P-307 Association between previous pulmonary tuberculosis and FHIT gene alterations in lung cancer

Poster Session 2/Molecular P 305 czl

Mutant DNA in Southwest Oncology Group (SWOG) SO003 non-small cell lung cancer (NSCLC) patient plasma: Potential for monitoring response to therapy

Tatsuo Kimura’, Will S. Holland’, Primo N. Lara Jr’, Tomoya Kawaguchi2, Wilbur A. Franklir?, Kari Chansky4, Jason McCoy4, John J. Crowley4, David R. Gandara’ , Paul H. Gumerlock’ 1 University of California, Davis, Cancer Center, Sacramento, USA; ‘National Kinki Central Hospital for Chest Disease, Sakai, Japan; 3 University of Colorado, Denver, USA; 4 Southwest Oncology Group Statistical Center, Seattle, USA

Biology 2

will be presented CA46441 .)

S169 at the meeting. (ROI

CA79099,

CA32102,

CA38926,

UIO

TUESDAY, 12 AUGUST 2003

Molecular

Biology

2

Purpose: Several studies have shown that genetic abnormalities

found in primary tumors can also be identified as shed DNA in cancer patient plasma. Our aims were to evaluate mutations of K-RAS (codon 12) in plasma DNA, and to assess the value of monitoring plasma for predicting clinical outcome. Materials and Methods: Training cohort: pre-treatment plasma specimens from 25 patients on a phase I trial (12 with paired post-treatment specimens) and 12 tumor blocks. Test cohort: plasma from 161 patients on the SO003 trial (NSCLC: carboplatin/paclitaxel i tirapazamine), 43 with paired post-treatment specimens. DNA was extracted and mutational analyses performed using a highly-sensitive two-step RFLP-PCR assay that can detect mutant DNA at a 5% frequency. Results: Training cohort: K-RAS mutations were found in plasma DNA of 5/25 patients (20%). Overall Median Survival (MS): 298 days: wild-type (wt) K-RAS group: 344 days; and mutant K-RAS group: 100 days (p=O.O563). To confirm the tumor as the source of the shed mutant DNA, the specific point mutation in a patient plasma DNA (GGT>TGT) was identical to the mutation in that patient’s tumor (control PBMCs are wt K-RAS). In 2 patients with K-RAS mutations pre-treatment, post-treatment plasmas were evaluated: One patient with progressive disease retained the mutant DNA, while in the patient with a complete response, the K-RAS mutation was no longer detectable. Test Cohort: In SO003 patients, K-RAS mutations were found in plasma DNA of 13/123 patients (10.6%). MS: wt K-RAS group: 9 months; mutant K-RAS group: 7 months. The 43 post-treatment plasma specimens are being evaluated. Correlations with survival and individual patient responses will be presented at the meeting. Conclusions: 1) Circulating, cell-free tumor DNA is present in NSCLC patient plasma, 2) Detection of the mutant DNA is feasible using sensitive molecular techniques; and 3) This approach shows promise for monitoring patient response to therapy and may allow assessment of tumor recurrence. Incorporating other tumor-specific markers (i.e., the methylated p16 promoter) in this assay will allow the extension of the approach to additional patients. Support: CA62505, CA32102, CA38926, CA46441, Hope Foundation, Sheeter Foundation, Sanofi-Synthelabo.

I P 306

Molecular abnormalities in anti-microtubule and DNA damage response pathways in non-small cell lung carcinomas (NSCLC): Southwest Oncology Group (SWOG) 9509 trial

Paul H. Gumerlock’, lrina V. Galvin’, Tatsuo Kimura’, Will S. Holland’, Wilbur A. Franklin2, Philip C. Mack’, Kari Chansky3, Jason McCoy3, John J. Crowley3, David R. Gandara’ ’ University of California, Davis Cancer Center, Sacramento, USA; ’ University of Colorado, Denver, USA; 3 Southwest Oncology Group Statistical Center, Seattle, USA SWOG 9509 was a phase Ill trial testing paclitaxel + carboplatin vs vinorelbine + cisplatin in advanced (stage lllb (pl eff), IV) NSCLC. Kelly et al (JCO, 2001) reported no survival differences between the treatment arms. This trial represents a well-characterized group of NSCLC patients in which to examine molecular markers for prognostic and predictive value in a retrospective fashion. We hypothesized that abnormalities in anti-microtubule (vinca alkaloid, taxane) and DNA damage (platinums) response pathways would be found at relatively high frequencies. The anti-microtubule pathway included the following genes: the tumor suppressor ~27, where stabilization of the protein is associated with taxane-induced apoptosis; the b-tubulin TUBB-III isoform, recently found to be abnormally expressed in NSCLC; and the microtubuleassociated protein MAP4, where high expression has been shown to correlate with vinca resistance and low expression with taxane resistance. The DNA damage response pathways examined included the repair inducer ~53, proapoptotic BAX, anti-apoptotic BCL2, and the excision repair gene ERCCI. Over 600 determinations of nine molecular markers have been made to date in 102 archival tumor specimens using immunohistochemistry and PCR-based assays. Anti-microtubule pathway abnormality rates: ~27: 78/91 (86%); TubbIll: 39/74 (53%); and Map4: low expression 15/35 (43%), high expression 13/35 (37%). DNA damage pathway abnormality rates: ~53: 54/82 (66%); Bax: 6/68 (9%); Bcl2: 6/64 (9%); and at least one of the three abnormalities in 58/84 (69%). High expression of ERCCI: 13/56 (23%). High proliferation (Ki-67) was found in 30/83 (36%) and K-RAS mutations in 2/l 1 (18%). Molecular analysis of additional specimens is continuing. Correlation of the data with patient demographics, histology, response, survival, and treatment arm is ongoing and

I P 307

Association Between Previous Pulmonary Tuberculosis and FHIT Gene Alterations in Lung Cancer

Lanyina Sonq’, Wenshenq Yan’, Min Deng3, Tonq Zhao’. ‘Dept. of Pathology The First Military Medical University, Guangzhou, China; ’ Cancer Center, Sun Yat-Sen University, Guangzhou, China

Aim: Epidemiologic data have strongly indicated previous pulmonary tuberculosis (PPT) is linked to the development of lung cancer, but little is known of the molecular targets of carcinogens contained in PPT. Recent studies have been taken as evidence that FHIT gene is a target for environmental carcinogens such as tobacco smoke carcinogens. The relationship between FHIT gene alterations and exposure to PPT has not yet been explored. Here, we undertook a molecular study of FHIT gene alteration in lung tumors with PPT and in tumors without PPT to seek genetic damage attributable to PPT. Materials and Methods: This analysis included 36 primary lung cancer cases (10 with PPT and 26 without PPT). The clinical-pathology features see Table 1. PPT was defined as subjects who had pulmonary tuberculosis 2 or more years before lung cancer was detected. DNA was prepared from microdissected paraffin-embedded pathology preparation, total RNA was isolated from frozen resected tumor. Point mutations and abnormal transcripts within encoding exons (exons 5,8) of FHIT gene were respectively measured by PCR-SSCP and RT-PCR. Loss of heterozygosity (LOH) of FHIT gene was analyzed at the following loci: D351234, D3S1300, D3S1481. The result of LOH was tested by using Fisher’s exact test and Logistic regression modeling. Table 1. Clinico-pathological features of tumor patients Sex Age Smoking

Female Male

7

29 56.911.7

YE. NO

25 11

Histology

Ad see other

PPT

Yes

11 17 8 10

NO PPT years Cancer family history

26

29.1*6.2 Ye5 NO

3 33

Data are counts or means i SD; Ad, adnocarcinoma; SCC, squeamous cell carcinoma.

Results: of 36 primary lung cancer analyzed, no point mutations were detected within FHIT gene. LOH affecting at least two locus of the FHIT gene was observed in 6 of IO tumors with PPT group (60%), but in only 3 of 26 tumors without PPT (11.5%), which was significantly different (Fisher’s exact test p = 0.006, logistic regression analysis p= 0.024), whereas difference in LOH rate

Poster Session 2/Molecular Biology 2

s170

I P 309

Table 2 (continued)

22d0* 29x3 6090 6239* 6213 6392 672X 3535 3296 632th 30 4883 5243 6192 1X96*

05x3* Total altmtiom

l l 0 ea l

0 0 0 l e

0 0 0 l 0

0

0

c

l

0

0 0

c) 0

0 l

l 0 0 e

e i: 0 Cl

0 e 0 e

0 0 e

Ci 0 0

0 e

17 of36(47.3%) ~--

3 of36(8 3%)

Shinichi Tovooka’, Narayan Shivapurkars, Kiyomi 0. Toyooka3, Kuniharu Miyajimas, Makoto Suzukis, Jyotsna Redds, Harvey I. Pass4, Jack A. Roths, John D. Minna6, Adi F. Gazdars. ’ Department of Surgery; Kagawa Prefectural Central Hospital, Takamatsu, Japan; s Hamon Center for Therapeutic Oncology Research and Departments of Pathology UT Southwestern Medical Center, Dallas, USA; 3 Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Da//as, USA; 4 Department of Thoracic Surgery, Karamanos Cancer Center, Detroit, USA; 5 Department of Thoracic Surgery, MD Anderson Cancer Center, Houston, USA; s Hamon Center for Therapeutic Oncology Research, Departments of internal Medicine and Pharmacology UT Southwestern Medical Center, Da//as, USA

0

l 13 uf36Ci6,1%)

5 of lS(33 3’%) --~---

*,fung cancertissueswith PPT; O,micms~telIiterctsid; l ,micmsatellitcLOH; O,nnrmal tmnscript;&transcriptsbscnce. affecting at any one of D3S1234, D3S1300 and D351481 locus was not observed in tumors with and without PPT (Fisher’s exact test is p=O.O99, p=O.O64, and p=O.212 respectively) (Table 2). 5 of 15 cases analyzed by RT-PCR showed absence of FHIT gene transcript and also was demonstrated with LOH at the FHIT locus (Table 2). Conclusion: These results highlighted that LOH of the FHIT gene is involved in the carcinogenesis of lung cancer in patients with PPT.

El308 P

Aberrant Methylation of the Tumor Necrosis factor related apoptosis inducing Ligand Decoy receptor Genes in Malignant Mesothelioma and Lung Cancer

Epigenetic Down regulation of Death associated Protein Kinase in Lung Cancers

Shinichi Tovooka’, Kuniharu Miyajima”, Narayan Shivapurkar3, Melvyn S. Tockman“, Stephen Lams, Nobuyoshi Shimizus, Adi F. Gazdar3. ’ Department of Surgery Kagawa Prefectural Central Hospifa/, Takamatsu, Japan; s Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Da//as, USA; 3 Hamon Center for Therapeutic Oncology Research and Departments of Pathology, UT Southwestern Medical Center, Dallas, USA; 4 H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, USA; s British Columbia Cancer Agency, Vancouver, Canada; 6 Cancer and Thoracic Surgery, Okayama University Graduate School of Medicine and Dentistry Okayama, Japan Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine kinase involved in apoptosis. Previously aberrant methylation of DAPK was reported in lung cancers by methylation specific PCR and related to poor prognosis. However, utilizing the previously developed methodology, we were unable to relate methylation with gene silencing. Our goals were to develop a methodology that related methylation with gene silencing, and utilize it to study the state of the gene in lung cancer cell lines and tumors. Utilizing a semi-quantitative real-time RT-PCR, DAPK expression was lower in lung cancers than in corresponding nonmalignant bronchial epithelial cells (NBECs) in five of six primary short term cultures. In continuous cell lines, mRNA expression was down regulated as well compared to NBECs and its protein was not detected by western blotting in 17 of 23 (74%) cell lines. We investigated the methylation status of 5 flanking region of DAPK by combined bisulfite restriction analysis and bisulfited DNA sequencing. Aberrant methylation was detected in 21 of 48 (44%) cell lines, two of six primary cultured lung tumors and 14 of 38 (37%) primary lung cancers, although varying degrees of methylation were noticed. In addition, bisufite sequence data suggested that aberrant methylation might occur selectively at some CpG dinucleotides in cell lines which had absent expression. Treatment with 5aza-2 L-deoxycytidine restored DAPK expression in heavily methylated cell lines tested and histone deacetylase inhibitor trichostatin A alone restored DAPK expression in some methylated cell lines as well. Our major findings are: a) DAPK expression is frequently down regulated in lung cancers; b) aberrant methylation of DAPK is frequent in lung cancers, although considerable heterogeneity of methylation is present and some specific CpG dinucleotides are often methylated in DAPK expression negative lung cancers and c) besides aberrant methylation and histone deacetylation, there may be other mechanisms for down regulation of DAPK expression.

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces programmed cell death (apoptosis) in various cancer cell lines but not in normal cells. TRAIL is known to bind to four different receptors, two proapoptotic (DR4 and DR5), and two antiapoptotic (DcRI and DcR2). Aberrant promoter methylation and resultant silencing of tumor suppressor genes plays an important role in the pathogenesis of many tumor types. Recently the aberrant methylation of TRAIL decoy receptors are reported in pediatric tumors. We examined the methylation and expression status of DcRl and DcR2 genes in malignant mesotheliomas (MMs) and lung cancer. Aberrant methylation of DcRl was present in 44 of 88 (66%) primary MMs and in 5 of 48 (10%) primary lung cancers. Aberrant methylation of DcR2 was present in 26 of 66 (39%) primary MMs and in 9 of 48 (20%) primary lung cancers and. No significant methylation was found in non-malignant tissue in case of lung or mesothelioma. In cell line, aberrant methylation of DcRl was present in 3 of 7 (43%) M M and 10 of 27 (37%) lung, while aberrant methylation of DcR2 was present in 5 of 7 (71%) M M and 13 of 27 (48%) lung cancer. The concordance between loss of gene expression and aberrant methylation ranged from 70% to 100%. Treatment with 5-aza-2 L-deoxycytidine restored DcRI and DcR2 expression in methylated cell lines confirming that aberrant methylation was the cause for silencing of DcR2 expression. Our results demonstrate that DcRl and DcR2 expression is frequently silenced by aberrant methylation in M M and lung cancer.

I P 310

Inhibitory effects of p53 with deletion of c-terminal residues 356-393 on human lung cancer cell line

Hui Wanq’, Bai Tana Lai’, Jinzhao Li3. ’ Deptof Cellular and Molecular Biology Beijing Thoracic Tumor Research Institute, Beijing, China; ‘same Beijing, China; s Chinese Academic Sience institute, Beijing, China

1,

Objective: To study inhibitory effects of extraneous p53 with deletion of cterminal residues 356-393 on malignant phenotype of human lung cancer cell line. Methods: Recombined plasmids pEGFP-p53 (wild type) and PEGFPp53(Del) in which p53 gene is deletion of codons of c-terminal residues 393-356 were constructed. The human lung cancer cell line 801 D having p53 one allele loss and another mutation at 248 codon was selected as a receipt celL801D cells were transfected by PEGFP-p53(wild type), PEGFP-p53(Del), PEGFP respectively and were selected by G418. Growing cells were cloned respectively. Extraneous p53 gene was detected by P.C.R and expression of green fluorescence protein (GFP)to monitor expression of extraneous p53 gene was carry out under fluorescence microscopy. Cloning formation rate and inhibitory rate was examined to evalute ability of cellular proliferation in vitro. Tumorgenesis was performed by transplanted those cell lines into the nude mice. Results: Transfected clone cell lines, PEGFP-p53(Del)-801 D, PEGFP-~53-801 D, pEGFP-801 D were established. These cell lines have presence of extraneous p53 gene and expression of GFP. Inhibitory rate of cloning formrmation was 99.6% for PEGFP-p53(Del)-801 D and 81% for PEGFP-~53-801 D. Inhibitory effect of extraneous p53(Del) on malignant proliferation was higher than p53(wild type) (p