Cytogenetics Core Achievements, 2006-2007

Effect of Core on Stimulating Research - Scientific Highlights Using Various Services from the Cytogenetics Core

Development of FISH probes to evaluate copy numbers of genes important in carcinogenesis

In 2006-2007, the Cytogenetics Core developed six new FISH probe sets: IGF1R/15, IGF2R/6, FGFR1/8, BRAF/8, MET/7, and 5’-3’TMPRSS2. These probes were developed for preliminary studies for a number of grants submissions and phase I and II clinical trials. Three examples involving numerous investigators of the UCCC are highlighted below.

IGF1R genomic status in NSCLC: Evaluation by FISH (Bunn, Hirsch, Chan, Helfrich, Heasley, Varella-Garcia)

Recently UCCC members Hirsch, Franklin, Bunn and Varella-Garcia reported that IGF1R1 expression was common in NSCLC (39% of  77 patients tested) and verified that this feature was not significantly associated with clinical characteristics (gender, histology, smoking history) nor biological markers (EGFR gene mutation, EGFR amplification, and p-Akt expression). It was also verified that in these NSCLC patients IGF1R expression has not impacted response and time to progression to the EGFR-TKI gefitinib treatment, although median survival was significantly shorter in IGF1R1-non expressors (Hazard Ratio 2.21, p=0.012). Because association of IGF1R with clinical stage and local or metastatic recurrence has been reported in melanoma, colorectal and breast carcinomas, these investigators decided to investigate the copy number status of the IGF1R gene in lung cancer in order to (a) verify occurrence of heterogeneity among specimens and (b) determine whether an association between the genomic content and sensitivity to IGF1R inhibitors could be detected.

The relevant conclusion from these studies was that the IGF1R genomic status in NSCLC is heterogeneous, thus it supports further investigation of the association of this biomarker with clinical outcome to IGF1R inhibitors, both antibodies against the receptor and small molecule tyrosine kinase blockers. These results are still unpublished but supported specific goals of a project included in the SPORE in Lung Cancer recently submitted. In addition, these studies also contributed for the selection of our core as the FISH reference laboratory in the correlative studies for CETP-sponsored IMC-A12 trials last August.

1. Cappuzzo F, Toschi L, Tallini G et al., Insulin-like growth factor receptor 1 (IGFR-1) and PTEN as determinants for intrinsic resistance to gefitinib therapy in non-small cell lung cancer (NSCLC) patients. Annals of Oncology, 17(7):1120-7, 2006.

Evaluation of rearrangements involving the TMPRSS2 genein prostate cancer by FISH (Glode, Flaig, van Bockhoven, Franklin, Varella-Garcia)

BAC clones containing DNA sequences distal (5’end, clone RP11-35C4) and proximal (3’ end, clone RP11-354C5) to the TMPRSS2 gene were obtained from CHORI (Oakland, CA). These clones were selected based on genomic information available at the Ensembl web site.

Our studies have supported the proposal of a specific project aiming to investigate the presence of fusions in prostate patients enrolled in the SWOG 9921 trial. The SWOG 9921 was initiated in 1999 to answer the question of whether 6 cycles of mitoxantrone and prednisone could improve the survival of “high risk” prostate cancer patients after radical prostatectomy. All patients received two years of adjuvant combined androgen blockade using goserelin acetate and bicalutimide at standard doses. This hormonal adjuvant treatment was based on the survival advantages seen in previous EORTC, RTOG and SWOG studies. Dr. Glode recently sponsored a conference to review the biomarkers for prostate cancer. Among the participants in the conference were representatives of the Inter-SPORE Prostate Biomarkers Study, the EDRN (www.cancer.gov/edrn), CaBIG (http://cabig.nci.nih.gov/), biotechnology companies, and individual investigators. Participants in the conference agreed that the specimen collection for SWOG 9921 could provide an invaluable resource for validating biomarkers that could guide analysis of this study as well as better selection of patients for future large prostate cancer trials. Dr. Glode is the PI in the proposal aiming to collect specimens and prospectively analyze established biomarkers for prostate, including the presence of TMPRSS rearrangements detected by FISH.

Biomarker for selection of NSCLC for EGFR-tyrosine kinase inhibitors  (Bunn, Hirsch, Franklin, Bemis, Robinson, Varella-Garcia)

The Cytogenetics Core has continued to work in the validation of the EGFR FISH assay for selection of NSCLC patients to treatment with EGFR tyrosine kinase inhibitors. Ten publications in 2007 have used and discussed results generated by this laboratory, as listed. In addition to two interesting case reports (1,6), these publications provided more details regarding the correlation between the EGFR genomic content in NSCLC and therapy. We verified that EGFR FISH and IHC data were independent markers and, when combined, defined distinct patient strata, double-negative and double-positive, who had, respectively, 2% and 41% of response rate, 30% and 77% of 1-year survival rate, and 6 and 21 months of median survival (8).  We also verified that presence of KRAS mutation impacts the sensitivity to EGFR-TKIs in the FISH positive patients. KRAS mutation has been known as a primary resistance factor to TKIs but this is the first time that KRAS mutation was specifically evaluated in FISH positive patients. The genomic status of EGFR was proved to be not predictive for chemotherapy treatment (in two independent cohorts) and survival (2, 7). The Core work has also assisted with the stratification of patients for the Oncobell trial, a prospective Phase II study in which the patients were selected for EGFR-TKIS by EGFR FISH,/AKT positive or never-smoking status (5). In FISH positive patients, the response rate (RR) was 68% and time to progression (TTP) was 7.6 months, while in the FISH negative patients RR was 9% and TTP was 2.7 months.

1. Bemis LT, Robinson WA, McFarlane R, Byers E, Kelly K, Varella-Garcia M, Mitchell JD, Franklin WA. EGFR-mutant lung adenocarcinoma in a patient with Li-Fraumeni syndrome. Lancet Oncology, 8(6):559-60, 2007.
2. Cappuzzo F, Ligorio C, Toschi L, Rossi E, Trisolini R, Paioli D, Magrini E, Finocchiaro G, Bartolini S, Cancellieri A, Hirsch FR, Crino L, Varella-Garcia M. EGFR and HER2 gene copy number and response to first-line chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). J. Thor Oncol, 2(5):423-9, 2007.
3. Cappuzzo F, Toschi L, Finocchiaro G, Ligorio C, Santoro A. Surrogate predictive biomarkers for response to anti-EGFR agents: state of the art and challenges. Int J Biol Markers, 22 (1 Suppl 4):S10-23, 2007.
4. Cappuzzo F. Predictive factors for response and for resistance to tyrosine kinase inhibitor therapy in lung cancer. J Thorac Oncol (5 Suppl):S12-4, 2007.
5. Cappuzzo F, Ligorio C, Jänne PA, Toschi L, Rossi E, Trisolini R, Paioli D, Holmes  AJ, Magrini E, Finocchiaro G, Bartolini S, Cancellieri A, Ciardiello F, Patelli M, Crino L, Varella-Garcia M. Prospective study of gefitinib in EGFRFISH positive/p-AKT positive or never smoker  patients with advanced non-small cell lung cancer (NSCLC): results of the ONCOBELL trial. J Clin Oncol., 25(16):2248-55, 2007.
6. Dziadziuszko R, Siemiatkoswska A, Limon J, Rzyman W, Jassem J, Bunn PA Jr, Varella-Garcia M, Hirsch FR. Unusual chemosensitivity of advanced bronchioalveolar carcinoma after gefitinib response and progression: a case report. J. Thor Oncology, 2:92-92, 2007.
7. Dziadziuszko R, Holm B, Skov BG, Osterlind K, Sellers MV, Franklin WA, Bunn PA Jr, Varella-Garcia M, Hirsch FR. Epidermal growth factor receptor gene copy number and protein level are not associated with outcome of non-small-cell lung cancer patients treated with chemotherapy. Ann Oncol. 18 (3):447-52, 2007.
8. Hirsch FR, Varella-Garcia M, Cappuzzo F, McCoy J, Bemis L, Xavier AC, Dziadziuszko R, Gumerlock P, Witta SE, West H, Crowley J, Gazdar AF, Crino L, Gandara D, Franklin WA, Bunn Jr. PA. Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib. Ann. Oncology, 18(4):752-60, 2007.
9. Massarelli E, Varella-Garcia M, Tang X, Xavier AC, Ozburn NC, Liu DD, B. Nebiyou Bekele BN, Herbst RS, Wistuba II KRAS Mutation is an Important Predictor of Resistance to Therapy with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients with Non-Small Cell Lung Cancer. Clin Cancer Res, 13(10):2890-2896, 2007.
10. Toschi L, Cappuzzo F Understanding the new genetics of responsiveness to epidermal growth factor receptor tyrosine kinase inhibitors. Oncologist 12(2):211-20, 2007.