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Clin Cancer Res. Author manuscript; available in PMC 2021 November 01.
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Published in final edited form as:

Clin Cancer Res. 2021 May 01; 27(9): 2435–2441. doi:10.1158/1078-0432.CCR-20-2409.

A randomized phase II study of coexpression extrapolation

(COXEN) with neoadjuvant chemotherapy for bladder cancer
(SWOG S1314; NCT02177695)
Thomas W. Flaig1, Catherine M. Tangen2, Siamak Daneshmand3, Ajjai Shivaram Alva4, Seth
P. Lerner5, M. Scott Lucia1, David James McConkey6, Dan Theodorescu7, Amir Goldkorn8,
Matthew I. Milowsky9, Rick Bangs10, Gary R. MacVicar11, Bruno R. Bastos12, Jared S.
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Fowles13, Daniel L. Gustafson13, Melissa Plets2, Ian Thompson Jr14

1University of Colorado, School of Medicine, University of Colorado, Aurora, CO
2Fred Hutchinson Cancer Research Center, Seattle, WA
3Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles,
CA
4University of Michigan, Ann Arbor, MI
5Scott Department of Urology, Baylor College of Medicine, Houston, TX
6Johns Hopkins School of Medicine, Baltimore, MD
7Cedars-Sinai CANCER, Los Angeles, CA
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Corresponding Author: Thomas W Flaig, MD ∣ Professor, University of Colorado, School of Medicine ∣ Department of Medicine, MS
C290, 13001 E. 17th Pl., Room E1342 ∣ Aurora, CO 80045, Telephone: 303-724-0499, Fax: 303-724-3889,
[email protected].
Thomas Flaig
• Research funding (Institutional recipient): Novartis, Bavarian Nordic, Dendreon, GTx, Janssen Oncology, Medivation, Sanofi, Pfizer,
Bristol-Myers Squibb, Roche/Genentech, Exelixis, Aragon Pharmaceuticals, Sotio, Tokai Pharmaceuticals, astrazeneca/MedImmune,
Lilly, Astellas Pharma, Agensys, Seattle Genetics, La Roche-Posay, Merck
• Aurora Oncology - I am a founder and hold intellectual property interests (no current clinical products or active clinical trials/
pending approvals)
Seth Lerner:
• Clinical trials: Endo, FKD, JBL (SWOG), Genentech (SWOG), UroGen, Vaxiion, Viventia
• Consultant/Advisory Board: C2i Genomics, FerGene, Genentech, Merck, Pfizer/EMD Serono, QED Therapeutics, UroGen, Vaxiion,
Verity
• Patent – TCGA classifier
• Honoraria – Grand Rounds Urology, UroToday
David McConkey:
• Grant support: Astra-Zeneca, Rainier Pharmaceuticals
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• Advisory boards: Janssen, H3 Biomedicine, Rainier Pharmaceuticals

Ajjai Alva:
• Consultant and/or Advisor for AstraZeneca; Bristol-Myers Squibb; Merck & Co., Inc; Pfizer, EMD Serono
• Paid DSMB member: Eisai
• Grant/Research Support from AstraZeneca; Bristol-Myers Squibb; and Merck & Co., Inc.Other Financial or Material Support from
Arcus Biosciences; Astellas Pharma US, Inc.; Celgene Corporation; Clovis Oncology; Pfizer, Inc.; Prometheus Biosciences; and
Seattle Genetics, Inc. in the form of research support to institution.
Matt Milowsky:
• Research Funding (Recipient - institutional): Merck, Roche/Genentech, Bristol-Myers Squibb, Seattle Genetics, Astellas Pharma,
Clovis Oncology, Inovio Pharmaceuticals, AstraZeneca, Mirati Therapeutics, Constellation Pharmaceuticals, Syndax, MedImmune,
Incyte
• --Other Relationship: Asieris (Institutional recipient)
Bruno Bastos: Honoria: Regeneron, Astellas, Seattle Genetics, Sanofi Genzyme
These data have been presented in part at the American Society of Clinical Oncology (ASCO) Annual meeting, Chicago IL, June 3,
2019.
 Flaig et al. Page 2

8Division
of Medical Oncology, Department of Medicine, Keck School of Medicine and Norris
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Comprehensive Cancer Center, University of Southern California, Los Angeles, CA

9University of North Carolina Lineberger Comprehensive Cancer Center, Chapel Hill, NC
10SWOG, San Antonio, TX
11Illinois CancerCare PC, Peoria, IL
12Cleveland Clinic Florida, Weston, FL; (previous); Miami Cancer Institute – Baptist Health South
Florida, Miami, Fl (current)
13Colorado State University, Fort Collins, CO
14CHRISTUS Medical Center Hospital, University of Texas Health Science Center at San Antonio,
San Antonio, TX
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Abstract
Purpose: Dose-dense Methotrexate-Vinblastine-Adriamycin-Cisplatin (ddMVAC) and
Gemcitabine-Cisplatin (GC) are accepted neoadjuvant regimens for muscle-invasive bladder
cancer (BC). The aim of this study was to validate the score from a Coexpression extrapolation
(COXEN) algorithm-generated gene expression model (GEM) as a biomarker in patients
undergoing radical cystectomy.

Experimental Design: Eligibility included cT2-T4a N0 M0, urothelial BC, ≥ 5 mm of viable

tumor, cisplatin eligible, with plan for cystectomy; 237 patients were randomized between
ddMVAC, given every 14 days for 4 cycles, and GC, given every 21 days for 4 cycles. The primary
objective assessed pre-specified dichotomous treatment specific COXEN score as predictive of
pT0 rate or ≤ pT1 (downstaging) at surgery.
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Results: Among 167 evaluable patients, the odds ratio for pT0 with the GC GEM score in GC-
treated patients was 2.63 (p=0.10; 95% CI [0.82,8.36]); for the ddMVAC COXEN GEM score
with ddMVAC treatment, the OR was 1.12 (p=0.82, 95% CI [0.42, 2.95]). The GC GEM score was
applied to pooled arms (GC and ddMVAC) for downstaging with an OR of 2.33 (p=0.02; 95% CI
[1.11, 4.89]). In an intention to treat analysis of eligible patients (n=227), pT0 rates for ddMVAC
and GC were 28% and 30% (p = 0.75); downstaging was 47% and 40% (p = 0.27), respectively.

Conclusion: Treatment-specific COXEN scores were not significantly predictive for response to
individual chemotherapy treatment. The COXEN GEM GC score was significantly associated with
downstaging in the pooled arms. Additional biomarker development is planned.

Introduction
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Bladder cancer is the fifth most common cancer with 80,470 new cases and 17,670 deaths
expected in the United States in 2019.1 The use of neoadjuvant, cisplatin-based combination
chemotherapy is optimal care in eligible patients with muscle-invasive disease planning for
radical cystectomy.2,3 Despite randomized data supporting this approach and increased
utilization over time, the application of pre-operative neoadjuvant chemotherapy remains
underutilized.4 No predictive biomarkers are currently used clinically to select
individualized patient chemotherapy or determine the appropriateness of chemotherapy.

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Currently, there are two acceptable regimens utilized in this setting: gemcitabine with
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cisplatin (GC) or dose-dense Methotrexate-Vinblastine-Adriamycin-cisplatin (ddMVAC)

chemotherapy.2,5-7

SWOG trial 8710 was pivotal in establishing the role of cisplatin-based combination therapy
in bladder cancer.3 The study enrolled 317 patients with cT2-T4aN0M0, who were
randomized between MVAC (traditional 28-day cycles) followed by cystectomy versus
cystectomy alone. The median survival improved from 46 to 77 months with the addition of
MVAC chemotherapy. The rate of pT0 was 15% without chemotherapy and 38% with the
addition of chemotherapy in those who underwent cystectomy (31% of all randomized).
Notably, regardless of treatment arm, those with pT0 had an 8-year overall survival estimate
of approximately 75%, while those with any residual disease at cystectomy had an 8-year
survival estimate of 30%. In a retrospective analysis of S8710, the overall survival was
evaluated by the pathologic response at surgery.8 The median overall survival was 13.6 years
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in those with pT0, 10.6 years with pT1/pTIS/pTa, and 3.7 years in those with >pT1.

Neoadjuvant ddMVAC has been utilized in two recent single arm studies. In a study of 54
bladder cancer patients with cT2-T4a N0 or N1 disease, three cycles of ddMVAC were given
preoperatively.9 Of the 40 evaluable patients, 38% had pT0 at the time of surgery and 52%
had residual, non-muscle invasive disease at the time of cystectomy. In a second study of
neoadjuvant ddMVAC in 39 patients with cT2-T4 and N0 or N1 disease, 49% achieved non-
muscle invasive disease.10

Coexpression extrapolation (COXEN) is a predictive biomarker approach developed by

Theodorescu and colleagues.11,12 Conceptually different than standard approaches, COXEN
is the first demonstration that development of predictive biomarkers is possible in the
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absence of clinical response data from patients. Technically, COXEN is based on an in vitro
assessment performed using the NCI-60, a bank of 60 well-characterized cell lines from a
wide range of cancer types, which include drug sensitivity and gene expression data among
other characterizations.13 Once the in vitro gene expression model (GEM) signature
associated with response is established, it is correlated with a histologically relevant gene
expression data set obtained from treatment naive human tumors to identify concordant
genes. GEMs developed for multiagent regimens are a compilation of GEMs for each
individual drug. Through this process, a correlation coefficient is derived for each individual
gene in the GEM signature with those showing concordance in the human sample.
Expression of this GEM signature can then be directly assessed to any transcriptionally
profiled individual patient sample. A “COXEN score” is then generated from the GEM
signature correlation coefficients and this is used to predict response (see Supplemental
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figure 1 for more details).

In S1314, we enrolled patients with muscle-invasive bladder cancer who were eligible for
cisplatin-based multi-agent chemotherapy and in whom cystectomy was planned. Patients
were randomized between neoadjuvant GC and ddMVAC chemotherapy with treatment
assignment independent of the GEM signature derived COXEN score. The primary endpoint
was pathologic response at the time of cystectomy. The purpose of the trial was to evaluate
whether either the pre-specified GC or the ddMVAC COXEN score dichotomies were

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associated with favorable response to neoadjuvant chemotherapy defined as pT0 or ≤pT1 at

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radical cystectomy. This strategy could be evaluated in a subsequent prospective trial. We

also assessed whether either score helped to predict which of the two regimens a patient
would be more likely to respond. As a phase II study, this trial was not designed to use
COXEN GEM scores to prospectively predict response to GC or ddMVAC chemotherapy.

Methods
Patients
The study was reviewed and received approval by the NCI Central Institutional Review
Board (CIRB), and patients provided written, informed consent; it was conducted according
to the Declaration of Helsinki guidelines. Eligible subjects had histologically-proven
urothelial carcinoma of the bladder, stage cT2-T4a N0 M0 disease and a Zubrod
performance status of 0 or 1. Those with mixed histology, including a component of
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urothelial carcinoma, were eligible. Those with small cell carcinoma, pure adenocarcinoma,
and pure squamous cell carcinoma were excluded. Pathologic confirmation of 5 mm of
viable tumor on the transurethral resection of bladder tumor (TURBT) specimen was
required to provide adequate tissue for COXEN testing. TURBT and pelvic exam under
anesthesia were performed within 56 days prior to registration. Patients with previous
systemic cytotoxic chemotherapy for urothelial carcinoma, peripheral neuropathy ≥ Grade 2,
Class III or IV heart failure, and hearing impairment > Grade 2 were excluded. Adequate
organ function for chemotherapy was required, including sufficient bone marrow reserve and
a calculated creatinine clearance of ≥ 60 mL/min, using the modified Cockcroft-Gault
formula. The study was listed on clinicaltrials.gov with identifier: NCT02177695.

The subset of eligible patients was considered evaluable for the primary COXEN GEM
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assessment if they received at least 3 of 4 cycles of chemotherapy (or progressed while

receiving chemotherapy), had adequate pretreatment tissue for COXEN GEM assessment,
and either underwent a cystectomy within 100 days after chemotherapy or progressed prior
to the expected cystectomy. The latter group was managed as non-responders.

Study design and treatment

S1314 was a phase II study with 1:1 randomization between GC and ddMVAC
chemotherapy conducted by SWOG and other member groups of the National Clinical Trials
Network (Figure 1). Randomization was balanced on two stratification factors: performance
status (0 vs 1) and clinical T-stage (cT2 vs cT3/4). GC was administered as Gemcitabine
1,000 mg/m2 on days 1 and 8 with Cisplatin 70 mg/m2 on day 1 every 21 days for 4 cycles,
with optional growth factor support. Dose-dense MVAC was administered as methotrexate
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(30 mg/m2) on day 1, vinblastine (3 mg/m2) on day 1 or 2, doxorubicin (30 mg/m2) on day 1
or 2, and cisplatin (70 mg/m2) on day 1 or 2 every 14 days for 4 cycles, with mandatory
growth factor support use. At minimum, a bilateral standard pelvic lymph node dissection,
including the external and internal iliac and obturator nodes, was to be performed at the time
of the radical cystectomy. Patients were removed from protocol treatment for unacceptable
toxicity, inability to receive adequate chemotherapy (e.g. a continuous delay more than 3
weeks, cumulative delay of 4 weeks, more than one dose reduction required, or less than 3

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cycles of chemotherapy administered). Radiographic assessments of the chest, abdomen and

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pelvis by CT or MRI imaging were performed at baseline, in the preoperative period, every
3 months for one year, then every 6 months for 2 years, then annually until 5 years from
registration.

The gene expression analysis for the COXEN determination was obtained via an Affymetrix
U133A GeneChip via ALMAC laboratory (Durham, North Carolina). RNA was isolated
from 10-micron slides and the samples were analyzed in 2 batches, with 7 patients having
their samples run in both batches, as a quality control measure.

COXEN development
For the development of the COXEN score, the process started with gene expression
modeling using the NCI60 panel for each agent (Methotrexate, Vinblastine, Adriamycin,
Cisplatin and Gemcitabine). These data were then evaluated with human gene expression
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data to yield between 20 and 60 probes per agent. Model training and evaluation was then
performed with additional data sets to develop the combined COXEN scores and the cut
point for positivity for ddMVAC and GC regimens. The COXEN algorithms for each
regimen and cut points for defining favorable versus unfavorable score were established
prior to the start of the trial (see Supplemental figure 1 for additional details). The Laval
cohort, comprised of bladder cancer patients treated with cystectomy and lymph node
dissection with chemotherapy, was used to develop the COXEN GEM in both cases and
consists of formalin-fixed paraffin embedded (FFPE) tissues of cystectomy specimens
obtained from l'Hôpital de l'Hôtel-Dieu at Laval University, Québec, Canada as previously
described (Supplemental Figure 1).14 Frozen robust multiarray analysis was used to
normalize the microarray samples.15 The information utilized to calculate the individual
drug scores is included as Supplemental Table 1.
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Statistical analysis
The primary objective of S1314 was to evaluate whether either the pre-specified ddMVAC-
or GC-specific COXEN GEM scores are associated with pathologic response at the time of
cystectomy in patients treated with the respective neoadjuvant chemotherapy regimen. This
was done by evaluating whether the treatment specific COXEN GEM score was associated
with a complete response (pT0) rate or downstaging (≤ pT1) in the corresponding treatment
group or in the pooled arms. Logistic regression was used to model pathologic response with
the respective dichotomous COXEN GEM score (favorable/unfavorable), managed as a
covariate with adjustment for the two stratification factors. All eligible, evaluable patients
were included. The protocol also specified that if no interaction was found between the
treatment specific GEM score and treatment arm, then data from both treatment arms could
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be combined to evaluate the predictive association of each GEM score with response to
neoadjuvant chemotherapy more generally.

Another component of the primary objective was to assess in a preliminary fashion whether
the COXEN GEM score is a regimen-specific predictive factor of pathologic response. This
was also evaluated in a logistic regression model, fit separately for each COXEN score. In
addition to stratification factors and dichotomous COXEN GEM score, an indicator for

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treatment arm and the interaction of treatment arm with COXEN GEM score was also
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included in the model, using the pooled sample. A significant interaction would suggest that
the respective COXEN GEM score was able to differentiate whether a patient was more
likely to respond to one chemotherapy regimen over another.

A secondary trial objective was to assess the difference in pT0 rate between the 21-day GC
and 14-day ddMVAC arms in an intent-to-treat (ITT) analysis of all eligible, randomized
patients, regardless of amount of treatment received, gene expression, or cystectomy status,
using a logistic regression model adjusted for stratification factors. All ITT analyses report
two-sided p values. We also assessed the safety and tolerability of 21-day GC and 14-day
ddMVAC chemotherapy when given in the neoadjuvant setting for bladder cancer. All
patients who received some protocol therapy were included in that analysis. Another
secondary objective was to evaluate the value of gene expression profiling in predicting
overall survival, but since survival data are not yet mature, that association will be evaluated
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in a subsequent report.

A sample size target of 184 eligible, evaluable patients was utilized to develop the statistical
plan. The score from the COXEN GEM had previously yielded a sensitivity and specificity
of 83% and 64%, respectively in a cohort of patients with muscle-invasive bladder cancer
treated with neo-adjuvant MVAC chemotherapy.11 Applying a one-sided alpha of 0.05 and
using 92 patients for each within-treatment arm analysis, there would be 99% statistical
power to detect differences in pT0 rates where the absolute difference in the pT0 rate is 50%
between the predicted responders and non-responders (based on favorable COXEN GEM
score dichotomy) and 92% power to detect differences in pT0 rates of 30%. Although
statistical power was known to be low, the ability of the regimen specific COXEN GEM
score to direct which of the two neoadjuvant treatment regimens the patient should receive
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was investigated.

Results
A total of 237 patients was enrolled between 7/11/14 and 12/1/17. Ten patients were
ineligible (6 without tissue, 2 without adequate disease assessment and 2 with kidney
function out of window), leaving 227 patients. Of those, 11 had tissue that was inadequate
for COXEN assessment, 23 received less than 3 cycles of chemotherapy, and among those
who received 3-4 cycles, 26 did not have a cystectomy performed within 100 days, yielding
167 evaluable patients (Figure 1) for the primary COXEN GEM assessment. Non-evaluable
patients were not included in the primary COXEN analysis, but were included in the ITT
efficacy and safety evaluation. The median age was 64 years and the majority of evaluable
patients were male, most common stage was cT2 and the majority of patents had a Zubrod
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performance status of 0 (Table 1). The proportion of favorable GC and ddMVAC COXEN
GEM scores was similar in each treatment arm, although the proportion of favorable GC and
ddMVAC GEM scores was higher in the ddMVAC treatment arm. The cross-classification of
the favorable proportion of each COXEN GEM score and pathologic response are shown in
Table 2 by treatment arm and in pooling both arms.

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For the primary analysis, we determined the relationship of GC and ddMVAC COXEN
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GEM scores to pT0. The odds ratio for pT0 with respect to the GC biomarker in patients
treated with GC was 2.63 (p=0.10; 95% CI [0.82,8.36]). This translates into an observed
difference in pT0 response rates (Table 2) between favorable and unfavorable GC GEM is
44% versus 31%. For the ddMVAC COXEN GEM score with the endpoint of pT0 in those
treated with ddMVAC, the odds ratio was 1.12 (p=0.82, 95% CI [0.42, 2.95]) (Table 3). The
GC COXEN GEM score was applied to both arms for the outcome of downstaging (≤ pT1)
with an odds ratio of 2.33 (p=0.02; 95% CI [1.11, 4.89]); when the ddMVAC COXEN GEM
score was applied to both arms for the outcome of downstaging, the odds ratio was 0.90
(p=0.76; 95% CI [0.46, 1.75]) (Table 3). In this pooled analysis for the GC COXEN score,
the sensitivity for pT0 and downstaging was 32% with a specificity of 81% (Table 3).

Seven subjects had their samples run in two batches with GEM scores calculated and
dichotomized for both GC and ddMVAC (14 determinations). One patient had a mismatch
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for both GC and MVAC GEM score classification, and one patient each had a mismatch in
the MVAC GEM or GC GEM among the seven patients resulting in 4 of the 14
determinations in 3 patients not matching in the duplicate runs.

The predictive ability of the GC and ddMVAC COXEN GEM scores to assign specific
chemotherapy treatments was assessed. No significant interaction between treatment arm
and either COXEN GEM score was detected. Interaction p-values for the four models
(complete response and downstaging outcomes for each of the two COXEN GEM scores)
ranged from 0.43-0.88. Both the estimated Spearman and Pearson correlation coefficient
between the GC and ddMVAC GEM scores were 0.39 (p=0.001), indicating moderate
correlation.
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Treatment and safety

The percentage of eligible subjects with adequate tissue receiving 3-4 cycles of
chemotherapy was 86% for GC and 92% for ddMVAC (Figure 1). The proportion of
pathologic complete response was comparable in both arms with 30% in the GC and 28% in
the ddMVAC arms (p = 0.75). Similarly, the downstaging (partial or complete response
combined) rate was similar in both arms with 40% in the GC and 47% in the ddMVAC
groups (p = 0.27) (supplemental Table 2).

A summary of the adverse events is included in Table 4, with a complete listing in

Supplemental Table 3. Febrile neutropenia was noted in 2 patients in the GC arm and 1 in
the ddMVAC arm. In the intention-to-treat population (n=227), 100% in the ddMVAC and
60% in the GC arm received growth factor support. There were some clinically relevant
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differences in toxicities between the arms of the study with a higher incidence of mucositis,
constipation, and diarrhea with ddMVAC, and higher incidence of thrombotic events,
peripheral edema and neutropenia in the GC arm. There was one case of grade 5 toxicity
categorized as related or likely related to therapy in the study, a patient treated with
ddMVAC who died from cardiac arrest. There was one additional patient with grade 5
toxicity in the GC and two in the ddMVAC arm, categorized as unrelated or unlikely to be
related to chemotherapy.

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Discussion
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The GC and ddMVAC COXEN scores were not significantly associated with response in
their respective treatment arms. There was no evidence of an interaction between COXEN
score and a specific treatment regimen in predicting response. Several factors may explain
the lack of association with the primary objective. The statistical power analysis was based
on 92 evaluable patients in each arm; however, despite increasing the overall accrual goal
during the conduct of the trial, the number of evaluable patients was slightly below the
number used in the power calculations, with 82 with GC and 85 with ddMVAC. This was
largely driven by delayed/abandoned cystectomy and inadequate chemotherapy (i.e. < 3
cycles) and this may have impacted the power to detect an association in the primary
analysis with respect to each COXEN marker.

It should be stressed that S1314 is a phase II, randomized study and was not powered to be a
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definitive study in this setting. Prospective phase II studies of this nature are a key aspect of
the clinical evaluation of predictive biomarkers and necessary to inform the next step of
development. As described, the GC marker has a signal of association with pathologic
response, while the ddMVAC marker does not. It may also be possible that the observed
modest statistical association with the GC COXEN score is a false positive. We observed an
absolute difference in pT0 rate between favorable GC GEM and unfavorable of only 13%
(44% vs. 31%, Table 2) while the study was adequately powered for differences in the range
of 30%. Samples from 7 individuals were assessed in each of the two cohorts for both the
MVAC and GC biomarkers; notably, 4 of 14 determinations were discrepant between the
cohorts, which is of concern. Based on this finding, a larger number of replicates should be
tested in future clinical application of this approach to better characterize the fidelity of these
determinations across assessments.
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Interestingly, the score from the GC GEM was associated with response when applied to the
pooled arms with respect to downstaging, including p≤T1 (odds ratio of 2.33; p=0.02; 95%
CT [1.11, 4.89]). This translates into a 65% downstaging for those with a favorable GC
GEM and 48% downstaging for those categorized as unfavorable. The potential differences
in performance of the GC versus ddMVAC COXEN GEM scores is not clear. It is possible
that differences in the performance of these biomarkers may reflect an intrinsic limitation of
COXEN principle, as GEMs developed for multiagent regimens are a compilation of GEMs
for each drug individually. By this virtue, a significant amount of statistical noise is likely
generated for regimens with the more drugs that are included (e.g. ddMVAC). There is
planned additional testing of this hypothesis post hoc by evaluating GEM models for each
individual drug used in each arm as well as in both arms and will report this separately.
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S1314 provides a platform to develop and validate additional biomarkers in the bladder
cancer neoadjuvant setting. Using the prospectively obtained clinical data and outcomes,
assessments of circulating tumor cells, miRNA, DNA and SNP-based markers in these
samples are ongoing. In addition, further refinement of the COXEN algorithm as well as the
utility of GEM combinations in multiagent drug combination therapies will be evaluated as a
future aim. While the overall survival (OS) data are not yet mature, we will evaluate the
association of these GEM scores with OS in the future as these results mature.

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While not primarily designed or powered to directly compare the clinical activity of GC and
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ddMVAC, this study does provide comparison data in a prospective randomized study.
Overall, there was no clinically meaningful difference in the pathologic response between
the arms and the observed pathologic response rates were similar to the rates observed in
S8710 and other recent neoadjuvant chemotherapy trials.3,9,10 With respect to the adverse
events, there were some regimen-specific differences observed between the treatment arms,
which are not unexpected for these regimens.

In summary, this study did not find that COXEN score were predictive of pT0 rate or ≤ pT1.
However, the GC COXEN score did show a statistically significant association with
downstaging when applied to a pooled group including both treatment arms. This positive
association of the COXEN derived GEM score and pathologic response in this setting
validates a conceptual advance in biomarker development, namely that predictive
biomarkers may be developed based on established cancer cell line drug response but in the
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absence of patient response data. This represents a novel translation of an in vitro-developed

biomarker into a clinical application. Future plans include additional investigations of
COXEN GEM uses and the evaluation of other biomarkers using this clinical data set.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgements:
Support from NIH/NCI grant CA180888, CA180819, CA180820, CA180821, CA189830, CA180830, CA180834,
CA189829, CA189971, CA180798, CA233230, CA180818, CA189822, CA189854, CA189972, CA180858,
CA189872, CA189858, CA189958, CA189848, CA180835, CA189808, CA180826, CA180855, CA46368,
CA13612 and CA46282.
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doxorubicin, and cisplatin with pegfilgrastim support in muscle-invasive urothelial cancer:
pathologic, radiologic, and biomarker correlates. J Clin Oncol 32:1889–94, 2014 [PubMed:
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12. Lee JK, Havaleshko DM, Cho H, et al.: A strategy for predicting the chemosensitivity of human
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Translational relevance
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The coexpression extrapolation (COXEN) algorithm represents a novel approach to

generate predictive gene expression models (GEMs) for cancer therapy response, based
on in vitro results. This methodology allows for the direct and rapid testing of new
biomarkers in the clinical setting. S1314 is a randomized trial conducted by the National
Clinical Trial Network of neoadjuvant GC and ddMVAC chemotherapy for bladder
cancer and was prospectively designed to test COXEN generated GEMs in this setting.
While neither chemotherapy specific COXEN GEM scores predicted for response in a
statistically significant manner for a complete pathologic response, there was a signal of
prediction when the GC score was tested across both treatment arms for pathologic
downstaging. S1314 adds important randomized data comparing GC and ddMVAC
neoadjuvant chemotherapy and the data generated from this study will be used as a
platform to further refine the COXEN approach and assess other prespecified biomarkers.
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Figure 1. Consort diagram.

*These patients are included in secondary analysis of intent-to-treat comparison of path
response rates between arms
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Table 1.

Patient Characteristics by Randomized Treatment Arm for the COXEN eligible and Intent-to-Treat groups
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COXEN-evaluable population ITT analysis population

GC (N=82) ddMVAC (N=85) GC (N=115) ddMVAC (N=112)

Age (median, range) 64.4 (34.5, 79.2) 64.8 (33.1, 78.4) 64.9 (34.5, 79.2) 64.8 (33.1, 86.5)

Sex

Male 64 (79%) 75 (88%) 92 (80%) 99 (88%)

Female 18 (21%) 10 (12%) 23 (20%) 13 (12%)

Clinical Stage

T2 74 (92%) 73 (87%) 102 (89%) 98 (88%)

T3 or T4a 8 (8%) 12 (13%) 13 (11%) 14 (12%)

Zubrod Performance Status

0 61 (75%) 67 (80%) 88 (77%) 86 (77%)

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1 21 (25%) 18 (20%) 27 (23%) 26 (23%)

GC Score

Favorable 18 (21%) 25 (30%) 19 (22%) * 29 (31%) *

Not Favorable 64 (79%) 60 (70%) 68 (78%) * 64 (69%) *

ddMVAC Score

Favorable 23 (28%) 30 (36%) 24 (28%) * 31 (33%) *

Not Favorable 59 (72%) 55 (64%) 63 (72%) * 62 (67%) *

*
n=47 patients included in the ITT analysis (n=28 on the GC arm, n=19 on the ddMVAC arm) were not evaluable for the primary COXEN analysis
and therefore did not have COXEN scores generated.

Abbreviations:
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GC - Gemcitabine, cisplatin

MVAC - Methotrexate, vinblastine, adriamycin, cisplatin

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Table 2.

Cross-Classification of Treatment Arm, COXEN Score Dichotomy Status and Pathologic Response from
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Cystectomy

Chemotherapy GC Arm ddMVAC Arm Pooled Treatment Arms

Response (n=82) (n=85) (n=167)

Favorable Unfavorable Favorable Unfavorable Favorable Unfavorable Favorable Unfavorable

GC Score GC Score MVAC MVAC score GC Score GC Score MVAC Score MVAC Score
score
pT0 8 (44%) 20 (31%) 10 (26%) 17 (38%) 18 (42%) 37 (30%) 17 (32%) 38 (33%)

≤pT1, but not pT0 2 (12%) 10 (16%) 6 (15%) 14 (31%) 10 (23%) 22 (18%) 9 (17%) 23 (20%)

Non-responders 8 (44%) 34 (57%) 14(59%) 24 (31%) 15 (35%) 65 (52%) 27 (51%) 53 (47%)

Total 18 (100%) 64 (100%) 30 (100%) 55 (100%) 43 (100%) 124 (100%) 53 (100%) 114 (100%)
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Table 3.

Results of Logistic Regression Modeling of COXEN Score and Pathologic Response at Cystectomy
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COXEN Outcome Arm N Odds Ratio P- Sensitivity Specificity PPV NPV

Score (95% CI)** value** (95% CI) (95% CI) (95% CI) (95% CI)

GC* pT0 GC 82 2.63 0.10 29% 81% 44% 69%

(0.82, 8.36) (13%, 49%) (69%, 91%) (22%, 69%) (56%, 80%)

GC* ≤pT1 GC 82 1.75 0.30 25% 81% 47% 53%

(0.60, 5.34) (13%, 41%) (66%, 91%) 34%, 60% 40%, 66%

ddMVAC* pT0 ddMVAC 85 1.12 0.82 37% 63% 33% 44%

(0.42, 2.95) (19%, 58%) (46%, 78%) (17%, 53%) (17%, 53%)

ddMVAC* ≤pT1 ddMVAC 85 0.92 0.86 34% 63% 53% 69%

(0.37, 2.27) (21%, 49%) (46%, 78%) (34%, 72%) (55%, 81%)

GC* pT0 Pooled 167 1.84 0.10 33% 78% 42% 70%
(0.88, 3.83) (21%, 47%) (69%, 85%) (27%, 58%) (61%, 78%)

GC* ≤pT1 Pooled 167 2.33 0.02 32% 81% 65% 52%
(1.11, 4.89) (23%, 43%) (71%, 89%) (49%, 79%) (43%, 61%)
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ddMVAC* pT0 Pooled 167 0.99 0.99 31% 68% 32% 67%
(0.49, 2.02) (19%, 45%) (58%, 76%) (20%, 46%) (58%, 76%)

ddMVAC* ≤pT1 Pooled 167 0.90 0.76 30% 66% 49% 46%
(0.46, 1.75) (21%, 41%) (55%, 76%) (35%, 63%) (37%, 56%)

*
favorable based on prespecified COXEN algorithm and cut point
**
adjusted for two stratification factors – clinical stage at baseline (T2 vs T3, T4a), PS (0 vs 1) in logistic regression model

pT0=complete pathologic response, ≤pT1 = downstaging

Pooled = GC + ddMVAC arms, PPV=positive predictive value, NPV = negative predictive value
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Table 4 -
Summary of Selected Adverse Events Number of Patients with a Given Type and Grade
of Adverse Event
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Adverse Events Unlikely or Not Related to Treatment Excluded

GC+CYST DDMVAC+CYST
(n=114) (n=109)
Grade Grade

ADVERSE EVENTS 0 1 2 3 4 5 0 1 2 3 4 5
Alopecia 95 16 3 0 0 0 62 27 20 0 0 0
Anemia 38 34 32 10 0 0 46 36 19 8 0 0
Anorexia 87 19 8 0 0 0 74 23 12 0 0 0
Constipation 86 21 7 0 0 0 74 29 6 0 0 0
Cardiac arrest 114 0 0 0 0 0 108 0 0 0 0 1
Creatinine increased 87 14 13 0 0 0 77 17 10 5 0 0
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Diarrhea 102 11 1 0 0 0 85 19 3 2 0 0
Edema limbs 103 11 0 0 0 0 104 5 0 0 0 0
Fatigue 35 57 19 3 0 0 31 47 26 5 0 0
Febrile neutropenia 112 0 0 2 0 0 108 0 0 1 0 0
Mucositis oral 102 10 2 0 0 0 73 24 7 5 0 0
Neutrophil count decreased 72 6 16 17 3 0 95 3 2 5 4 0
Peripheral sensory neuropathy 103 8 3 0 0 0 96 10 3 0 0 0
Platelet count decreased 66 34 3 7 4 0 73 22 10 2 2 0
Sinus tachycardia 106 8 0 0 0 0 107 1 1 0 0 0
Thromboembolic event 104 1 5 3 1 0 106 0 2 1 0 0
Tinnitus 95 15 3 1 0 0 87 20 2 0 0 0
Vomiting 95 15 4 0 0 0 86 14 6 3 0 0
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White blood cell decreased 74 17 18 5 0 0 97 5 2 4 1 0

MAX. GRADE ANY ADVERSE EVENT* 4 16 44 41 9 0 1 20 47 30 10 1

*
including AE’s not highlighted in this particular table
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