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Thymidylate Synthase as a Predictor of Response

Thymidylate Synthase as a Predictor of Response

ABSTRACT: 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 in 46 patients with disseminated or recurrent colorectal cancer testing whether expression of TS within the primary tumor, as assessed by quantitative polymerase chain reaction (PCR) methodology, would predict the responsiveness of that cancer to fluoropyrimidine-based therapy. This trial demonstrated that intratumoral TS/beta-actin messenger RNA (mRNA) ratio can accurately predict which metastatic colorectal tumors will be resistant to a leucovorin-modulated 5-FU infusion and which have a high likelihood of responding to such a regimen. Results of other studies of adjuvant therapy in gastric cancer and colorectal cancer also indicated that TS expression within the tumor is predictive of response to 5-FU-based therapy. It may be possible to use this parameter prospectively to decide which patients should receive fluorinated pyrimidine therapy: Patients whose tumors express low TS levels would be likely to benefit from such therapy, whereas limited preliminary data suggest that patients whose tumors express high TS levels may benefit from irinotecan (CPT-11 [Camptosar]). [ONCOLOGY 12(Suppl 6):43-47, 1998]

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 (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
).

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