Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma

BACKGROUND: Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be 'major', characterized by a single tumor showing two different histologic types, and 'minor', due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas. METHODS: 590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR. RESULTS: 10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components. CONCLUSION: Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes

Aberrant signaling through the HER2-ERK1/2 pathway is predictive of reduced disease-free and overall survival in early stage non-small cell lung cancer (NSCLC) patients

Background: Purpose of this study was to evaluate the contribution of the Extracellular-regulated protein kinase (ERK)-1/2 pathway to oncogenic signaling elicited by the tyrosine kinase receptor HER2 in Non-Small Cell Lung Cancer (NSCLC) and to assess the prognostic value of these oncoproteins in NSCLC patients. Methods: Immunohistochemistry was performed to determine expression and activation of HER2 and ERK1/2 (detected by phosphorylation of Y1248 and T202/Y204, respectively) using Tissue Micro Arrays (TMA) containing matched normal and neoplastic tissues from 132 NSCLC patients. Survival analysis was carried out using the Kaplan-Meier method. Univariate and multivariate analysis were used to evaluate the prognostic value of pERK1/2, pHER2 and a combination thereof with clinical-pathological parameters such as age, lymph node status (N), size (T), stage (TNM) and grade. Results: We found that HER2 was overexpressed in 33/120 (27%) and activated in 41/114 (36%) cases; ERK1/2 was activated in 44/102 (43%) cases. A direct association was found between pERK1/2 and pHER2 (23/41; p=0.038). In addition, patients positive for pERK1/2 and for both pHER2 and pERK1/2 showed significantly worse overall survival (OS) and disease-free survival (DFS) compared with negative patients. Univariate and multivariate analysis of patients' survival revealed that positivity for pHER2-pERK1/2 and for pERK1/2 alone were independent prognostic factors of poor survival in NSCLC patients. In particular, this association was significantly important for DFS in stage I+II patients. Conclusion: This study provides evidence that activated ERK1/2 and/or the combined activation of HER2 and ERK1/2 are good indicators of poor prognosis in NSCLC patients, not only in unselected patients but also in early stage disease

Clinical and Histological features of ALK-rearranged and EGFR-mutated patients.

<p>NA*: not available; ALK: Anaplastic Lymphoma Kinase, EGFR: Epidermal Growth Factor Receptor, R: ALK-rearranged, a R: ALK-rearranged with atypical FISH pattern.</p><p><b>(*</b>^<b>1</b><b>): % of neoplastic cells with ALK-R.</b></p><p>Clinical and Histological features of ALK-rearranged and EGFR-mutated patients.</p

Clinical and pathological features of patients harboring mut-EGFRand ALK-R.

<p>NA: not available; Wt: wild type, mADCs: mixed adenocarcinomas, AdSqLCs: adenosquamous lung carcinoma,ALK: Anaplastic Lymphoma Kinase, EGFR: Epidermal Growth Factor Receptor.</p><p>Clinical and pathological features of patients harboring mut-EGFRand ALK-R.</p

Clinical and pathological features of patients.

<p>NA: not available; Wt: wild type, mADCs: mixed adenocarcinomas, AdSqLCs: adenosquamous lung carcinoma,ALK: Anaplastic Lymphoma Kinase, EGFR: Epidermal Growth Factor Receptor.</p><p>Clinical and pathological features of patients.</p

Oncogenic Role of the E3 Ubiquitin Ligase NEDD4-1, a PTEN Negative Regulator, in Non-Small-Cell Lung Carcinomas

Loss of the PTEN tumor suppressor gene occurs frequently in non-small-cell lung carcinoma (NSCLC), although neither genetic alterations nor epigenetic silencing are significant predictors of PTEN protein levels. Since recent reports implicated neural precursor cell expressed, developmentally down-regulated 4-1 (NEDD4-1) as the E3 ubiquitin ligase that regulates PTEN stability, we investigated the role of NEDD4-1 in the regulation of PTEN expression in cases of NSCLC. Our findings indicate that NEDD4-1 plays a critical role in the development of NSCLC and provides novel insight on the mechanisms that contribute to inactivate PTEN in lung cancer. Immunohistochemical analysis on tissue microarrays containing 103 NSCLC resections revealed NEDD4-1 overexpression in 80% of tumors, which correlated with the loss of PTEN protein (n = 98; P < 0.001). Accordingly, adoptive NEDD4-1 expression in NSCLC cells decreased PTEN protein stability, whereas knock-down of NEDD4-1 expression decreased PTEN ubiquitylation and increased PTEN protein levels. In 25% of cases, NEDD4-1 overexpression was due to gene amplification at 15q21. In addition, manipulation of NEDD4-1 expression in different lung cell systems demonstrated that suppression of NEDD4-1 expression significantly reduced proliferation of NSCLC cells in vitro and tumor growth in vivo, whereas NEDD4-1 overexpression facilitated anchorage-dependent and independent growth in vitro of nontransformed lung epithelial cells that lack pRB and TP53 (BEAS-2B). NEDD4-1 overexpression also augmented the tumorigenicity of lung cancer cells that have an intact PTEN gene (NCI-H460 cells)

Representative results of the different areas of a mADC with coexistence of del E746-A750 EGFR and ALK-R (case 4).

<p>Tumoral area of standard H&E slide with microphotographs of each single growth pattern is identified by different colored arrows/box. Acinar pattern: A, B, C, D. Papillary pattern: E, F, G, H. Lepidic pattern: I, L, M, N. Solid pattern: O, P, Q, R. Solid-signet ring cell pattern: S, T, U, V. In the first column: Hematoxylin and Eosin staining for each histological pattern (20X). In the second column: del E746-A750 EGFR immunopositivity in all patterns. (20X) The black arrows indicate the positive staining: acinar (B), papillary (F), lepidic (L), solid (P), signet (T). In the third column: ALK IHC results, acinar (C), papillary (G) and lepidic (M) patterns are negative; solid(Q) and solid-signet ring cell (U) patterns are positive, indicated by the black arrows. In the fourth column: ALK FISH results, acinar (D), papillary(H) and lepidic(N) are negative; solid pattern (R) shows the classic break-apart pattern with one fusion signal and two separated orange and green signals, indicated by the white arrows. Solid-signet ring cell pattern (V) shows the atypical FISH pattern with fused signals and one single red signal without a corresponding green signal, indicated by the white arrows.</p

Adjuvant anastrozole versus exemestane versus letrozole, upfront or after 2 years of tamoxifen, in endocrine-sensitive breast cancer (FATA-GIM3): a randomised, phase 3 trial

Background: Uncertainty exists about the optimal schedule of adjuvant treatment of breast cancer with aromatase inhibitors and, to our knowledge, no trial has directly compared the three aromatase inhibitors anastrozole, exemestane, and letrozole. We investigated the schedule and type of aromatase inhibitors to be used as adjuvant treatment for hormone receptor-positive early breast cancer. Methods: FATA-GIM3 is a multicentre, open-label, randomised, phase 3 trial of six different treatments in postmenopausal women with hormone receptor-positive early breast cancer. Eligible patients had histologically confirmed invasive hormone receptor-positive breast cancer that had been completely removed by surgery, any pathological tumour size, and axillary nodal status. Key exclusion criteria were hormone replacement therapy, recurrent or metastatic disease, previous treatment with tamoxifen, and another malignancy in the previous 10 years. Patients were randomly assigned in an equal ratio to one of six treatment groups: oral anastrozole (1 mg per day), exemestane (25 mg per day), or letrozole (2·5 mg per day) tablets upfront for 5 years (upfront strategy) or oral tamoxifen (20 mg per day) for 2 years followed by oral administration of one of the three aromatase inhibitors for 3 years (switch strategy). Randomisation was done by a computerised minimisation procedure stratified for oestrogen receptor, progesterone receptor, and HER2 status; previous chemotherapy; and pathological nodal status. Neither the patients nor the physicians were masked to treatment allocation. The primary endpoint was disease-free survival. The minimum cutoff to declare superiority of the upfront strategy over the switch strategy was assumed to be a 2% difference in disease-free survival at 5 years. Primary efficacy analyses were done by intention to treat; safety analyses included all patients for whom at least one safety case report form had been completed. Follow-up is ongoing. This trial is registered with the European Clinical Trials Database, number 2006-004018-42, and ClinicalTrials.gov, number NCT00541086. Findings: Between March 9, 2007, and July 31, 2012, 3697 patients were enrolled into the study. After a median follow-up of 60 months (IQR 46–72), 401 disease-free survival events were reported, including 211 (11%) of 1850 patients allocated to the switch strategy and 190 (10%) of 1847 patients allocated to upfront treatment. 5-year disease-free survival was 88·5% (95% CI 86·7–90·0) with the switch strategy and 89·8% (88·2–91·2) with upfront treatment (hazard ratio 0·89, 95% CI 0·73–1·08; p=0·23). 5-year disease-free survival was 90·0% (95% CI 87·9–91·7) with anastrozole (124 events), 88·0% (85·8–89·9) with exemestane (148 events), and 89·4% (87·3 to 91·1) with letrozole (129 events; p=0·24). No unexpected serious adverse reactions or treatment-related deaths occurred. Musculoskeletal side-effects were the most frequent grade 3–4 events, reported in 130 (7%) of 1761 patients who received the switch strategy and 128 (7%) of 1766 patients who received upfront treatment. Grade 1 musculoskeletal events were more frequent with the upfront schedule than with the switch schedule (924 [52%] of 1766 patients vs 745 [42%] of 1761 patients). All other grade 3–4 adverse events occurred in less than 2% of patients in either group. Interpretation: 5 years of treatment with aromatase inhibitors was not superior to 2 years of tamoxifen followed by 3 years of aromatase inhibitors. None of the three aromatase inhibitors was superior to the others in terms of efficacy. Therefore, patient preference, tolerability, and financial constraints should be considered when deciding the optimal treatment approach in this setting. Funding: Italian Drug Agency