Person:

Chirieac, Lucian

Mesothelioma is diagnosed in approximately 2,500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die from their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intra-peritoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, based on tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established which showed loss of both Tsc1 and Tp53, with mTORC1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival, and a 95–99% reduction in mesothelioma tumor volume, in comparison to vehicle-treated mice. To see if TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined 9 human mesothelioma cell lines, and found that 4 of 9 showed persistent activation of mTORC1 though none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma and suggesting that it is a potential therapeutic target.

Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.

INTRODUCTION

ALK gene rearrangements occur in ~5% of lung adenocarcinomas (ACA), leading to ALK overexpression and predicting response to targeted therapy. Fluorescence in situ hybridization (FISH) is the gold standard for detection of ALK rearrangements in lung ACA but requires specialized equipment and expertise. Immunohistochemistry (IHC) for ALK protein overexpression is a promising screening modality, with reports of newer antibodies showing excellent sensitivity and specificity for ALK-rearranged lung ACA.

METHODS

In this study, we analyze ALK IHC (5A4 clone) in 186 cases from our clinical service and compare with ALK FISH and EGFR and KRAS mutation status.

RESULTS

Twelve cases had concordant ALK protein overexpression and ALK rearrangement by FISH. Three ALK-rearranged cases lacked ALK protein expression. Of these discrepant cases, one had a coexisting EGFR mutation and a subtle “atypical” ALK rearrangement with a break in the 5’ centromeric portion of the FISH probe. One case had a concurrent BRAF mutation; followup testing on a metastasis revealed absence of the ALK-rearrangement with persistent BRAF mutation. In one ALK-rearranged, protein negative case, very limited tissue remained for ALK IHC, raising the possibility of false negativity due to protein expression heterogeneity. Importantly, ALK protein expression was detected in one case initially thought not to have an ALK rearrangement. In this case, FISH was falsely negative due to interference by benign reactive nuclei. After correcting for these cases, ALK IHC was 93% sensitive and 100% specific as compared to FISH.

CONCLUSIONS

ALK IHC improves the detection of ALK rearrangements when used together with FISH, and its use in lung adenocarcinoma genetic testing algorithms should be considered.

In a subset of lung adenocarcinomas the epidermal growth factor receptor (EGFR) is activated by kinase domain mutations and/or gene amplification, but the interaction between the two types of abnormalities is complex and unclear. We selected to study 99 consecutive never-smoking women of East Asian origin with lung adenocarcinomas that were characterized by histologic subtype. We analyzed EGFR mutations by PCR-capillary sequencing, EGFR copy number abnormalities by fluorescence and chromogenic in situ hybridization and quantitative PCR, and EGFR expression by immunohistochemistry with both specific antibodies against exon 19 deletion-mutated EGFR and total EGFR. We compared molecular and clinicopathologic features with disease-free survival. Lung adenocarcinomas with EGFR amplification had significantly more EGFR exon 19 deletion mutations than adenocarcinomas with disomy, low and high polysomy (100% v 54%, P=0.009). EGFR amplification occurred invariably on the mutated and not the wildtype allele (median mutated:wildtype ratios 14.0 v .33, P=0.003), was associated with solid histology (P=0.008), and advanced clinical stage (P=0.009). EGFR amplification was focally distributed in lung cancer specimens, mostly in regions with solid histology. Patients with EGFR amplification had a significantly worse outcome in univariate analysis (median disease-free survival 16 v 31 months, P=0.01) and when adjusted for stage (P=0.027). Lung adenocarcinomas with EGFR amplification have a unique association with exon 19 deletion mutations and demonstrate distinct clinicopathologic features associated with a significantly worsened prognosis. In these cases, EGFR amplification is heterogeneously distributed, mostly in areas with a solid histology.

Lineage survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development.1,2 Here we show that a peak of genomic amplification on chromosome 3q26.33, found in squamous cell carcinomas (SCCs) of the lung and esophagus, contains the transcription factor gene SOX2—which is mutated in hereditary human esophageal malformations3 and necessary for normal esophageal squamous development4, promotes differentiation and proliferation of basal tracheal cells5 and co-operates in induction of pluripotent stem cells.6,7,8 SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These observations identify SOX2 as a novel lineage survival oncogene in lung and esophageal SCC.

Many patients with stage I non-small cell lung carcinoma will develop recurrence following surgical excision. Sox2 is a marker of embryonic stem cell pluripotency that is associated with aggressive tumor behavior and is expressed in a subset of lung adenocarcinomas. We hypothesized that Sox2 expression may provide prognostic information in early stage lung adenocarcinomas. We evaluated formalin-fixed paraffin embedded tissue from 104 stage I lung adenocarcinomas resected between 1997 and 2000. Sox2 expression was analyzed by immunohistochemistry and compared to clinicopathologic features, time-to-progression (TTP) and overall survival (OS). Sox2 expression was detected in 50% of cases and was more frequent in tumors from older and male patients but not significantly associated with smoking status, tumor stage, grade, or histologic subtype. Compared to Sox2-negative tumors, Sox2 expression predicted a shorter TTP (49% versus 82% at 5 years; P = 0.0006) and shorter OS (54% versus 79% at 5 years; P = 0.004). By multivariate analysis, Sox2 expression predicted a greater risk of progression among men (hazard ratio [HR] 5.6; 95% CI 2.3 to 13.8) and women (HR 2.1; 95% CI 0.8 to 5.7). Sox2 expression was associated with significantly shorter OS among men (HR 2.5; 95% CI 1.2 to 5.1), but not in women. Sox2 appears to be an independent predictor of poor outcome in stage I lung adenocarcinomas and may help stratify patients at increased risk for recurrence.

Background Although grading has prognostic significance for many tumor types, a prognostically significant grading system for lung adenocarcinoma has not yet been established. The aim of this study was to evaluate histologic characteristics included in tumor grading systems, establish optimal cutoff values that have the strongest association with overall survival, and develop a grading system incorporating the histopathologic characteristics that we found to have prognostic significance in patients with lung adenocarcinoma.

Methods We studied lung adenocarcinomas from 85 consecutive patients, and evaluated the percentage of solid pattern (as a reflection of tumor architecture), the degree of cytologic atypia, and the mitotic count.

Results In univariate analysis, overall survival was associated significantly with sex (P=0.045), age (P=0.0008), tumor status (P<0.0001), node status (P=0.02), solid pattern (P=0.046) and cytologic atypia (P=0.01), but not with mitotic count (P=0.26). Based on optimal cutoff values, we found that a solid pattern ≥ 90% and severe cytologic atypia were the best discriminators of worse outcome. A grading score, computed as the sum of the architecture score and cytologic atypia score (2=well differentiated, 3=moderately differentiated, 4=poorly differentiated), was a significant predictor of overall survival in univariate analysis (median overall survival times 72.4, 39.5, and 8.7 months for well, moderately and poorly differentiated adenocarcinoma, respectively P=0.0001). Moreover, grading was an independent predictor of survival in multivariate analysis (P=0.002).

Conclusions We describe a grading system that incorporates the percentage of solid pattern and degree of the cytologic atypia that is an independent predictor of survival in patients with lung adenocarcinoma.

Somatic alterations in cellular DNA underlie almost all human cancers1. The prospect of targeted therapies2 and the development of high-resolution, genome-wide approaches3–8 are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumors (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ~12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.

BACKGROUND There are no reliable markers able to identify patients with non-small cell lung cancer (NSCLC) likely to metastasize to the brain. We investigated associations between immunohistochemical markers and development of brain metastases in patients with NSCLC.

METHODS We performed a hospital-based, case-control study of patients with newly diagnosed NSCLC between 1989 and 2003 that developed brain metastases who had available pathology material from both primary NSCLC and brain metastases. The control patients had NSCLC and no evidence of brain metastases. We examined NSCLC for expression of Ki-67, caspase-3, VEGF-A, VEGF-C, E-cadherin and EGFR in 54 surgical pathology specimens using immunohistochemistry and evaluated associations with development of brain metastases.

RESULTS Brain metastases developed after a median time of 12.5 months (range 1.7-89.4 months) from the diagnosis of NSCLC. A significantly increased risk of developing brain metastases was associated with patients who had high Ki-67 (adjusted odds ratio 12.2, 95% CI, 2.4 to 70.4, P<0.001), low caspase-3 (adjusted odds ratio 43.0, 95% CI, 5.3 to >100, P<0.001), high VEGF-C (adjusted odds ratio 14.6, 95% CI, 2.0 to >100, P<0.001), and low E-cadherin (adjusted odds ratio 3.6, 95% CI, 0.9 to 16.4, P=0.05), in the primary NSCLC. No significant risk was observed with VEGF-A and EGFR. A high Ki-67 was also associated with a shorter overall survival (P=0.04).

CONCLUSIONS Patients with NSCLC and high Ki-67, low caspase-3, high VEGF-C, and low E-cadherin in their tumors may benefit from close surveillance since they may have an increased risk of developing brain metastases.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.