR or K-ras mutations. The present study reports the case of a patient possessing the EML4-ALK rearrangement that was initially treated with erlotinib and achieved a lasting clinical response. To the best of our knowledge, the current study is the first report of a clinical response to EGFR-TKI in a patient with lung adenocarcinoma harbouring the EML4-ALK fusion gene, but no EGFR mutations. However, as the disease progressed, the ALK gene status of the tumour was investigated, and based upon a positive result, the patient was treated with crizotinib and achieved a complete response. In conclusion, the present study suggests that the EML4-ALK rearrangement is not always associated with resistance to EGFR-TKIs. Further studies are required to clarify the biological features of these tumours and to investigate the mechanisms underlying the primary resistance to EGFR-TKIs when the EML4-ALK rearrangement is present.

Therapeutic effect of diagnostic ultrasound on enzymatic thrombolysis. An in vitro study on blood of normal subjects and patients with coronary artery disease.

If delivered at elevated intensity, ultrasound potentiates enzymatic clot dissolution; however, an elevated acoustic intensity damages vascular wall and favors reocclusion. This study's aim was to investigate whether exposure to high-frequency, low-intensity ultrasound - generated by a diagnostic scanner -enhances enzymatic thrombolysis, and if this effect differs in clots from blood of normal subjects and of patients with coronary artery disease (CAD). Venous blood samples were drawn from 10 healthy volunteers and from 10 CAD patients on chronic medical treatment, which also included aspirin. Each sample generated 2 radiolabelled clots, which were positioned in 2 in vitro models filled with human plasma recirculating at 37 degrees. One clot was exposed to acetyl salicylic acid (60 microg/ml), tissue plasminogen activator (3 microg/ml) and heparin (1 IU/ml), while the other was exposed to the same medications plus ultra-sound (2.5 MHz, mechanical index = 1.0) for 3 hours. Enzymatic thrombolysis was measured as solubilization of radiolabel. Normal subjects and patients did not significantly differ as to coagulation parameters, weight, volume and density of the clots, and fibrinolytic activity (p = 0.794). Ultrasound exposure did not influence thrombolysis in clots of normal subjects (p = 0.367), while it enhanced the dissolution of clots of CAD patients (p = 0.013). The enhancement was equal to 51% at 5 minutes, 32% at 15 minutes, 27% at 30 minutes, 20% at 1 hour and 19% at 3 hours (p < 0.05). Diagnostic ultrasound enhances enzymatic dissolution of clots generated from the blood of CAD patients, likely due to chronic treatment and in particular to aspirin.

Anaplastic lymphoma kinase gene rearrangements in cytological samples of non-small cell lung cancer: comparison with histological assessment.

BACKGROUND: Anaplastic lymphoma kinase (ALK) gene rearrangements are detected in approximately 5% of cases of non-small cell lung cancer (NSCLC). Patients who are positive for ALK rearrangements may be successfully treated with the ALK inhibitor crizotinib. Because advanced-stage lung cancers are not suitable for surgical resection, approximately 70% of patients are diagnosed via preoperative specimens. In the current study, the authors evaluated the suitability of stained cytologic direct smears and cell blocks for fluorescence in situ hybridization (FISH) to determine ALK status compared with small biopsies. METHODS: A total of 493 consecutive patients with NSCLC were analyzed by FISH for ALK gene rearrangements. The analyzed sample comprised 180 cytological samples, 94 direct smears, 86 cell blocks, and 313 preoperative small biopsies. Moreover, in the same series, 426 patients and 369 patients, respectively, were evaluated for epidermal growth factor receptor and KRAS mutations, respectively. RESULTS: Of the total of 493 patients, 18 patients who were positive for a gene rearrangement (4.4%) demonstrated ALK FISH rearrangements, whereas 387 patients (95.6%) were negative. All other cases were classified as inadequate (7.7%) and insufficient (10.1%). A strong statistical association was found between the cytology and the small biopsy with respect to ALK rearrangements (P=.0048). Fifty-three patients (12.4%) demonstrated an epidermal growth factor receptor mutation, whereas 90 patients (24.4%) were found to have KRAS mutations. None of these patients presented with ALK gene rearrangements. CONCLUSIONS: ALK gene rearrangements may be easily detected in cytological samples and particularly in direct smears, thereby yielding important improvements in the diagnostic approach to patients with advanced NSCLC. Cytological samples may be useful for ALK analysis when insufficient material is available in cell blocks or small biopsies.

FoxP3 expression in papillary thyroid carcinoma: a possible resistance biomarker to iodine 131 treatment.

BACKGROUND: The forkhead transcription factor FoxP3 plays an important role in regulatory T cell (Treg) functions. Tregs are critical in maintaining immunologic tolerance. It has been shown that vaccination against FoxP3-expressing cells is associated with enhancement of tumor immunity. Tregs appear to be increased in blood and in the tumor microenvironment of patients with different cancer types. Tumor cells themselves can express FoxP3. The present study investigates the possible role of FoxP3 expression in a series of human papillary thyroid cancers with a mean follow-up time of 15 years. METHODS: One hundred five cases of papillary thyroid carcinoma (PTC) were investigated, and FoxP3 expression was evaluated in both tumor cells and tumor-associated infiltrates. For all patients, clinical/pathologic features were considered and the results analyzed by statistical tests. RESULTS: Of the 105 PTC cases, 45 (43%) scored FoxP3-positive and 60 (57%) were negative. FoxP3 staining was localized predominantly in the cytoplasm of tumor cells. In some cases, both nuclear and cytoplasmic staining was seen in infiltrating cells. FoxP3 expression in tumor cells was correlated with the presence of extrathyroid invasion (p=0.04) and distant metastasis (p=0.04), but not with overall survival. Interestingly, FoxP3 expression in neoplastic cells was significantly associated with a resistance phenotype to radioiodine treatment (p=0.041). CONCLUSIONS: The data show an association of FoxP3 expression with features of PTC that seem to have a specific impact on radioiodine sensitivity.

KRAS and BRAF genotyping of synchronous colorectal carcinomas.

v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) genotyping is required prior to anti-epidermal growth factor receptor monoclonal antibody therapy administered in cases of metastatic colorectal carcinoma (CRC). Thus, KRAS mutation screening is required for patient management. The present study reported the experience of KRAS/v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational screening on synchronous CRC pairs from 26 patients, which were defined as index lesions (ILs) and concurrent lesions (CLs) on the basis of tumor grade and dimension and their respective lymph node and distant metastases. Overall, KRAS mutations were present in 38.4% of patients, whereas BRAF mutations were present at a frequency of 11.5%. The genotyping of paired synchronous carcinomas indicated that 11 patients (42.3%) exhibited discordant KRAS mutational statuses in terms of the presence of a mutation in only one lesion of the pair or of two different mutations harbored by each lesion. BRAF mutations were present in the synchronous tumors of two cases, whereas in two other cases, only the IL or CL harbored mutant BRAF. Overall, the mutational statuses of distant and lymph node metastases confirm the genetic heterogeneity of synchronous primary tumors. These results highlighted the fact that adequate sampling and comprehensive testing, when feasible, is likely to optimize the decision-making process for treatment approaches, even in the relatively rare event of multiple synchronous lesions.

ALK rearrangement in a large series of consecutive non-small cell lung cancers: comparison between a new immunohistochemical approach and fluorescence in situ hybridization for the screening of patients eligible for crizotinib treatment.

CONTEXT: Echinoderm microtubule associated proteinlike 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application. OBJECTIVE: To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC. DESIGN: We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement. RESULTS: Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK(+) by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK(+) cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY. CONCLUSIONS: Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.

EGFR and K-Ras mutations in women with lung adenocarcinoma: implications for treatment strategy definition.

BACKGROUND: We aimed at investigating the outcomes of female patients with stage IIIB-IV adenocarcinoma of the lung according to EGFR and K-Ras mutational status. METHODS: One hundred and three consecutive female patients genotyped at a single Italian Institution were analyzed. Patients were planned to receive first-line platinum-based chemotherapy (CT) and a salvage treatment with anti-EGFR tyrosine-kinase inhibitors (TKIs) was proposed irrespective of tumor mutational status. EGFR (exons 18-21) and K-Ras (exon 2, codons 12-13) mutations were evaluated by real-time PCR and pyrosequencing. The association of mutational status with clinical variables and treatment benefit was investigated by chi-square test and log-rank test. RESULTS: EGFR and K-Ras mutations were found in 31 (30%) and 13 (15%) cases, respectively. Sixty-six patients received platinum CT: no correlation was observed between EGFR or K-Ras mutational status and response rate (RR) (p > 0.05). However, patients treated with first-line CT harboring EGFR activating mutations experienced a significantly reduced progression-free survival (PFS) in comparison with wild-type ones (4.4 vs. 6.4 months, respectively; HR 0.597, 95% CI 0.287-0.975; p = 0.048). Thirty-nine patients received salvage treatment with erlotinib: EGFR activating mutations were significantly correlated with RR (60% vs. 12.5%; p = 0.004) and PFS (11.4 vs. 4.5 months; HR 0.491, 95% CI 0.216-0.936; p = 0.044). Responses to erlotinib were not reported among women with K-Ras mutant tumors, while 50% of those with wild-type K-Ras achieved an objective remission (p = 0.296). Median PFS (3.5 vs. 8.8 months; HR 0.284, 95% CI 0.015-0.510; p = 0.010) and OS (3.9 vs. 19.8 months; HR 0.158, 95% CI 0.001-0.075; p < 0.001) were significantly shorter among K-Ras mutant patients treated with TKI. CONCLUSIONS: In our population of Caucasian women with advanced lung adenocarcinoma we observed that the presence of EGFR activating mutations correlates with a significant reduction in the benefit from first-line platinum-based CT, emphasizing the importance of an upfront use of anti-EGFR TKIs in this patient subset. K-Ras mutations seem to correlate with a detrimental effect from anti-EGFR TKI, but this finding deserves further investigation.