Abstract

Background: Preclinical studies show that mitomycin-C (MMC) followed by irinotecan (CPT-11) is synergistic. Therefore, we evaluated the toxicity and efficacy of sequentially administered low-dose MMC and CPT-11 in patients (pts) with pretreated metastatic breast cancer (MBC). Secondary objective was to evaluate the correlation between MMC-induced topoisomerase I (TOPO I) expression and NAD(P)H:quinone oxireductase 1 (NQO1) genotypes in peripheral blood mononuclear cells (PBMC) and efficacy or toxicity of the regimen.

Design: Thirty-two pts received MMC i.v. 6 mg/m2 day 1 and CPT-11 i.v. 125 mg/m2 days 2 and 8 every 28 days for maximum of six cycles. TOPO I expression and NQO1 reductase genotyping in 23 of 32 (72%) pts were assayed by PCR.

Results: The median time to progression (TTP) was 4.7 months (95% confidence interval 4.0–5.4 months). TOPO I expression was increased 5- to 10-fold and 20- to 30-fold in PBMC at 24 and 168 h, respectively. There was no relationship between these markers and efficacy or toxicity of the regimen.

Conclusions: Sequential low-dose MMC and CPT-11 was active and tolerable by pretreated MBC pts. Future trials should focus on less pretreated MBC pts and sequential tumor biopsies to test the hypothesis that increased intratumoral expression of TOPO I is related to efficacy.

introduction

For patients (pts) with metastatic breast cancer (MBC) that is hormone receptor negative, resistant to hormonal therapy or presents as a life-threatening visceral disease chemotherapy is the only option [1, 2]. Anthracyclines and taxanes are the most active drugs in breast cancer; however, a significant proportion of MBC pts will have prior exposure to these drugs in the adjuvant or neoadjuvant setting. Thus, the development of novel combinations for MBC pts is a high priority.

Several phase II trials of irinotecan (CPT-11) demonstrate antitumor activity in MBC [3, 4]. MBC pts with prior anthracycline and taxane treatment received either 100 mg/m2 i.v. weekly for 4 weeks followed by 2-week rest or 240 mg/m2 i.v. every 3 weeks of CPT-11 in a randomized phase II trial [3]. In the weekly arm, which was superior in terms of efficacy and decreased toxicity, the overall response rate was 23% with a median time to progression (TTP) of 2.8 months (range 1.6–3.5 months). The most frequent non-hematologic toxicity was grade 3 or 4 diarrhea occurring in 13% and 4% of pts, respectively.

The mitomycin-C (MMC) dose in MBC is 12–20 mg/m2 i.v. every 6 weeks with response rates of 12%–25% [5] MMC, however, at these doses was associated with severe myelosuppression and rare life-threatening complications such as pulmonary toxicity and hemolytic uremic syndrome.

Preclinical studies show that MMC followed by CPT-11 is synergistic in cultured leukemia cells [6]. The potential mechanism is that MMC increases expression and activity of topoisomerase (TOPO) I, the target of CPT-11 [7, 8]. Villalona-Calero at our institution performed a phase I dose-escalation trial of sequentially administered low-dose MMC followed by CPT-11 in 37 solid tumor pts [8]. The results showed no pharmacokinetic interaction between these drugs when administered 24 h apart; acceptable side-effects at the maximum tolerated doses and evidence of antitumor activity. The induction of TOPO I expression in peripheral blood mononuclear cells (PBMC) was associated with response to the regimen. Included were five heavily pretreated inflammatory MBC pts, three of whom had either an objective response or a clinical benefit.

We hypothesized that sequentially administered low-dose MMC and CPT-11 would have acceptable side-effects and antitumor activity in MBC pts with prior treatment with anthracyclines and taxanes. This hypothesis was tested in a phase II trial with the primary end points of TTP and toxicity. Secondary end points were to evaluate TOPO I gene expression and NAD(P)H:quinone oxireductase 1 (NQO1) genotypes in PBMC and see whether this relates to TTP or toxicity. NQO1 reductase is thought necessary for the activation of MMC [9]. A single nucleotide polymorphism (SNP) at base pair 609 has been identified in human cancer lines and human colon and lung cancers [10–12]. This SNP results in the conversion of proline to serine at position 187 of the NQO1 enzyme, and homozygotes for this SNP generally have no enzyme activity.

patients and methods

Eligible pts were 18 years or older with histologically confirmed breast cancer, Eastern Cooperative Oncology Group (ECOG) performance status of two or less, measurable and/or non-measurable metastatic disease, and life expectancy of >3 months. The following laboratory studies were required: absolute neutrophil count ≥1500 cells/mm3, platelets ≥100 000 cells/mm3, hemoglobin ≥9 g/dl, serum creatinine ≤1.5 mg/dl or calculated creatinine clearance ≥60 ml/min, serum bilirubin ≤1.5 × upper limit normal (ULN), aspartate aminotransferase (AST) or alanine aminotransferase (ALT) and/or alkaline phosphatase <3 × ULN if no liver mets; AST or ALT <5 × ULN in the presence of liver metastasis. Pts must have received prior chemotherapy including taxane and anthracycline as adjuvant, neoadjuvant or the treatment of metastatic disease. No more than two prior chemotherapy regimens for MBC were permitted. Prior treatments with MMC, CPT-11 or nitrosoureas were not allowed. The study protocol was approved by the Clinical Scientific Review Committee and Institutional Review Board at The Ohio State University Medical Center and Comprehensive Cancer Center, and written informed consent was obtained from all pts.

treatment plan

MMC 6 mg/m2 i.v. was administered by slow intravenous push over 10–20 min through a free-flowing intravenous solution on day 1 of each 28-day cycle. CPT-11 i.v. 125 mg/m2 was administered as a 90-min infusion 24 h after the administration of MMC (day 2) and repeated on day 8 of each 28-day cycle. Pts were assessed for tumor response after the completion of every two cycles. Chemotherapy was continued for a maximum of six cycles or a total cumulative dose of MMC of 36 mg/m2. In pts with complete response (CR), partial response (PR) or stable disease (SD) after six cycles, CPT-11 alone was continued until disease progression.

Toxic effects were graded according to the Common Toxicity Criteria Version 2.0. Toxicity assessments and complete blood counts with differential were obtained on days 1 and 8 of each treatment cycle. Physical examination and serum chemistry studies were obtained on day 1 of each treatment cycle. Blood samples for correlative studies were obtained during cycle 1 just before MMC infusion (baseline) and then 24 and 168 h after MMC just before CPT-11 treatment on day 8.

dose modifications

On day 1 of a new treatment cycle, the ANC and platelets were required to be ≥1500/mm3 and ≥100 000/mm3, respectively, and any other treatment-related toxic effects that developed during the previous cycle had to resolve to ≤grade 1. Otherwise, treatment was held for up to 2 weeks. If the toxic effects did not resolve to ≤grade 1 after the 2-week delay, the pt was removed from the study. For pts experiencing toxicity within a cycle, the dose of CPT-11 on day 8 was omitted for grade >2 hematologic and non-hematologic toxic effects. Dose modifications at the start of subsequent courses of therapy were on the basis of toxic effects developing during the prior treatment cycle. The dose of MMC was reduced to 4 mg/m2 for neutropenic fever, grade 4 hematologic toxicity, and grade ≥3 non-hematologic toxic effects (with the exception of fatigue). The dose of CPT-11 was reduced by 25% for neutropenic fever, grade 4 nausea, and grade 3 non-hematologic toxic effects (with the exception of fatigue). The dose of CPT-11 was reduced by 50% for grade 4 non-hematologic toxic effects. Prophylactic use of colony-stimulating factors was not permitted.

tumor evaluation and criteria for response

Imaging studies and/or physical exam measurements of tumor sites were obtained within 28 days before starting first cycle of treatment. Pts were evaluated for tumor response every two treatment cycles by the same methods used at baseline. Response assessment was carried out according to response evaluation criteria in solid tumors criteria in pts with measurable disease [13].

correlative studies

RNA was extracted and complementary DNA (cDNA) was synthesized by standard methods. TOPO I and NQO1 were amplified from cDNA as previously described [14, 15]. Briefly, all PCR was carried out in a total volume of 100 μl containing 10 μl of Gibco 10× buffer, 2 μl of 10 mM dNTPs, 3 μl of 50 mM MgCl2, 50 pmol of each primer, 5 μl of 10−15 internal standard sequence and 250 ng of template cDNA. Samples were denatured in thermal cycler (MJ Research, San Francisco, California) at 92°C for 2 min before adding 2 U Taq polymerase. The samples were amplified by 92°C for 30 s, 58°C for 60 s and 72°C for 90 s for 32 cycles. Samples were then subjected to a final extension step at 72°C for 5 min and stored at −20°C for further analysis. All RT-PCR samples were analyzed by capillary electrophoresis with laser-induced fluorescence (CE-LIF). Separations were carried out on a P/ACE 2050 CE system using CE-LIF in the reversed-polarity mode at excitation of 488 nm and emission of 520 nm. Samples were introduced hydrodynamically by 10-s injections at 3.4 kPa across a 65-cm, 100-μm internal diameter coated eCAP double-stranded DNA (dsDNA) capillary filled with replaceable linear polyacrylamide. The capillary was conditioned with eCAP dsDNA 1000 gel buffer that contained 60 μl of LiFluor dsDNA 1000 EnhanceCE (thiazole orange) intercalator per 20 ml. Separations were carried out under constant voltage at 7.0 kV for 15–30 min.

Specific oligonucleotide primers for amplification by PCR of NQO1 gene fragments from genomic DNA were derived from known sequences (GenBank accession no. AC005068) using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). PCR for pyrosequencing was carried out in 40 μl reactions containing 20 μl PCR Master Mix (Promega, Madison, WI), 10 pmol forward and reverse primer (IDT Technology, Coraville, IA), 14 μl of nuclease-free water and 10–100 ng of genomic DNA. PCR amplification was carried out under the following conditions: initial denaturation at 95°C for 5 min, 50 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s followed by a final extension step at 72°C for 5 min.

The pyrosequencing primers were designed using SNP Primer Design Software Version 1.01 (http://www.pyrosequencing.com). Briefly, 35 μl of biotinylated PCR product was immobilized on streptavidin-coated Sepharose beads (Amersham Biosciences, Piscataway, NJ) with binding buffer (10 mmol/l Tris–HCl, 2 mol/l NaCl, 1 mmol/l EDTA, and 0.1% Tween 20, pH 7.6). After room temperature incubation with constant agitation for 10 min, the strands were separated and treated with 70% ethanol, denaturation solution (0.2 mol/l NaOH) and washing buffer (10 mmol/l Tris–acetate, pH 7.6). The beads, containing the biotinylated template, were released into wells with a 40-μl mixture of annealing buffer (20 mmol/l Tris–acetate, 2 mmol/l magnesium acetate tetrahydrate, pH 7.6) and 21 pmol of sequencing primer (IDT Technology). Incubation was carried out at 80°C for 2 min. Genotyping was subsequently carried out using a PSQ 96 SNP Reagent Kit and PSQ 96 MA System (Biotage AB, Uppsala, Sweden). Genotypes were resolved on the basis of peak height measurements using PSQ 96 SNP Software, Version 1.2 AQ. This software automatically performs genotyping and quality assessment of the raw data utilizing an algorithm. Genotype is scored automatically on the basis of pattern recognition. Quality value is assigned on the basis of several parameters such as the difference in match between the best and next best choice genotypes, agreement between expected and obtained sequence around the SNP, signal-to-noise ratios, variance in peak heights around the SNP and peak width.

statistical design

In anthracycline- and taxane-pretreated MBC pts, the median TTP is between 2 and 4 months for single-agent capecitabine, vinorelbine, gemcitabine, pegylated liposomal doxorubicin and CPT-11 [3, 16–23]. A Fleming single-stage phase II design with a sample size of 32 pts (p1 = 0.75, p0 = 0.50, α = 0.05 and β = 0.10) was chosen for this trial. The regimen would be recommended for further study if 75% of pts remained progression free at 4 months, whereas the regimen would be deemed uninteresting if <50% of pts remained progression free at 4 months.

The definition of TTP was from day 1 of treatment to the first evidence of disease progression or death in pts without evidence of disease progression. All analyses were on the basis of intention-to-treat population. The correlative studies were hypothesis generating secondary end points and as such no formal statistical tests were carried out.

results

patient population

Between July 2002 and May 2005 thirty-two pts enrolled (Table 1). The median age was 50 years (range 29–73 years). Twenty (63%) pts had ECOG performance status 0, 29 (91%) were postmenopausal and 13 (41%) were hormone receptor negative. Ten (31%) pts had estrogen receptor (ER) negative, progesterone receptor negative and HER2-neu negative tumors and seven (22%) had HER2 overexpressing tumors either standard immunohistochemical methods or by fluorescence in situ hybridization. All 32 (100%) pts received prior anthracyclines and taxanes in either the adjuvant or the metastatic setting. Twenty-eight (87%) pts received adjuvant or neoadjuvant chemotherapy and 19 (59%) pts received two prior chemotherapy regimens for metastatic disease.

Table 1.

Patient characteristics (n = 32)

 n (%) 
Median age (range) 50 (29–73) years 
ECOG performance status  
    0 20 (63) 
    1 11 (34) 
    2 1 (3) 
Race  
    White 28 (88) 
    African-American 3 (9) 
    Asian 1 (3) 
Menopausal status  
    Premenopausal 3 (9) 
    Postmenopausal 29 (91) 
Receptor status  
    ER and/or PR positive 19 (59) 
    ER/PR negative 13 (41) 
    ER/PR/HER2-neu negative 10 (31) 
    HER2-neu 3+ 7 (22) 
Prior treatment  
    Adjuvant chemotherapy 28 (88) 
    Prior anthracyclines 27 (84) 
    Prior taxanes 14 (44) 
Number of chemotherapy regimens for metastases  
    0 6 (19) 
    1 7 (22) 
    2 19 (59) 
Metastatic sites  
    Lung 17 (53) 
    Liver 14 (44) 
    Bone 13 (41) 
    Lymph nodes 12 (38) 
    Chest wall 5 (16) 
 n (%) 
Median age (range) 50 (29–73) years 
ECOG performance status  
    0 20 (63) 
    1 11 (34) 
    2 1 (3) 
Race  
    White 28 (88) 
    African-American 3 (9) 
    Asian 1 (3) 
Menopausal status  
    Premenopausal 3 (9) 
    Postmenopausal 29 (91) 
Receptor status  
    ER and/or PR positive 19 (59) 
    ER/PR negative 13 (41) 
    ER/PR/HER2-neu negative 10 (31) 
    HER2-neu 3+ 7 (22) 
Prior treatment  
    Adjuvant chemotherapy 28 (88) 
    Prior anthracyclines 27 (84) 
    Prior taxanes 14 (44) 
Number of chemotherapy regimens for metastases  
    0 6 (19) 
    1 7 (22) 
    2 19 (59) 
Metastatic sites  
    Lung 17 (53) 
    Liver 14 (44) 
    Bone 13 (41) 
    Lymph nodes 12 (38) 
    Chest wall 5 (16) 

ER, estrogen receptor; PR, progesterone receptor; ECOG, Eastern Cooperative Oncology Group.

treatment summary

Pts received a median of four cycles (range 1–12 cycles). Nine (28%) pts completed all six treatment cycles and six (19%) of them continued treatment with CPT-11 alone. Eight (25%) pts received only one treatment cycle for the following reasons: two (6%) had disease progression confirmed by imaging studies after cycle 1 and six (19%) pts developed grade ≥3 toxicity during cycle 1 and were removed from the study.

toxicity

Table 2 describes treatment-related adverse events. The majority of adverse events were grade ≤2. Removal from study for toxicity occurred in 13 (41%) pts, including the six who were removed during cycle 1. Toxic effects that necessitated removal during cycle 1 were diarrhea (two pts), neutropenia (one pt), abdominal pain (one pt) and vomiting (two pts). Nine (28%) pts required dose reductions, and there were no treatment-related deaths.

Table 2.

Toxicity per patient

 n (%)
 
Grade 0–1 Grade 2 Grade 3 Grade 4 
Hematologic     
    Neutropenia 16 (50) 3 (9) 12 (38) 1 (3)a 
    Anemia 24 (75) 8 (25) 
    Thrombocytopenia 24 (75) 6 (19) 2 (6) 
Non-hematologic     
    Fatigue 9 (27) 18 (57) 5 (16) 
    Diarrhea 14 (41) 11 (34) 6 (19) 1 (3) 
    Nausea 16 (50) 9 (28) 7 (22) 
    Vomiting 25 (78) 3 (9) 4 (13) 
    Infectionb 27 (84) 5 (16) 
    Anorexia 15 (47) 17 (53) 
    Dyspnea 19 (59) 13 (41) 
    Pain 22 (69) 7 (22) 2 (6) 1 (3) 
    Thromboembolism 31 (97) 1 (3) 
 n (%)
 
Grade 0–1 Grade 2 Grade 3 Grade 4 
Hematologic     
    Neutropenia 16 (50) 3 (9) 12 (38) 1 (3)a 
    Anemia 24 (75) 8 (25) 
    Thrombocytopenia 24 (75) 6 (19) 2 (6) 
Non-hematologic     
    Fatigue 9 (27) 18 (57) 5 (16) 
    Diarrhea 14 (41) 11 (34) 6 (19) 1 (3) 
    Nausea 16 (50) 9 (28) 7 (22) 
    Vomiting 25 (78) 3 (9) 4 (13) 
    Infectionb 27 (84) 5 (16) 
    Anorexia 15 (47) 17 (53) 
    Dyspnea 19 (59) 13 (41) 
    Pain 22 (69) 7 (22) 2 (6) 1 (3) 
    Thromboembolism 31 (97) 1 (3) 
a

Febrile neutropenia.

b

Infection without neutropenia.

efficacy

Table 3 describes the treatment efficacy. Sixty-two percent of pts were progression free at 4 months corresponding to a median TTP of 4.7 months [95% confidence interval (CI) 4.0–5.4 months]. In the pts with measurable disease there were no CRs, 10 (31%) pts had a PR and 13 (41%) pts had SD. Three (9%) pts had SD lasting for >6 months, for an overall clinical benefit rate of 13 (40%). In the group of 10 pts with triple negative (estrogen, progesterone, HER2 negative) breast cancers, four pts had PR and the median TTP was 4.6 months.

Table 3.

Response rate

 n (%) 95% confidence interval 
Complete response  
Partial response 10 (31)  
Overall response rate 10 (31)  
Stable disease 13 (38)  
Stable disease > 6 months 3 (9)  
Progressive disease 9 (28)  
Clinical benefit rate 14 (40)  
Median time to progression 4.7 months 4.0–5.4 months 
 n (%) 95% confidence interval 
Complete response  
Partial response 10 (31)  
Overall response rate 10 (31)  
Stable disease 13 (38)  
Stable disease > 6 months 3 (9)  
Progressive disease 9 (28)  
Clinical benefit rate 14 (40)  
Median time to progression 4.7 months 4.0–5.4 months 

correlative studies

Figure 1 describes the induction of TOPO I gene expression in PBMC during cycle 1 in 23 (72%) pts with available specimens at baseline (pre-MMC), 24 and 168 h. Overall, gene expression of TOPO I increased 10- and 29-fold at 24 and 168 h, respectively, after MMC. Table 4 describes the expected and observed frequency of NQO1 genotypes. The observed and expected frequency of the genotypes CC wild-type, TC heterozygotes and TT homozygotes were similar. Figure 2 describes TOPO I gene expression in CC, CT and TT genotypes. In CC, the baseline levels of TOPO I expression were increased and after MMC, they further increased ∼5- and 20-fold at 24 and 168 h, respectively. This is in contrast to heterozygotes and homozygotes where expression was not increased. However, neither overall TOPO I gene expression nor within NQO1 genotypes was related to toxicity or TTP.

Table 4.

NAD(P)H:quinone oxireductase 1 genotypes in peripheral blood mononuclear cells (n = 23)

 CC (wild-type) CT (heterozygote) TT (homozygote) 
Expected frequency 0.735 0.265 0.520 0.431 0.049 
Observed frequency 0.761 0.239 0.565 0.391 0.044 
 CC (wild-type) CT (heterozygote) TT (homozygote) 
Expected frequency 0.735 0.265 0.520 0.431 0.049 
Observed frequency 0.761 0.239 0.565 0.391 0.044 
Figure 1.

Topoisomerase I gene expression as measured by RT-PCR in peripheral blood mononuclear cells at baseline, 24 and 168 h after mitomycin-C administration.

Figure 1.

Topoisomerase I gene expression as measured by RT-PCR in peripheral blood mononuclear cells at baseline, 24 and 168 h after mitomycin-C administration.

Figure 2.

Topoisomerase I (TOPO I) gene expression as measured by RT-PCR in peripheral blood mononuclear cells at baseline, 24 and 168 h after mitomycin-C administration by NAD(P)H:quinone oxireductase 1 genotype. Pts with the CC (wild-type) have increased TOPO I gene expression at baseline and expression increases further at 24 and 168 h.

Figure 2.

Topoisomerase I (TOPO I) gene expression as measured by RT-PCR in peripheral blood mononuclear cells at baseline, 24 and 168 h after mitomycin-C administration by NAD(P)H:quinone oxireductase 1 genotype. Pts with the CC (wild-type) have increased TOPO I gene expression at baseline and expression increases further at 24 and 168 h.

discussion

Sequential administration of low-dose MMC and CPT-11 in MBC has antitumor activity with grade 1 and 2 toxic effects in the majority of pts. Grade 3 toxic effects including fatigue, nausea, vomiting and diarrhea were observed in up to 22% of pts, and grade 4 toxic effects were infrequent (Table 2). Six (19%) pts were removed from the trial during cycle 1 including one pt for prolonged neutropenia and two pts for severe diarrhea. Although uridine diphosphate glucuronosyltransferase (UGT)1A1 polymorphism was not tested, it is of interest to speculate whether some of these pts were homozygous or heterozygous for UGT1A1*28 alleles, which predisposed them to neutropenia and diarrhea [24, 25].

The combination of MMC and CPT-11 has been evaluated in solid tumors [26–28]; however, this is the first trial in MBC pts. In terms of efficacy, the median TTP of 4.7 months (95% CI 4.0–5.4 months) is comparable to the results of other trials with single-agent docetaxel, capecitabine, vinorelbine, gemcitabine, CPT-11, pegylated liposomal doxorubicin and MMC with vinblastine in similar MBC pts [16–23]. Other phase II trials have combined MMC and capecitabine or CPT-11 and docetaxel in similar anthracycline- and taxane-pretreated pts with an apparent improvement in TTP relative to the results observed in the current trial [29–32]. These indirect comparisons between phase II trials are limited by the pt selection criteria, individual host and tumor heterogeneity and the small sample sizes with resultant wide CI surrounding estimates of benefit or toxicity. Only direct comparisons of new regimens in adequately powered randomized trials can answer questions of efficacy and toxicity.

There is an ongoing debate over benefit of combination chemotherapy versus sequentially administered single drugs [2]. Whether MMC and CPT-11 is a novel two-drug combination or MMC is acting mainly as a modulator enhancing the activity of CPT-11 is still an unanswered question. The underlying hypothesis of this clinical trial was that MMC by increasing the expression and activity of TOPO I, the target enzyme of CPT-11, would enhance the antitumor effect [7, 8]. There is a precedent for MMC as a modulator to enhance activity of other drugs. Low-dose MMC also induces thymidine phosphorylase in human rectal cancers [33], and the phase II trials of low-dose MMC and capecitabine showed antitumor activity in MBC pts who received prior anthracyclines and taxanes [29, 30].

In this study, we confirmed the increased TOPO I expression in PBMC after MMC (Figure 1) as it was observed in the prior phase I trial [8]. We were not able to demonstrate correlation between increased efficacy of the regimen and increased TOPO I expression in PBMC. The lack of this correlation can be related to the fact that changes observed in PBMC may not reflect the changes occurring in the tumor. PBMC are relatively easy to obtain as opposed to invasive biopsies. We planned to obtain tumor biopsies before and after MMC. Unfortunately, many pts did not have metastases amenable to sequential biopsies, and in other cases, the pt refused them as it was optional. In addition, since only 23 pts provided sequential PBMC for analyses, the power to detect association with toxicity or TTP was diminished. We confirmed that individuals who were wild-type for NQO1 (and were able to activate MMC) had increased TOPO I induction compared with those who were NQO1 heterozygotes or variants (Figure 2).

Sixty-two percent of pts were progression free at 4 months, which did not meet the a priori criteria for recommending further study of regimen (i.e. 75% pts would be progression free at 4 months). However, we think that this regimen may merit further study for the following reasons: (i) the pts were heavily pretreated receiving a median of 2 (range 0–2) prior regimens for metastases; (ii) about one-third of the pt tumors were phenotypically triple negative (ER negative, progesterone receptor negative and HER2 non-overexpressing) and most of these tumors will be basaloid subtype [34] with a poorer prognosis [35] and (iii) in the planning stages of this trial, we may have overestimated the expectation of efficacy as the median TTP. In similarly pretreated pts, the median progression-free survival is between 2 and 4 months [16–23]. To detect smaller differences in progression-free survival with adequate power would have required increasing the sample size and was not feasible at a single institution.

The results in this trial justify using this novel regimen in less pretreated MBC pts. We would propose a neoadjuvant trial with sequential tumor biopsies to test the hypothesis that low-dose MMC increases the expression of TOPO I leading to enhanced antitumor effects of CPT-11. Additional preclinical xenograft models may possibly better characterize the synergism between MMC and CPT-11 and identify more robust pharmacodynamic end points.

funding

National Comprehensive Cancer Network.

This study was approved by the National Comprehensive Cancer Network from general research support provided by Pfizer, Inc.

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