Feasibility of RNA collection for micro-array gene expression analysis in the treatment of cervical carcinoma: a scientific correlate of RTOG C-0128.
OBJECTIVE
To determine the feasibility of RNA collection in a multi-institutional cooperative group setting to be utilized for micro-array gene expression analysis, and to describe the methodology.


METHODS
RTOG C0128, a phase I-II, protocol was designed to look at the safety and efficacy of external beam radiation therapy to 45 Gy with concomitant 5-FU and cisplatin chemotherapy, brachytherapy to deliver 85 Gy to point A, and Celecoxib at 400 mg twice daily for 1 year. Patients had the option of participating in a tissue collection portion of the protocol to be utilized for micro-array gene expression analysis before treatment and at the time of the first implant. RNA quality was determined by two parameters: the absorbance ratio at 260 nm/280 nm, and by the ratio of the integrated peak of 28S RNA to 18S RNA after gel electrophoresis.


RESULTS
From August 2001 to March 2004, 84 patients were accrued to the trial, and tissue was obtained prior to initiation of therapy on 34 patients (40%). FIGO stages for the patients who provided tissue were IB (23%), II (57%), and IIIA-IVA (20%). Additionally, biopsies were obtained at the time of the first implant from 22 of the accrued patients making paired samples available on 26% for RNA extraction and micro-array gene expression analysis. The mean +/- SEM amount of tissue obtained pretreatment was 97 +/- 13 mg compared with 51 +/- 8 mg for tissue obtained at the time of the first implant (P = 0.009). The mean total RNA extracted from the samples prior to treatment was 119 +/- 19 microg versus 35 +/- 6 microg at the time of the first procedure (P = 0.001). The RNA quality was assessed via the absorbance ratio at 260 nm divided by 280 nm. The mean values pretreatment and at first implant were 1.87 +/- 0.07 versus 1.66 +/- 0.11, respectively (P = 0.002); however, the integrated peak of 28S RNA to 18S RNA after gel electrophoresis was not significantly different (P = 0.26).


CONCLUSIONS
RNA extraction for gene expression analysis can be successfully performed in the multi-institutional cooperative group setting. Fresh tissue samples were obtained on 40% of accrued patients prior to treatment. The amount of biopsy material and the quantity of RNA extracted were greater prior to treatment compared with the first implant. The quality of RNA was superior prior to treatment as measured by the ratio of absorbance at 260/280 nm. These results indicate that gene expression analysis is feasible in the cooperative group setting utilizing amplification techniques for the RNA. Hopefully, this will allow for improvement in prognosis, therapeutic development, and correlation with acute and late toxicities in patients with cancer.
Gaffney
D K
DK
University of Utah and Huntsman Cancer Institute, Salt Lake City, UT 84108, USA. david.k.gaffney@hci.utah.edu
Winter
K
K
Fuhrman
C
C
Flinner
R
R
Greven
K
K
Ryu
J
J
Forbes
A
A
Kerlin
K
K
Nichols
R C
RC
Zempolich
K
K
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000009:adverse effects / Q000627:therapeutic use; D001918:Brachytherapy / Q000009:adverse effects / Q000379:methods; D000068579:Celecoxib; D002945:Cisplatin / Q000008:administration & dosage / Q000009:adverse effects; D003131:Combined Modality Therapy; D005240:Feasibility Studies; D005260:Female; D005472:Fluorouracil / Q000008:administration & dosage / Q000009:adverse effects; D020869:Gene Expression Profiling; D006801:Humans; D009367:Neoplasm Staging; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011720:Pyrazoles / Q000008:administration & dosage / Q000009:adverse effects; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D013449:Sulfonamides / Q000008:administration & dosage / Q000009:adverse effects; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000532:radiotherapy / Q000628:therapy*
D011720:Pyrazoles; D013449:Sulfonamides; D012313:RNA; D000068579:Celecoxib; D002945:Cisplatin; D005472:Fluorouracil