Thymidylate Synthase as a Predictor of Response

Introduction

If a molecular marker for a particular tumor can predict response or resistance to specific treatment, therapy could focus not only on drugs targeting that marker but also on newer agents with different molecular targets. Specifically, it has been hypothesized that intratumoral thymidylate synthase (TS) gene expression might be used to select therapy for patients with disseminated colorectal cancer. We recently reported the results of a clinical trial testing the hypothesis that expression of TS within a metastatic colorectal cancer, as assessed by quantitative polymerase chain reaction (PCR) methodology, would predict the responsiveness of that cancer to fluoropyrimidine-based therapy.[1]

This article summarizes the design and results of that trial, as well as other data suggesting that TS may be useful as a predictor of response to adjuvant therapy for gastric and colorectal cancer.

Clinical Trial: Correlation of TS and Response to 5-FU

Patients and Methods

Eligibility criteria were typical for enrollment in a phase II colon cancer trial. Patients were required to have a diagnosis of disseminated or recurrent colorectal cancer that was measurable, a Southwest Oncology Group (SWOG) performance status of 0 to 2, and adequate hematologic, hepatic, and renal function. The exception to standard eligibility criteria was that each patient was required to have a measurable lesion accessible for biopsy. Prior therapy with fluorouracil(Drug information on fluorouracil) (5-FU) was allowed, but patients were excluded if they had received prior infusional 5-FU.

All patients signed an institutional review board (IRB)-approved written informed consent for a core-needle biopsy of their tumor, performed specifically for the purpose of measuring TS expression prior to the initiation of chemotherapy. The lesion assayed for TS expression had to be bidimensionally measurable either by physical or radiologic examination.

Treatment Regimen--The treatment regimen used in this trial, developed in previously reported phase I and II studies conducted at the University of Southern California, consisted of 5-FU, 200 mg/m²/d administered as a continuous infusion by ambulatory infusion pump (Pharmacia Deltec CADD pump), with leucovorin, 20 mg/m² given by intravenous push weekly.[2,3] The initial treatment cycle was 4 weeks, followed by a 1-week rest, with subsequent cycles consisting of 3 weeks of continuous therapy and 1 week of rest.

After two cycles (8 weeks) of treatment, measurable disease was reassessed for tumor response by the same test used at baseline. Response criteria for this trial were the standard definitions used for national cooperative group trials.[4] Upon disease progression, additional tumor biopsies were taken from the measurable disease site for quantitation of TS expression when patients consented to a second procedure.

Laboratory Methods--In this trial, all specimens analyzed for TS messenger RNA (mRNA) expression were obtained by core-needle biopsy. The technique employs a coaxial system in which fine-needle aspiration is used to confirm cytologic evidence of cancer within moments of the aspiration, with the core-needle material being used for TS analysis.

Our laboratory’s methodology for determining a relative TS mRNA ratio, which has been extensively detailed in previous publications,[5-7] involves the use of an internal standard gene (beta-actin) for the measurement of relative gene expression by determining a ratio between the amplified internal standard complementary DNA (cDNA) and target cDNA within a linear range.

Response Definitions and Statistical Methods--Bidimensional measurement of the lesion sampled for TS/beta-actin mRNA ratio, referred to as the "indicator lesion," had to meet established, standard criteria for the determination of response or progression after administration of two cycles of protocol therapy. A complete response (CR) was defined as disappearance of the indicator lesion, as well as all other clinically and radiologically detectable disease. A partial response (PR) required a 50% reduction in the sum of the products of the perpendicular diameters of the indicator lesion, as well as other disease measured at baseline, without growth of other disease or appearance of new lesions.

Failure of therapy was defined as a 25% increase in the size of the sum of the product of the perpendicular diameters of the indicator lesion or other disease, or appearance of new disease, during the initial 8 weeks of therapy. Patients who showed a response or stable disease continued on protocol therapy until progression was documented.

Investigators were blinded to the results until complete data were available for each patient: Clinicians were not informed of TS mRNA levels until response data were known, and laboratory investigators were not informed of response data until after patients’ TS mRNA levels were determined.

In a preliminary analysis (with fewer patients), a TS/beta-actin mRNA ratio of 3.5 ×10^-3 was identified as the "optimal" cut-off for predicting patients who might respond and those who definitely would not respond; this ratio was significantly associated with response (P < .01).[8] In the current cohort, although a TS/beta-actin mRNA ratio of 3.5 ×10^-3 was the sample median, and a TS/beta-actin mRNA ratio of 4.1 ×10^-3 was found to be the "optimal" cut-off, a TS/beta-actin mRNA ratio of 3.5 ×10^-3 was still statistically significant (P < .01) when compared to the simulated maximal chi-square distribution.

Demographics--A total of 46 patients (29 men and 17 women) were entered into the trial. Median age was 61 years (range, 34 to 80 years). Liver metastases represented the most common site of disease that was biopsied and assessed for response (28 patients). Nodal metastases were the disease sites evaluated in six patients and peritoneal or retroperitoneal metastases were evaluated in eight.

Results

Ratios of TS/beta-actin mRNA were available for 42 patients (12 responders, 30 nonresponders); RNA extraction on the remaining 4 specimens was insufficient in quantity to perform the PCR assay. The range of TS/beta-actin mRNA ratios measured in tumor biopsies for the 42 patients was 0.3 × 10^-3 to 18.2 ×10^-3, with a median TS/beta-actin mRNA ratio of 3.5 × 0^-3 . Analyses revealed no significant associations of TS/beta-actin mRNA ratios with age, sex, ethnicity, or tumor site.

Of the 46 patients, 12 (26%) had partial responses to protocol therapy; 34 patients demonstrated resistance (disease progression) to treatment. For patients responding to therapy, the range of tumor TS/beta-actin mRNA ratios was 0.5 ×10^-3 to 4.1 × 10^-3, with a median value of 1.9 ×10^-3. Nonresponders had TS/beta-actin mRNA ratios ranging from 0.3 ×10 ^-3 to 18.2 ×10 ^-3, with a median value of 5.6 ×10 ^-3 . The difference between responders and nonresponders in baseline TS/beta-actin mRNA ratios was statistically significant (P < .001, based on a two-sided Wilcoxon test.)

TS/Beta-Actin mRNA Ratios as Response Predictors--The median TS/beta-actin mRNA ratio (3.5 ×10^-3) significantly distinguished responders and nonresponders based on the distribution of a maximal chi-square statistic (P = .001). Of the 21 patients with TS/beta-actin mRNA ratios of 0.3 ×1^-3 to 3.5 ×10^-3 (below the median), 11 (52%) responded, while only 1 (5%) patient with a TS/beta-actin mRNA ratio > 3.5 ×10^-3 showed a response. The TS/beta-actin mRNA ratios for the 30 nonresponding patients spanned the entire range of values seen, although none of the 19 patients with a TS/beta-actin mRNA ratio of > 4.1 ×10 ^-3 responded.

Six patients who responded initially consented to a second tumor biopsy after their disease progressed during therapy. All of these patients had higher TS/beta-actin mRNA ratios in their cancers at progression than at baseline, with increases ranging from 3- to 25-fold.

TS/Beta-Actin mRNA Ratios and Survival--Patients with a relatively low TS/beta-actin mRNA ratio treated with the 5-FU infusion had a better median survival than those with a relatively high TS/beta-actin mRNA ratio treated with the same dose and schedule of 5-FU. The median survival for the whole cohort of 46 patients was 8.9 months.

When survival is stratified by median TS/beta-actin mRNA ratios, patients with tumors expressing a TS/beta-actin mRNA ratio £ 3.5 ×10^-3 have a median survival of 13.6 months, whereas patients whose tumors have a TS/beta-actin mRNA ratio > 3.5 ×10^-3 have a median survival of 8.2 months (P = .02, based on the log-rank test) (Figure 1).