The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance.

Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were 'primed for death'. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ∼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.

AXL regulates mesothelioma proliferation and invasiveness.

Mesothelioma is an asbestos-associated and notoriously chemotherapy-resistant neoplasm. Activation of the receptor tyrosine kinases (RTKs), epidermal growth factor receptor and MET, has been described in subsets of mesothelioma, suggesting that TKs might represent therapeutic targets in this highly lethal disease. We employed proteomic screening by phosphotyrosine immunoaffinity purification and tandem mass spectrometry to characterize RTK activation in mesothelioma cell lines. These assays demonstrated expression and activation of the AXL protein, which is an RTK with known oncogenic properties in non-mesothelial cancer types. AXL was expressed and activated strongly in 8 of 9 mesothelioma cell lines and 6 of 12 mesothelioma biopsies, including each of 12 mesotheliomas with spindle-cell histology. Somatic AXL mutations were not found, but all mesotheliomas expressed an alternatively spliced AXL transcript with in-frame deletion of exon 10, and six of seven mesothelioma cell lines expressed the AXL ligand, growth arrest-specific 6 (GAS6). GAS6 expression appeared to be functionally relevant, as indicated by modulation of AXL tyrosine phosphorylation by knockdown of endogeneous GAS6, and by administration of exogenous GAS6. AXL silencing by lentivirus-mediated short hairpin RNA suppressed mesothelioma migration and cellular proliferation due to G1 arrest. The AXL inhibitor DP-3975 inhibited cell migration and proliferation in mesotheliomas with strong AXL activation. DP-3975 response in these tumors was characterized by inhibition of PI3-K/AKT/mTOR and RAF/MAPK signaling. AXL inhibition suppressed mesothelioma anchorage-independent growth, with reduction in colony numbers and size. These studies suggest that AXL inhibitors warrant clinical evaluation in mesothelioma.

Mutations in the LKB1 tumour suppressor are frequently detected in tumours from Caucasian but not Asian lung cancer patients.

Somatic mutations of LKB1 tumour suppressor gene have been detected in human cancers including non-small cell lung cancer (NSCLC). The relationship between LKB1 mutations and clinicopathological characteristics and other common oncogene mutations in NSCLC is inadequately described. In this study we evaluated tumour specimens from 310 patients with NSCLC including those with adenocarcinoma, adenosquamous carcinoma, and squamous cell carcinoma histologies. Tumours were obtained from patients of US (n=143) and Korean (n=167) origin and screened for LKB1, KRAS, BRAF, and EGFR mutations using RT-PCR-based SURVEYOR-WAVE method followed by Sanger sequencing. We detected mutations in the LKB1 gene in 34 tumours (11%). LKB1 mutation frequency was higher in NSCLC tumours of US origin (17%) compared with 5% in NSCLCs of Korean origin (P=0.001). They tended to occur more commonly in adenocarcinomas (13%) than in squamous cell carcinomas (5%) (P=0.066). LKB1 mutations associated with smoking history (P=0.007) and KRAS mutations (P=0.042) were almost mutually exclusive with EGFR mutations (P=0.002). The outcome of stages I and II NSCLC patients treated with surgery alone did not significantly differ based on LKB1 mutation status. Our study provides clinical and molecular characteristics of NSCLC, which harbour LKB1 mutations.

A microaliquoting technique for precise histological annotation and optimization of cell content in frozen tissue specimens.

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and "front and back"' section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.

Insulin receptor activation in solitary fibrous tumours.

Solitary fibrous tumours (SFTs) are known to overexpress insulin-like growth factor 2 (IGF-2). The down-stream oncogenic pathways of IGF-2, however, are not clear. Here we report uniform activation of the insulin receptor (IR) pathway in SFTs, which are mesenchymal tumours frequently associated with hypoglycaemia. Whereas the IR and its downstream signalling pathways were constitutively activated in SFTs, insulin-like growth factor 1 receptor (IGF-1R) was not expressed in these tumours. We also find that SFT cells secrete IGF-2 and proliferate in serum-free medium, consistent with an IGF-2/IR autocrine loop. The aetiological relevance of IGF-2 is supported by expression of IR-A, the IR isoform with high affinity for IGF-2, in all SFTs. Our studies suggest that IR activation plays an oncogenic role in SFTs.

Expression patterns of inhibitor of apoptosis proteins in malignant pleural mesothelioma.

Inhibitor of apoptosis proteins (IAPs) comprise a family of structurally similar proteins, five of which are widely studied in the context of cancer: IAP-1/MIHC/cIAP2, IAP-2/MIHB/cIAP1, livin/ML-IAP/KIAP, survivin, and XIAP/MIHA/hILP. IAPs are overexpressed by most neoplasms, promote tumour cell survival after a wide variety of apoptotic stimuli, and frequently have gene and/or protein expression patterns associated with a relatively poor prognosis. However, many IAPs are also expressed by normal tissues, can facilitate apoptotic cell death, and have expression patterns associated with a relatively favourable prognosis in some cases. The result is that the precise role(s) of IAPs in human tumours is not exactly known. It has been previously reported that IAP-1 is overexpressed in malignant pleural mesothelioma (MPM) and is responsible for a large degree of the resistance of cultured MPM cells to cisplatin. Given the high homology of IAP family members, it is likely that other IAPs will be important in MPM. In the present study, the gene and protein expression patterns of IAP-1, IAP-2, survivin, livin, and XIAP have been determined in MPM cell lines (n=9) and a large number of MPM tumours using high-density oligonucleotide microarrays (n=40) and an MPM tissue array (n=66). Human tumours were linked to clinical data and it was found that IAP-1 and survivin mRNA expression patterns were associated with a relatively shorter patient survival, while those of XIAP and livin were associated with a relatively longer patient survival. Abundant protein for all IAPs was also detected in MPM tumours, where they were expressed primarily in the cytoplasm. Only IAP-1 and livin protein was expressed in the nucleus of MPM tumours. These results provide the rationale for additional study of this gene family in MPM and cancer in general.

Inhibitor of apoptosis proteins are regulated by tumour necrosis factor-alpha in malignant pleural mesothelioma.

Inhibitor of apoptosis proteins (IAPs) are overexpressed by most neoplasms and promote tumour cell survival after a wide variety of apoptotic stimuli elicited via intrinsic (ie mitochondrial) and extrinsic (ie death receptor) pathways. It has previously been reported that one of these proteins, IAP-1(MIHC/cIAP2), is overexpressed in malignant pleural mesothelioma (MPM) and is responsible for a large degree of the resistance of cultured MPM cells to cisplatin. Subsequent analysis in a larger number of human tumours revealed that additional IAPs (eg IAP-2/MIHB/cIAP1, livin/ML-IAP/KIAP, survivin, and XIAP/MIHA/hILP) are also overexpressed in MPM and, with the exception of IAP-2, have expression patterns that correlate with prognosis. In the present study, potential regulatory mechanisms of IAP genes in MPM were investigated and it was found that tumour necrosis factor-alpha (TNF-alpha) can increase mRNA and protein levels of IAP-1, IAP-2, and XIAP, but not livin or survivin in MPM cell lines (n=4). It was also found that IAP gene expression levels are increased concomitantly with translocation to the nucleus of the TNF-responsive transcription factor NF-kappaB. Co-incubation of MPM cells with TNF-alpha and pyrrolidine dithiocarbamate (PDTC), an NF-kappaB inhibitor, prevented TNF-mediated up-regulation of IAP gene expression levels. In survival studies, TNF-alpha was not toxic to MPM cells at any concentration examined. However, MPM cells exposed to TNF-alpha were twice as resistant to cisplatin in dose response survival assays compared with unstimulated controls and were found to have a significantly greater fraction of surviving cells at multiple cisplatin concentrations (p<0.0087). Finally, it was found that levels of circulating TNF-alpha were statistically significantly (p=0.031) (median 312.5 pg/ml) higher in MPM patients (n=6) prior to surgical tumour debulking compared with those after surgery (median 0 pg/ml). These results when combined with previous observations by our laboratory and others strongly suggest that IAPs act synergistically with TNF family members to promote survival of MPM tumour cells after exposure to cisplatin and possibly other chemotherapeutic drugs.

K-ras activation generates an inflammatory response in lung tumors.

Activating mutations in K-ras are one of the most common genetic alterations in human lung cancer. To dissect the role of K-ras activation in bronchial epithelial cells during lung tumorigenesis, we created a model of lung adenocarcinoma by generating a conditional mutant mouse with both Clara cell secretory protein (CC10)-Cre recombinase and the Lox-Stop-Lox K-ras(G12D) alleles. The activation of K-ras mutant allele in CC10 positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately adenocarcinoma. Surprisingly, K-ras activation in the bronchiolar epithelium is associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response with a median survival of 8 weeks. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that increased significantly with age. Cell lines derived from these tumors directly produced MIP-2, LIX and KC. This model demonstrates that K-ras activation in the lung induces the elaboration of inflammatory chemokines and provides an excellent means to further study the complex interactions between inflammatory cells, chemokines and tumor progression.

Histone deacetylation is directly involved in desilencing the expression of the catalytic subunit of telomerase in normal lung fibroblast.

The regulation of telomerase expression in normal cells is poorly understood. Moreover, the molecular mechanism underlying tumor-specific expression of telomerase remains unclear. We investigated the link between histone deacetylation and telomerase activity in normal lung and lung tumor cells. Four non-small-cell lung cancer (NSCLC) lines and one normal lung fibroblast line were tested for telomerase activity with or without Trichostatin A(TSA). The telomerase activity and the expression of telomerase associated components were determined by TRAP assay, RT-PCR analysis and Western blot analysis. All 4 NSCLC cell lines exposed to 1 microM TSA for 24 h had no change in telomerase activity or hTERT mRNA level. Telomerase activity was very low in normal lung fibroblasts (mrc-9) until exposed to 1 microM TSA for 24 h, at which time telomerase activity was readily detectable, with concomitant upregulation of hTERT mRNA (10-fold). The level of other telomerase associated components (hTER and TP1) were unaltered. Furthermore, 1 microM TSA exposure for 24 h did not alter the level of c-Myc or p21 mRNA. Immunodetection reveled that hTERT protein expression increased (approximately 6 fold) compared to c-Myc, p21, or gelsolin. The effect of TSA on hTERT expression is independent of DNA methylation as judged by 5-azacytidine (5aza) treatment. TSA effect on mrc-9 cells is unaltered even in the presence of 200 microg/ml cyclohexamide, suggesting a direct inhibition of histone deacetylation. Collectively, our study indicates that inhibition of histone deacetylation selectively regulates the transcriptional derepression of telomerase catalytic subunit in normal lung fibroblast cells compared to lung tumor cells.

Metabolic significance of the pattern, intensity and kinetics of 18F-FDG uptake in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma is an aggressive neoplasm with a highly variable course. This pilot study evaluated the significance of the pattern, intensity and kinetics of 18F-FDG uptake in mesothelioma in the context of histopathology and surgical staging. METHODS: Sixteen consecutive patients with pleural disease on CT scan underwent 18F-FDG imaging. Imaging was performed with a dual detector gamma camera operating in coincidence mode. Semiquantitative image analysis was performed by obtaining lesion-to-background ratios (18F-FDG uptake index) and calculating the increment of 18F-FDG lesion uptake over time (malignant metabolic potential index (MMPi)). RESULTS: Twelve patients had histologically proven malignant mesotheliomas (10 epithelial, two sarcomatoid). Thirty two lesions were positive for tumour. Patterns of uptake matched the extent of pleural and parenchymal involvement observed on CT scanning and surgery. Mean (SD) 18F-FDG uptake index for malignant lesions was 3.99 (1.92), range 1.5-9.46. Extrathoracic spread and metastases had higher 18F-FDG uptake indices (5.17 (2)) than primary (3.42 (1.52)) or nodal lesions (2.99 (1)). No correlation was found between histological grade and stage. The intensity of lesion uptake had poor correlation with histological grade but good correlation with surgical stage. 18F-FDG lesion uptake increased over time at a higher rate in patients with more advanced disease. The MMPi was a better predictor of disease aggressiveness than the histological grade. CONCLUSIONS: This pilot study suggests that the pattern, intensity, and kinetics of 18F-FDG uptake in mesothelioma are good indicators of tumour aggressiveness and are superior to the histological grade in this regard.

Lung cancer. 6: The case for limited surgical resection in non-small cell lung cancer.

The choice between conservative resection or standard anatomical resection for stage I NSCLC depends on the size and biology of the tumour and the age and state of health of the patient.

Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses.

We have generated a molecular taxonomy of lung carcinoma, the leading cause of cancer death in the United States and worldwide. Using oligonucleotide microarrays, we analyzed mRNA expression levels corresponding to 12,600 transcript sequences in 186 lung tumor samples, including 139 adenocarcinomas resected from the lung. Hierarchical and probabilistic clustering of expression data defined distinct subclasses of lung adenocarcinoma. Among these were tumors with high relative expression of neuroendocrine genes and of type II pneumocyte genes, respectively. Retrospective analysis revealed a less favorable outcome for the adenocarcinomas with neuroendocrine gene expression. The diagnostic potential of expression profiling is emphasized by its ability to discriminate primary lung adenocarcinomas from metastases of extra-pulmonary origin. These results suggest that integration of expression profile data with clinical parameters could aid in diagnosis of lung cancer patients.

Combined antegrade and retrograde dilation: a new endoscopic technique in the management of complex esophageal obstruction.

BACKGROUND: Esophageal strictures that cause complete obstruction are often difficult to dilate with standard bougienage techniques. METHODS: A new technique was developed and applied, combined antegrade and retrograde dilation, for dilatation of complex esophageal strictures. The stomach is accessed and an endoscope (9.8 mm diameter) is directed under fluoroscopy in a retrograde fashion into the distal esophagus. A guidewire with a hydrophilic coating is advanced through the stricture and then pulled through the mouth with a simultaneously placed proximal endoscope. The guidewire is then used as a guide for antegrade esophageal dilatation. RESULTS: Ten patients with complex esophageal strictures (with and without fistulas) were treated with this technique. Three required a second combined antegrade and retrograde dilation procedure. All strictures were dilated and no perforations occurred. CONCLUSIONS: Combined antegrade and retrograde dilation is a safe and effective technique for dilation of complex obstructing esophageal lesions.

Lung cancer in patients under age 40.

A retrospective review of patients <40 years (n=91) seen at the Dana-Farber Cancer Institute and Brigham and Women's Hospital from January 1, 1983-January 1, 1993 was carried out. Of 91 patients, there were 43 men and 48 women with a median age of 36 years (range 28-39). Eighty percent of patients were cigarette smokers for a median of 25 pack years (range 2-68). Ninety-one percent were symptomatic at presentation. The ECOG performance status (PS) was 0 or 1 in 83%. At the time of diagnosis 15% had stage I/II, 17% stage IIIA, 22% stage IIIB and 45% stage IV disease. The most common histopathology was adenocarcinoma (46%), followed by small cell carcinoma (14%), squamous cell carcinoma (12%), large cell undifferentiated (8%) and other types (20%). The median survival for all 91 patients was 1 year with 2 and 5 year survivals of 30% and 18% respectively. Five year survival was related to stage of disease: 60% for patients with stage I, 58% for stage II, 36% for stage IIIA, 10% for stage IIIB, and 3% for stage IV disease. Factors that had no significant effect on overall survival included gender, histologic subtype, degree of differentiation, presence or absence of symptoms, and sites of metastases. Factors that adversely affected survival by univariate analysis included advanced stage of disease, poor PS, duration of symptoms for more than 3 months, and 5% or greater body weight loss. By multivariate analysis only stage (P<0.001) and weight loss (P=0.02) affected survival. This data plus results of other published studies show that young patients under age 40 with lung cancer, compared to the more common older patients, have an increased percentage of women, have a longer duration of symptoms, more often have adenocarcinoma with lower frequency of squamous cell carcinoma and sometimes small cell carcinoma, and more often present with advanced disease. Despite these differences, overall patient survival remains poor and is similar to that of older patients.

Treatment of malignant mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare tumor that predominantly afflicts men over 50 years of age. Nearly 3000 MPMs are reported annually in the United States with the incidence expected to rise into the new millenium. Over the past 40 years, MPM has been unequivocally linked to asbestos exposure worldwide. Recently, however, a new theory on the carcinogenesis of this tumor has been proposed with the isolation of a simian virus (SV 40)-like gene sequence in mesothelioma tumor cells. The clinical presentation of MPM is variable, although most patients typically present with dyspnea, chest pain, or pleural effusion. Obtaining a diagnosis of MPM has been greatly assisted by video-assisted surgery and the use of immunohistochemistry and electron microscopic techniques, which help distinguish MPM from other tumor pathologies such as adenocarcinoma. Computed tomography and magnetic resonance imaging have been also useful for determining tumor burden and resectability. Traditionally, strategies for the treatment of MPM have included supportive care, surgery, radiotherapy, and chemotherapy. Survival with supportive care alone ranges between 4 and 12 months. Single-modality therapy using traditional approaches (surgery, radiotherapy, chemotherapy) alone has failed to improve patient survival significantly. Recently, results using a multimodality approach have been favorable. In particular, cytoreductive surgery (pleuropneumonectomy) followed by sequential chemotherapy and radiotherapy have demonstrated improved survival, especially for patients with epithelial histology, negative resection margins, and no metastases to extrapleural lymph nodes. Innovative therapies such as the use of photodynamic, targeted cytokines and gene therapy are currently being investigated for management of MPM.

CALGB 9380: a prospective trial of the feasibility of thoracoscopy/laparoscopy in staging esophageal cancer.

BACKGROUND: The staging of esophageal cancer is imprecise. Thoracoscopic/laparoscopic (TS/LS) staging has been proposed as a more accurate lymph node (LN) staging method. We report the experience of an Intergroup NCI trial (CALGB 9380) evaluating the feasibility and accuracy of this staging modality. PATIENTS AND METHODS: From February 1995 to September 1999, 134 patients were entered in the study. This study represents the analysis of final data on 113 patients. TS/LS was considered feasible if TS and 1 LN sampled at least 3 LN by LS; a confirmed positive node was found; or T4 or M1 disease was documented. If this was accomplished in more than 70% of patients, TS/LS was believed to be feasible. RESULTS: The LN stations most frequently sampled in the thorax (134 patients) were levels 2 (33%), 3 (38%), 4 (40%), 7 (76%), 8 (69%), 9 (55%), and 10 (43%) and in the abdomen levels 17 (70%) and 20 (55%). The frequency of positive LN by level were as follows: 2 (10%), 3 (8%), 4 (10%), 7 (10%), 8 (25%), 9 (10%), 10 (10%), 17 (34%), and 20 (27%). Noninvasive tests (computed tomographic scan, magnetic resonance imaging, esophageal ultrasound scan) each incorrectly identified TN staging as noted by missed positive or false-negative LN or metastatic disease found at TS/LS staging in 50%, 40%, and 30% of patients, respectively. Median operating time was 210 minutes (range, 40 to 865 minutes). Median postoperative hospital stay was 3 days (range, 1 to 35 days). There were no deaths or major complications. Seventy-three percent of patients met the definition for feasibility. In 30 patients TS was not feasible. Positive LN disease was found in 43 patients; 32 were deemed N0. Ten patients had T4/M1 disease. Of the 32 potentially resectable N0 patients, 14 patients had preoperative induction therapy; 13 patients went directly to operation with N0 confirmed in 9 patients, NX in 1 and N1 in 3. Three patients were unresectable, 1 patient died, and 1 was lost to follow-up. CONCLUSIONS: In summary, the feasibility of TS/LS was confirmed. It doubled the number of positive LNs identified by conventional, noninvasive staging. The overall accuracy remains to be defined by analysis of the LN negative group in follow-up. Although the positive predictive value was high, further study is warranted to confirm the role of TS/LS in the staging algorithm of esophageal cancer.

Sequential thoracic metastasectomy prolongs survival by re-establishing local control within the chest.

OBJECTIVE: The value of sequential thoracic metastasectomies is unknown. We evaluate repeat metastasectomy for limited recurrences within the thorax. METHODS: From July 1988 to September 1998, 54 patients underwent 2 to 6 separate sequential procedures to excise metastases after recurrence isolated to the thorax. Kaplan-Meier survival and Cox modeling determined prognostic variables. RESULTS: Thirty-three men and 21 women, 22 to 76 years underwent 2 (100%, n = 54), 3 (50%), 4 (22%), or 5 to 6 (11%) metastasectomies. Fifty-four percent of patients had carcinoma, 35% sarcoma, 9% germ cell, and 2% melanoma. There were no operative deaths; all late deaths occurred from cancer. Median follow-up was 48 months. Cumulative 5-year survival from the second procedure was 57%. After the second, third, fourth, and fifth procedures, respectively, permanent control was achieved in 15 (27%) of 54 patients, 5 (19%) of 27, 1 (8%) of 12, and 0 of 7. Recurrence amenable to additional surgery occurred in 27 (50%) of 54, 12 (44%) of 27, 6 (50%) of 12, and 1 (17%) of 6. Mean hazard for the development of unresectable recurrence increased from 0.21 after the second procedure to 0.91 after the fifth procedure. The 5-year survival for the 27 patients undergoing only 2 metastasectomies was 60% (median not yet reached), 33% for the 15 patients undergoing only 3 metastasectomies (median 34.7 months), and 38% for the 12 patients undergoing 4 or more (median 45.6 months). From the time a recurrence was declared unresectable, patients had a 19% 2-year survival (median 8 months). CONCLUSIONS: Multiple attempts to re-establish intrathoracic control of metastatic disease is justified in carefully selected patients, but the magnitude of benefit decays with each subsequent attempt.

The alpha folate receptor is highly activated in malignant pleural mesothelioma.

OBJECTIVE: To determine whether the folate receptor gene is overexpressed in malignant pleural mesothelioma. METHODS: Differential display analysis was performed with fresh frozen RNA obtained from normal lung, pleura, and mesothelioma. Sixty differentially expressed genes were identified and characterized. One gene that was over-expressed in mesothelioma versus normal tissue was the human alpha folate receptor. In situ hybridization with antisense probes designed on the basis of the sequence of the folate receptor was performed with frozen sections from 61 patients (33 epithelial and 28 mixed or sarcomatoid tumors) with malignant pleural mesothelioma. The controls included normal pleura, normal lung, other cancers, and sense controls for all of the tumors. Northern analysis with a folate receptor probe and immunohistochemical analysis with anti-alpha folate receptor antibodies were also performed. RESULTS: Forty-four (72%) of the 61 mesothelioma tumors were found to have between 2-fold and 4-fold higher mRNA expression of the folate receptor when compared with the control tissues. The histologic type of the tumor did not affect the rate of folate receptor activation. Northern analysis and immunohistochemical experiments confirmed these findings. CONCLUSIONS: A majority of mesothelioma tumors examined overexpress the alpha folate receptor protein when compared with normal adjacent tissues. This finding may help explain the observations that antifolate drugs have activity in the treatment of mesothelioma. It also encourages further study of folate receptor-related treatment strategies in this malignancy.

Malignant pleural mesothelioma.

Malignant pleural mesothelioma remains a difficult tumor to treat, much less cure. Currently, the best chance for long-term survival lies with early diagnosis and aggressive surgical extirpation, but given the typically long delay between the onset of symptoms and diagnosis, this is only possible with a high index of suspicion and an aggressive diagnosis workup. Early referral to a tertiary center experienced in the treatment of MPM may be important for several reasons: (1) decreased risk of tumor spread along multiple thoracenesis/biopsy tracts, (2) the availability of specialized pathologic assays for definitive diagnosis, (3) the availability of critical staging modalities (aggressive mediastinoscopy +/- thoracoscopy, MRI scans performed according to specific mesothelioma protocols, and perhaps PET scans), (4) surgical experience with pleurectomy/decortication and/or extrapleural pneumonectomy, that may decrease morbidity and mortality, and (5) the availability of novel adjuvant protocols. Single-modality therapy is unlikely to result in long-term survival. Aggressive surgery is required for optimal debulking, and extrapleural pneumonectomy may offer better local control compared with pleurectomy/ecortication. Delivery of optimal radiation schedules, which may involve large fractions as well as large total doses, is limited by the presence of nearby dose-limiting structures. Current chemotherapy is severely lacking in producing objective responses and improved survival although gemcitabine and IL-2 may be active agents to be combined with radiation and/or other agents. Hyperthermia, photodynamic therapy, intracavitary therapy, and gene therapy are all relatively new techniques under active investigation that should be supported by enrollment in on-going protocols. Predictably, many of these techniques provide greater benefit when used in the setting of adjuvant protocols or minimal residual disease, emphasizing the importance of multimodality therapy.