Purpose

Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those that do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in ALK+ non-small cell lung cancer (NSCLC) patients.

Experimental Design

We analyzed tissue obtained from 14 ALK+ NSCLC patients demonstrating evidence of radiologic progression while on crizotinib in order to define mechanisms of intrinsic and acquired resistance to crizotinib.

Results

Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK kinase domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient demonstrated outgrowth of EGFR mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient demonstrated the emergence of an ALK gene fusion negative tumor compared to the baseline sample, but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism.

Conclusions

Crizotinib resistance in ALK+ NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers.

Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein–coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ~27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.

Chronic human immunodeficiency virus (HIV) infection is associated with higher incidence of pulmonary complications including hypertension, vasculopathy, lymphocytic alveolitis, and interstitial pneumonitis not attributed to either opportunistic infections or presence of the virus. The Tat (TransActivator of Transcription) protein, a required transactivator for expression of full-length viral genes, is pleiotropic and influences expression of cellular inflammatory genes. Tat-dependent transactivation of cellular genes requires specific mediators, including NF-κB, widely recognized as sensitive to changes in cellular oxidant burden. We hypothesized that overproduction of Tat leads to increased oxidant burden and to alterations of basal inflammatory status as measured by NF-κB activation. We engineered transgenic mouse lines that express Tat (86-amino acid isoform) in the lung under the control of the surfactant protein C promoter (SP-C). Tat-transgenic mice exhibit increased pulmonary cellular infiltration, increased nitrotyrosine and carbonyl protein modifications, increased levels of NF-κB, MnSOD and thioredoxin interacting protein (TxNIP). These data indicate that Tat increases oxidant burden and resets the threshold for inflammation, which may increase susceptibility to secondary injuries.

Introduction

The detection of circulating tumor cells (CTC) is prognostic in several cancer types. This trial examines the incidence and prognostic value of CTCs in urothelial cancer (UC).

Materials and Methods

44 subjects with UC were assessed for CTCs using CellSearch® Technology (Veridex, LLC, Raritan, NJ), using 7.5 ml of peripheral blood, sorted by magnetic separation (EpCaM positive, and immunofluorescent staining, (Cytokeratin 8, 18, or 19 positive, CD 45 negative, DAPI positive cells) to identify CTCs.

Results

Five of 30 (17%) subjects with clinically localized and 7 of 14 (50%) patients with metastatic UC had at least 1 detectable CTC, with a range of 1–177 CTCs. Six subjects had 5 or more CTCs. FISH analysis was performed in 20 samples from 18 unique subjects, using the UroVysion probe set. Copy number gains consistent with neoplasm were observed in cases with measurable CTCs, but not in any of the CTC-negative samples tested. With a median follow-up of 337 days, all 7 metastatic subjects with detectable CTCs had died, versus 3 of 7 (43%) metastatic subjects without detectable CTCs.

Conclusions

CTCs were observed in 50% of the metastatic UC patients tested. FISH analysis confirmed the aneusomic chromosomal content in the CTCs. The findings suggest measurable CTCs may be prognostic for shortened survival in metastatic UC patients, although the optimal threshold for a “positive” finding is unknown. CTCs were also detected in a subset of patients with clinically-localized disease, identifying a potential high-risk, pre-operative group for future study.

Purpose

Erlotinib prolongs survival in patients with advanced non–small-cell lung cancer (NSCLC). We report the results of a randomized, phase II study of erlotinib alone or intercalated with chemotherapy (CT + erlotinib) in chemotherapy-naïve patients with advanced NSCLC who were positive for epidermal growth factor receptor (EGFR) protein expression and/or with high EGFR gene copy number.

Patients and Methods

A total of 143 patients were randomly assigned to either erlotinib 150 mg daily orally until disease progression (PD) occurred or to chemotherapy with paclitaxel 200 mg/m2 intravenously (IV) and carboplatin dosed by creatinine clearance (AUC 6) IV on day 1 intercalated with erlotinib 150 mg orally on days 2 through 15 every 3 weeks for four cycles followed by erlotinib 150 mg orally until PD occurred (CT + erlotinib). The primary end point was 6-month progression-free survival (PFS); secondary end points included response rate, PFS, and survival. EGFR, KRAS mutation, EGFR fluorescent in situ hybridization and immunohistochemistry, and E-cadherin and vimentin protein levels were also assessed.

Results

Six-month PFS rates were 26% and 31% for the two arms (CT + erlotinib and erlotinib alone, respectively). Both were less than the historical control of 45% (P = .001 and P = .011, respectively). Median PFS times were 4.57 and 2.69 months, respectively. Patients with tumors harboring EGFR activating mutations fared better on erlotinib alone (median PFS, 18.2 months v 4.9 months for CT + erlotinib).

Conclusion

The feasibility of a multicenter biomarker-driven study was demonstrated, but neither treatment arms exceeded historical controls. This study does not support combined chemotherapy and erlotinib in first-line treatment of EGFR-selected advanced NSCLC, and the patients with tumors harboring EGFR mutations had a better outcome on erlotinib alone.

Vascular endothelial growth factor-2 (VEGFR-2 or KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected NSCLC (n=248). KDR copy number gains (CNGs), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR=5.16; P=0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear HIF-1α in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11 and 21 by PCR-based sequencing; however, two variant SNP genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.

Purpose

Anaplastic lymphoma kinase (ALK) rearrangements, associated with sensitivity to an experimental ALK/MET inhibitor, occur in 3% to 5% of non-small cell lung cancers. Intratumoral fluorescence in situ hybridization (FISH) heterogeneity has been reported. We explored the heterogeneity basis, the requirements for accurately determining ALK FISH positivity, and the effect of enriching the tested population using clinical and molecular factors.

Experimental Design

Lung cancer patients were screened by ALK and MET FISH and for EGFR and KRAS mutations.

Results

Thirteen ALK-positive cases were identified from 73 screened patients. Gene copy number increases occurred together with classic rearrangements. All positive cases were adenocarcinomas, 12 were EGFR/KRAS wild-type, and 1 had a coexistent EGFR exon 20 mutation. No association with MET amplification occurred. ALK positivity was associated with <10-pack-year smoking status (P = 0.0004). Among adenocarcinomas, without KRAS or EGFR mutations, with <10-pack-year history, 44.8% of cases were ALK positive. ALK FISH positivity was heterogeneous, but mean values in tumor areas from ALK-positive patients (54% of cells; range, 22-87%) were significantly higher than in adjacent normal tissue or tumor/ normal areas from ALK-negative patients (mean, 5-7%). Contiguous sliding field analyses showed diffuse heterogeneity without evidence of focal ALK rearrangements. One hundred percent sensitivity and specificity occurred when four or more fields (~60 cells) were counted.

Conclusions

Intratumoral ALK FISH heterogeneity reflects technique, not biology. The clinical activity of ALK/MET inhibitors in ALK-positive patients probably reflects ALK, but not MET, activity. Prescreening by histology, EGFR/KRAS mutations, and smoking status dramatically increases the ALK-positive hit rate compared with unselected series.

Background

Sensitivity to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and frequency of activation mutations in EGFR is lower in Caucasian than Asian non small-cell lung cancer (NSCLC) patients. Increased EGFR gene copy numbers evaluated by fluorescence in situ hybridization (FISH) has been reported as predictor of clinical benefit from EGFR-TKIs in Caucasian NSCLC patients. This study was carried out to verify whether EGFR FISH had similar performance in Japanese patients.

Methods

A cohort of 44 Japanese patients with recurrent NSCLC after surgery was treated with gefitinib 250 mg daily. The cohort included 48% females and 52% never-smokers; 73% had prior chemotherapy and 57% had stage III-IV at the time of surgery. Adenocarcinoma was the most common histology (86%). FISH was performed using the EGFR/Chromosome Enumeration Probe 7 and PathVysion DNA probes (Abbott Molecular). Specimens were classified as FISH positive when showing gene amplification or high polysomy (≥4 copies of the gene in ≥40% of tumor cells). Tumor response to gefitinib was assessed by RECIST for 33 patients with measurable diseases.

Results

Twenty-nine tumors (66%) were EGFR FISH+ and 23 (53%) were HER2 FISH+. Overall response rate was 52%, representing 65% of EGFR FISH+ patients and 29% of EGFR FISH+ patients (p = 0.0777). Survival was not impacted by the EGFR FISH (p = 0.9395) or the HER2 FISH (p = 0.0671) status. EGFR FISH= was significantly associated with HER2 FISH+ (p = 0.015) and presence of EGFR mutation (p = 0.0060). EGFR mutation significantly correlated with response (p < 0.0001) and survival after gefitinib (p = 0.0204). EGFR and HER2 FISH status were not associated with KRAS mutation.

Conclusion

Frequency of EGFR FISH+ status was higher and its predictive power for TKI sensitivity was lower in this Japanese cohort than in Western NSCLC cohorts. These findings support differences in the mechanisms of EGFR pathway activation in NSCLC between Asian and Caucasian populations. Confirmation of these results in larger cohorts is warranted.

Purpose

To compare the characteristics of HER receptors and their ligands deregulation between primary tumor and corresponding brain metastases of non-small cell lung carcinoma (NSCLC).

Experimental design

Fifty five NSCLC primary tumors (PT) and corresponding brain metastases (BM) specimens were examined for the immunohistochemical expression of EGFR, phosphorylated (p)–EGFR, Her2, Her3, and p-Her3, and their ligands EGF, TGF-α, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, and neuregulins-1 and -2. Analysis of EGFR copy number using fluorescent in situ hybridization and mutation by PCR-based sequencing was also performed.

Results

Metastases showed significantly higher immunohistochemical expression of EGF (membrane, BM 66.0 vs. PT 48.5; P=0.027; and nucleus, BM 92.2 vs. 67.4; P=0.008), amphiregulin (nucleus, BM 53.7 vs. PT 33.7; P=0.019), p-EGFR (membrane, BM 161.5 vs. PT 76.0; P<0.0001; and cytoplasm, BM 101.5 vs. PT 55.9; P=0.014), and p-Her3 (membrane, BM 25.0 vs. PT 3.7; P=0.001) than primary tumors (PT) did. Primary tumors showed significantly higher expression of cytoplasmic TGF–α (PT 149.8 vs. BM 111.3; P=0.008) and neuregulin-1 (PT 158.5 vs. BM 122.8; P=0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time points.

Conclusions

Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptors-related abnormalities from primary lung tumors.

To identify the characteristics and sequence of epidermal growth factor receptor (EGFR) abnormalities relevant to the pathogenesis and progression of lung adenocarcinoma, we performed a precise mapping analysis of EGFR mutation, gene copy number and total and phosphorylated EGFR (pEGFR) protein expression for the same tissue sites. We examined normal bronchial and bronchiolar epithelium (NBE) and tumor tissues obtained from 50 formalin-fixed lung adenocarcinomas, including 24 EGFR-mutant primary tumors with nine corresponding lymph node metastases and 26 wild-type primary tumors. NBE in 12/24 (50%) mutant and 3/26 (12%) wild-type tumors harbored EGFR mutation; these NBE also showed lack of EGFR copy number increase and frequent EGFR (69%) and pEGFR (33%) overexpression. EGFR mutation and protein overexpression were more frequent in NBE sites within tumors than in NBE sites adjacent to and distant from tumors, suggesting a localized field effect. Sites with high and low EGFR copy numbers were heterogeneously distributed in six of nine primary tumors and one of eight metastases. EGFR protein overexpression was significantly higher in metastasis sites than in primary tumors. We conclude from our findings that EGFR mutations and protein overexpression are early phenomena in the pathogenesis of lung adenocarcinoma and that EGFR mutation precedes an increase in gene copy number. In EGFR-mutant adenocarcinoma metastases, the higher levels of EGFR overexpression and more homogeneously distributed high gene copy numbers suggest tumor progression. Our findings have important implications for the development of new strategies for targeted chemoprevention and therapy in lung adenocarcinoma using EGFR inhibitors.

Purpose

Epidermal growth factor receptor (EGFR) gene copy number detected by fluorescent in situ hybridization (FISH) has proven to be useful for selection of non–small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors. Here, we evaluate EGFR FISH as a predictive marker in NSCLC patients receiving the EGFR monoclonal antibody inhibitor cetuximab plus chemotherapy.

Patients and Methods

Two hundred twenty-nine chemotherapy-naive patients with advanced-stage NSCLC were enrolled onto a phase II selection trial evaluating sequential or concurrent chemotherapy (paclitaxel plus carboplatin) with cetuximab.

Results

EGFR FISH was assessable in 76 patients with available tumor tissue and classified as positive (four or more gene copies per cell in ≥ 40% of the cells or gene amplification) in 59.2%. Response (complete response/partial response) was numerically higher in FISH-positive (45%) versus FISH-negative (26%) patients (P = .14), whereas disease control rate (complete response/partial response plus stable disease) was statistically superior (81% v 55%, respectively; P = .02). Patients with FISH-positive tumors had a median progression-free survival time of 6 months compared with 3 months for FISH-negative patients (P = .0008). Median survival time was 15 months for the FISH-positive group compared with 7 months for patients who were FISH negative. (P = .04). Furthermore, survival favored FISH-positive patients receiving concurrent therapy.

Conclusion

These results are the first to suggest that EGFR FISH is a predictive factor for selection of NSCLC patients for cetuximab plus chemotherapy. Prospective validation of these findings is warranted.

Purpose

TRIBUTE was a phase III trial evaluating the addition of erlotinib to carboplatin and paclitaxel as a first-line treatment for advanced non – small cell lung cancer that did not meet its primary end point of improving overall survival. Here, we assess the value of using epidermal growth factor receptor (EGFR) gene copy number in tumor biopsy samples, as determined by fluorescence in situ hybridization (FISH), as a predictor of treatment outcome.

Methods

EGFR FISH analysis was done using LSI EGFR SpectrumOrange/CEP7 Spectrum-Green probe.

Results

Of 275 samples, 245 (89.1%) were successfully analyzed by FISH. One hundred (40.8%) of patients were EGFR FISH(+). Median overall survival was not different between FISH(+) and FISH(−) patients in either the chemotherapy+erlotinib arm or the chemotherapy+placebo arm. In FISH(+) patients, median time to progression (TTP) was 6.3 months in the erlotinib arm versus 5.8 months in the placebo arm (hazard ratio, 0.59; 95% confidence interval, 0.35–0.99; P = 0.0430); in FISH(−) patients, median TTP was 4.6 months versus 6.0 months (hazard ratio,1.42; 95%confidence interval, 0.95–2.14; P = 0.0895; treatment interaction test, P = 0.007). After 6 months of treatment, a notable separation of the TTP curves in favor of erlotinib emerged. Objective response rates were11.6% versus 29.8% in FISH(+) patients (chemotherapy+erlotinib arm versus chemotherapy+placebo arm; P = 0.0495) and 21.8% versus 25.4%, respectively, for FISH(−) patients (P = 0.6954).

Conclusions

EGFR gene copy number by FISH did not predict survival benefit. However, among EGFR FISH(+) patients, TTP was longer in patients who received erlotinib and continued to receive it after completing first-line therapy.

Introduction

This phase I/II study evaluated the safety and anti-tumor effect of the combination of erlotinib with cixutumumab, a recombinant fully humanized anti-insulin-like growth factor-1 receptor IgG1 monoclonal antibody, in advanced non-small cell lung cancer (NSCLC).

Methods

Patients with advanced NSCLC were treated in an initial safety-lead and drop-down cohorts using erlotinib 150 mg/d with cixutumumab 6 or 5 mg/kg on days 1, 8, 15, and 22 in 28-day cycles (cohorts 1 and 2). Emerging pharmacokinetic data led to an additional cohort (3 + 3 design) with cixutumumab at 15 mg/kg on day 1 in 21-day cycles (cohort 3).

Results

Eighteen patients entered the study (6 at 6 mg/kg, 8 at 5 mg/kg, and 4 at 15 mg/kg), with median age of 65 years. Four of six patients at 6 mg/kg experienced dose-limiting toxicities (DLTs), whereas at 5 mg/kg, one of eight patients experienced DLT but three of eight patients still required a dose delay during cycle 1. At 15 mg/kg every 21 days, two of four patients experienced DLTs. In all cohorts, DLTs were either G3 rash or fatigue. Five patients had stable disease as best response and 14 patients had progressive disease. The median progression-free survival was 39 days (range 21–432+ days). Biomarkers analyses showed a trend toward better progression-free survival seen with higher free baseline insulin-like growth factor-1 levels as seen with other insulin-like growth factor-1R inhibitors.

Conclusions

The combinations of cixutumumab at 6 mg/kg every 7 days and 15 mg/kg every 21 days and full-dose erlotinib are not tolerable in unselected patients with NSCLC, as measured by DLT. Cixutumumab at 5 mg/kg every 7 days was tolerable per DLT, but dose delays were common. Efficacy in unselected patients with NSCLC seems to be low.

Summary

Background

ALK gene rearrangement defines a new molecular subtype of non-small-cell lung cancer (NSCLC). In a recent phase 1 clinical trial, the ALK tyrosine-kinase inhibitor (TKI) crizotinib showed marked antitumour activity in patients with advanced, ALK-positive NSCLC. To assess whether crizotinib affects overall survival in these patients, we did a retrospective study comparing survival outcomes in crizotinib-treated patients in the trial and crizotinib-naive controls screened during the same time period.

Methods

We examined overall survival in patients with advanced, ALK-positive NSCLC who enrolled in the phase 1 clinical trial of crizotinib, focusing on the cohort of 82 patients who had enrolled through Feb 10, 2010. For comparators, we identified 36 ALK-positive patients from trial sites who were not given crizotinib (ALK-positive controls), 67 patients without ALK rearrangement but positive for EGFR mutation, and 253 wild-type patients lacking either ALK rearrangement or EGFR mutation. To assess differences in overall survival, we assessed subsets of clinically comparable ALK-positive and ALK-negative patients.

Findings

Among 82 ALK-positive patients who were given crizotinib, median overall survival from initiation of crizotinib has not been reached (95% CI 17 months to not reached); 1-year overall survival was 74% (95% CI 63–82), and 2-year overall survival was 54% (40–66). Overall survival did not differ based on age, sex, smoking history, or ethnic origin. Survival in 30 ALK-positive patients who were given crizotinib in the second-line or third-line setting was significantly longer than in 23 ALK-positive controls given any second-line therapy (median overall survival not reached [95% CI 14 months to not reached] vs 6 months [4–17], 1-year overall survival 70% [95% CI 50–83] vs 44% [23–64], and 2-year overall survival 55% [33–72] vs 12% [2–30]; hazard ratio 0·36, 95% CI 0·17–0·75; p=0·004). Survival in 56 crizotinib-treated, ALK-positive patients was similar to that in 63 ALK-negative, EGFR-positive patients given EGFR TKI therapy (median overall survival not reached [95% CI 17 months to not reached] vs 24 months [15–34], 1-year overall survival 71% [95% CI 58–81] vs 74% [61–83], and 2-year overall survival 57% [40–71] vs 52% [38–65]; p=0·786), whereas survival in 36 crizotinib-naive, ALK-positive controls was similar to that in 253 wild-type controls (median overall survival 20 months [95% CI 13–26] vs 15 months [13–17]; p=0·244).

Interpretation

In patients with advanced, ALK-positive NSCLC, crizotinib therapy is associated with improved survival compared with that of crizotinib-naive controls. ALK rearrangement is not a favourable prognostic factor in advanced NSCLC.

PURPOSE

Emerging evidence suggests that aberrant expression of oncogenes contributes to development of lung malignancy. The thyroid transcription factor 1 (TITF-1) gene functions as a lineage survival gene abnormally expressed in a significant fraction of NSCLCs, in particular lung adenocarcinomas.

EXPERIMENTAL DESIGN

To better characterize TITF-1 abnormality: patterns in NSCLC, we studied TITF-1’s gene copy number using fluorescent in situ hybridization (FISH) and quantitative PCR, as well as its protein expression by immunohistochemistry analysis in a tissue microarray comprised of surgically resected NSCLC (N=321) including 204 adenocarcinomas and 117 squamous cell carcinomas (SCCs). TITF-1 copy number and protein expression were correlated with patients’ clinicopathologic characteristics, and in a subset of adenocarcinomas with EGFR and KRAS mutation status.

RESULTS

We found that increased TITF-1 protein expression was prevalent in lung adenocarcinomas only and was significantly associated with female gender (p<0.001), never smokers (p=0.004), presence of EGFR mutations (p=0.05) and better overall survival (all stages, p=0.0478. stages I and II, p=0.002). TITF-1 copy number gain (CBG) was detected by FISH analysis in both adenocarcinomas (18.9%; high CNG, 8.3%) and SCCs (20.1%; high CNG, 3.0%), and correlated significantly with the protein product (p=0.004) and presence of KRAS mutations (p=0.008) in lung adenocarcinomas. Moreover, multivariate analysis revealed that TITF-1 copy number gain was an independent predictor of poor survival of NSCLC (p=0.039).

CONCLUSIONS

Our integrative study demonstrates that the protein versus genomic expression patterns of TITF-1 have opposing roles in lung cancer prognosis and may occur preferentially in different subsets of NSCLC patients with distinct oncogene mutations.

The etiologic agent of Chagas Disease is the Trypanosoma cruzi, transmitted through blood-sucking insect vectors of the Triatominae subfamily, representing one of the most serious public health concerns in Latin America. There are geographic variations in the prevalence of clinical forms and morbidity of Chagas disease, likely due to genetic variation of the T. cruzi and the host genetic and environmental features. Increasing evidence has supported that inflammatory cytokines and chemokines are responsible for the generation of the inflammatory infiltrate and tissue damage. Moreover, genetic polymorphisms, protein expression levels, and genomic imbalances are associated with disease progression. This paper discusses these key aspects. Large surveys were carried out in Brazil and served as baseline for definition of the control measures adopted. However, Chagas disease is still active, and aspects such as host-parasite interactions, genetic mechanisms of cellular interaction, genetic variability, and tropism need further investigations in the attempt to eradicate the disease.

The epidermal growth factor receptor directed antibody, cetuximab, is an effective clinical therapy for patients with colorectal, head and neck and non-small cell lung cancer patients particularly for those with KRAS and BRAF wild type cancers. Treatment in all patients is limited eventually by the development of acquired resistance but little is known about the underlying mechanism. Here we show, that activation of ERBB2 signaling, either through ERBB2 amplification or through heregulin upregulation, leads to persistent ERK 1/2 signaling and consequently cetuximab resistance. Inhibition of ERBB2 or disruption of ERBB2/ERBB3 heterodimerization restores cetuximab sensitivity in vitro and in vivo. A subset of colorectal cancer patients that exhibit either de novo or acquired resistance to cetuximab based therapy possess ERBB2 amplification or high levels of circulating heregulin. Collectively, these findings identify two distinct resistance mechanisms, both of which promote aberrant ERBB2 signaling, that mediate cetuximab resistance. Moreover, these results suggest that ERBB2 inhibitors, in combination with cetuximanb, represent a rational therapeutic strategy that should be assessed in cetuximab-resistant cancers.

Rationale: Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies have indicated that the endothelial cell proliferation is quasineoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences.

Objectives: To assess whether there is larger-scale genomic instability.

Methods: We performed genome-wide microarray copy number analysis on pulmonary artery endothelial cells and smooth muscle cells isolated from the lungs of patients with PAH.

Measurements and Main Results: Mosaic chromosomal abnormalities were detected in PAEC cultures from five of nine PAH lungs but not in normal (n = 8) or disease control subjects (n = 5). Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female subjects with mosaic loss of the X chromosome, methylation analysis showed that the active X was deleted. One subject also showed completely skewed X-inactivation in the nondeleted cells, suggesting the pulmonary artery endothelial cell population was clonal before the acquisition of the chromosome abnormality.

Conclusions: Our data indicate a high frequency of genetically abnormal subclones within PAH lung vessels and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH.

Background

A plethora of agents are in early stages of development for colorectal cancer, including those that target the insulin-like growth factor receptor (IGF1R) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGF1R tyrosine kinase inhibitor, OSI-906, that could be applied in colorectal cancer (CRC)-specific studies of this agent.

Methods

Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC50 value as sensitive (< 1.5μM), or resistant (>5μM). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by FISH, KRAS/BRAF/PI3K mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were utilized for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against 8 human CRC explants in vivo.

Results

Baseline gene array data from cell lines and xenografts was used to develop a k-Top Scoring Pair (k-TSP) classifier, which in combination with IGFIR FISH and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts.

Conclusions

These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials.

Purpose

The purpose of this study was to characterize insulin-like growth factor-1 receptor (IGF1R) protein expression, mRNA expression, and gene copy number in surgically resected non–small-cell lung cancers (NSCLC) in relation to epidermal growth factor receptor (EGFR) protein expression, patient characteristics, and prognosis.

Patients and Methods

One hundred eighty-nine patients with NSCLC who underwent curative pulmonary resection were studied (median follow-up, 5.3 years). IGF1R protein expression was evaluated by immunohistochemistry (IHC) with two anti-IGF1R antibodies (n = 179). EGFR protein expression was assessed with PharmDx kit. IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 114 corresponding fresh-frozen samples. IGF1R gene copy number was assessed by fluorescent in situ hybridization using customized probes (n = 181).

Results

IGF1R IHC score was higher in squamous cell carcinomas versus other histologies (P < .001) and associated with stage (P = .03) but not survival (P = .46). IGF1R and EGFR protein expression showed significant correlation (r = 0.30; P < .001). IGF1R gene expression by qRT-PCR was higher in squamous cell versus other histologies (P = .006) and did not associate with other clinical features nor survival (P = .73). Employing criteria previously established for EGFR copy number, patients with IGF1R amplification/high polysomy (n = 48; 27%) had 3-year survival of 58%, patients with low polysomy (n = 87; 48%) had 3-year survival of 47% and patients with trisomy/disomy (n = 46; 25%) had 3-year survival of 35%, respectively (P = .024). Prognostic value of high IGF1R gene copy number was confirmed in multivariate analysis.

Conclusion

IGF1R protein expression is higher in squamous cell versus other histologies and correlates with EGFR expression. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number harbors positive prognostic value.

BACKGROUND

Oncogenic fusion genes consisting of EML4 and anaplastic lymphoma kinase (ALK) are present in a subgroup of non–small-cell lung cancers, representing 2 to 7% of such tumors. We explored the therapeutic efficacy of inhibiting ALK in such tumors in an early-phase clinical trial of crizotinib (PF-02341066), an orally available small-molecule inhibitor of the ALK tyrosine kinase.

METHODS

After screening tumor samples from approximately 1500 patients with non–small-cell lung cancer for the presence of ALK rearrangements, we identified 82 patients with advanced ALK-positive disease who were eligible for the clinical trial. Most of the patients had received previous treatment. These patients were enrolled in an expanded cohort study instituted after phase 1 dose escalation had established a recommended crizotinib dose of 250 mg twice daily in 28-day cycles. Patients were assessed for adverse events and response to therapy.

RESULTS

Patients with ALK rearrangements tended to be younger than those without the rearrangements, and most of the patients had little or no exposure to tobacco and had adenocarcinomas. At a mean treatment duration of 6.4 months, the overall response rate was 57% (47 of 82 patients, with 46 confirmed partial responses and 1 confirmed complete response); 27 patients (33%) had stable disease. A total of 63 of 82 patients (77%) were continuing to receive crizotinib at the time of data cutoff, and the estimated probability of 6-month progression-free survival was 72%, with no median for the study reached. The drug resulted in grade 1 or 2 (mild) gastrointestinal side effects.

CONCLUSIONS

The inhibition of ALK in lung tumors with the ALK rearrangement resulted in tumor shrinkage or stable disease in most patients.

Lung cancer is usually disseminated at diagnosis making prognosis poor. Smokers are at high risk for lung cancer and are targets for prevention and early detection strategies. Sputum is a potential source for lung cancer biomarkers, but no test is currently available with sufficient sensitivity and specificity for clinical screening utility. Chromosomal aneusomy (CA) was measured in sputum samples collected prospectively from 100 incident lung cancer cases and 96 controls matched on age, gender, and date of collection. The CA-FISH assay was performed using a four-target DNA FISH probe including EGFR, MYC, 5p15 and CEP6. Sensitivity for a positive CA-FISH assay (abnormal for ≥ 2 of the 4 markers) was substantially higher for samples collected within 18 months (76%) than >18 months before lung cancer diagnosis (31%). Specificity for a positive FISH by this same definition was 85%. Among subjects providing sputum sample within 18 months before diagnosis, sensitivity was higher for squamous cell cancers (94%) than for other histologic types (69%). The adjusted odds ratios for specimens collected within 18 months of cancer diagnosis were higher using the CA-FISH assay (OR=27.2, 95% CI 7.8 to 94.1) than previous studies assessing cytologic atypia (OR=2.3, CI 0.8 to 6.4) or gene promoter methylation (OR=6.5; CI 1.2 to 35.5). In conclusion, chromosomal aneusomy in sputum is a promising biomarker for prediction of lung cancer risk. Evaluation of the 4-DNA targets was more effective than any single marker and had highest sensitivity for samples collected ≤ 18 months to lung cancer diagnosis and patients diagnosed with squamous cell carcinoma.

Lung cancer is a complex spectrum of diseases characterized by extensive genomic instability, which can be detected among both histological subtypes and different foci within a tumor. Conventional and cutting edge investigative technologies have uncovered scores of genomic changes in individual specimens that have been used to characterize specific molecular subtypes. Oncogenes with predominant roles in lung cancer include EGFR, MYC and RAS family members, PIK3CA, NKX2-1 and ALK; tumor suppressor genes include TP53, RB1, CDKN2, and a cluster of genes mapped at 3p. MicroRNA regulators also have been linked to lung cancer. The functional role of the recurrent genomic changes in lung tumors has been explored, which has led to a better understanding of cell growth, differentiation and apoptotic pathways. Additionally, this knowledge has supported the development of novel therapeutics and translational tools for selection of patients for personalized therapy.

Purpose

Previous studies in non–small-cell lung cancer (NSCLC) have demonstrated a wide variation in responsiveness to epidermal growth factor receptor (EGFR) –targeting agents and in genetic aberrancies of the EGFR pathway according to ethnic background, most notably a higher frequency of activating EGFR mutations among East-Asian patients. We investigated the frequency of EGFR pathway aberrancies among African American patients with NSCLC, for whom limited information presently exists.

Patients and Methods

EGFR fluorescent in situ hybridization (FISH) was performed on archived tissues from 53 African American patients. Extracted DNA was sequenced for mutational analysis of EGFR exons 18 to 21 and KRAS exon 2. Results were compared by multivariate analysis to an historical control cohort of 102 white patients with NSCLC.

Results

African Americans were significantly less likely to harbor activating mutations of EGFR than white patients (2% v 17%; P = .022). Only one EGFR mutation was identified, a novel S768N substitution. EGFR FISH assay was more frequently positive for African Americans than for white patients (51% v 32%; P = .018). KRAS mutational frequency did not differ between the groups (23% v 21%; P = .409).

Conclusion

African American patients with NSCLC are significantly less likely than white counterparts to harbor activating mutations of EGFR, which suggests that EGFR tyrosine kinase inhibitors (TKIs) are unlikely to yield major remissions in this population. Our findings add to a growing body of evidence that points to genetic heterogeneity of the EGFR pathway in NSCLC among different ethnic groups and that underscores the need for consideration of these differences in the design of future trials of agents that target the EGFR pathway.

This review summarizes the chromosomal changes detected by molecular cytogenetic approaches in esophageal squamous cell carcinoma (ESCC), the ninth most common malignancy in the world. Whole genome analyses of ESCC cell lines and tumors indicated that the most frequent genomic gains occurred at 1, 2q, 3q, 5p, 6p, 7, 8q, 9q, 11q, 12p, 14q, 15q, 16, 17, 18p, 19q, 20q, 22q and X, with focal amplifications at 1q32, 2p16-22, 3q25-28, 5p13-15.3, 7p12-22, 7q21-22, 8q23-24.2, 9q34, 10q21, 11p11.2, 11q13, 13q32, 14q13-14, 14q21, 14q31-32, 15q22-26, 17p11.2, 18p11.2-11.3 and 20p11.2. Recurrent losses involved 3p, 4, 5q, 6q, 7q, 8p, 9, 10p, 12p, 13, 14p, 15p, 18, 19p, 20, 22, Xp and Y. Gains at 5p and 7q, and deletions at 4p, 9p, and 11q were significant prognostic factors for patients with ESCC. Gains at 6p and 20p, and losses at 10p and 10q were the most significant imbalances, both in primary carcinoma and in metastases, which suggested that these regions may harbor oncogenes and tumor suppressor genes. Gains at 12p and losses at 3p may be associated with poor relapse-free survival. The clinical applicability of these changes as markers for the diagnosis and prognosis of ESCC, or as molecular targets for personalized therapy should be evaluated.