Abstract
Background
Poly(ADP-ribose) polymerase (PARP) inhibitors, such as veliparib are potent sensitizing agents and have been safely combined with DNA-damaging agents such as temozolomide. The sensitizing effects of PARP inhibitors are magnified when cells harbor DNA repair defects.
Methods
We performed a single arm, open label Phase II study to investigate the disease control rate (DCR) after 2 cycles of veliparib plus temozolomide in patients with metastatic colorectal cancer (mCRC), refractory to all standard therapies. 50 patients received temozolomide (150mg/m2/day) days 1-5, and veliparib (40mg BID) days 1-7 of each 28-day cycle. An additional 5 patients with mismatch repair enzyme deficient (dMMR) tumors were also enrolled. Twenty additional patients were then treated with 200mg/m2/day of temozolomide. Archived tumor specimens were used for immunohistochemistry to assess for MMR, PTEN, and MGMT protein expression levels.
Results
The combination was well tolerated, although some patients required dose reductions for myelosuppression. The primary endpoint was successfully met with a DCR of 24%, with two confirmed PRs. The median PFS was 1.8 months, and the median OS was 6.6 months. PTEN protein and MGMT protein expression were not predictors of DCR. There was also a suggestion of a worse outcome in patients with dMMR tumors.
Conclusions
In this heavily pretreated mCRC population, combining veliparib and temozolomide was well-tolerated at doses up to 200mg/m2/day of temozolomide, and was clinically active. PARP inhibitor-based therapy merits further exploration in patients with mCRC.
Keywords: veliparib, temozolomide, colorectal cancer, PTEN, MGMT, mismatch repair genes
INTRODUCTION
Treatment options for metastatic colorectal cancer (mCRC) have improved significantly over the last 2 decades, but most patients ultimately experience disease progression, and are left without established treatment options. A novel approach to the treatment of mCRC may be targeting a critical DNA repair pathway through the inhibition of Poly(ADP-ribose) polymerase (PARP). PARP is a nuclear enzyme that plays a critical role in DNA damage repair(1-3). Inactive PARP is autoactivated upon binding to damaged DNA and subsequently poly(ADP-ribosyl)ates many nuclear target proteins, including those that facilitate the repair of both single- and double-stranded DNA breaks. Thus, PARP inhibition results in less efficient DNA repair following a cytotoxic insult. Colon cancer cells are characteristically genetically unstable, and thus, should be more dependent on PARP for DNA repair(3-5). Based on this notion, PARP inhibitors are proposed as sensitizing agents for a variety of DNA-damaging chemotherapeutic agents(6).
Veliparib (ABT-888, Abbvie, Inc) is a PARP inhibitor that has proven in vivo activity(7), and has been shown to increase tumor cell sensitivity to chemotherapy and radiation(8-11). In humans, veliparib is safe and demonstrates inhibition of PARP activity in tumor biopsies(7). Temozolomide is a potent DNA alkylating agent approved for therapy of CNS tumors and melanoma, and is one of the most studied alkylating agents used with PARP inhibitors(12). Several prior publications have evaluated the use of single-agent temozolomide in mCRC patients, with mixed results. Initially, in one small Phase II study of the combination of temozolomide plus the O(6)-methylguanine-DNA methyltransferase (MGMT)-inhibitor, lomeguatrib, three patients had prolonged stable disease (SD)(13). Additionally, a small case series demonstrated activity of temozolomide in mCRC patients with low MGMT expression(14). In 2013, two publications evaluated the use of temozolomide in mCRC patients, and MGMT promoter methylation. Hochhauser, et al, evaluated 37 mCRC patients as part of a larger Phase II trial, with 1/37 patients responding to therapy(15). By contrast, Pietrantonio et al, enrolled 32 mCRC patients with MGMT promoter methylation in a Phase II trial and demonstrated a 12% response rate with temozolomide (16).
Importantly, cancer cells with one or more DNA repair pathway defects have been shown to be exquisitely sensitive to a PARP inhibitor, alone or combined with chemotherapy (i.e. synthetic lethality)(17,18). This has been classically demonstrated in pre-clinical studies and human trials in the context of BRCA1/2 and homologous-repair deficiency(19-22). Additionally PARP inhibitor efficacy has been described in the setting of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency(23,24). The connection between PTEN deficiency and PARP inhibitor effectiveness is rational since PTEN was functionally established as a phosphatase that can regulate the phophoinositide 3 kinase (PI3K) signaling pathway(25) which has also been linked to maintaining genomic integrity(26,27). In fact, it was shown that tumors lacking PTEN exhibited a defect in homologous recombination(28). Thus, PTEN-deficient cells were hypersensitive to PARP-inhibitors in both in vitro and in vivo models(28). Clinically, PTEN deficiency is relevant to this study since estimates show that up to 40% of CRC patients have an absence of PTEN cytoplasmic expression in tumor cells(29,30).
Furthermore, 5-7% of spontaneous mCRCs are characterized by high levels of microsatellite instability (MSI), which is a marker for, and occurs as a result of a loss of expression or mutation of mismatch repair (MMR) genes, also labeled mismatch repair deficient (dMMR) tumors. Mismatch repair enzyme expression levels can be detected with a very high sensitivity from paraffin-embedded tumor samples, and correlates well with microsatellite instability status(31). In fact, testing for MSI status and/or MMR-protein deficiency has become standard practice for all colon cancer patients(32). Pre-clinical data have demonstrated that dMMR cells are significantly more sensitive to the combination of veliparib and cisplatin, irinotecan, and temozolomide compared to MMR-proficient cells(10,33). These clinical studies together with the premise that severe DNA damage, coupled by an inhibition of DNA repair mechanisms, would have a significant cytotoxic effect on cancer cells providing activity in patients (6,34,35).
Herein, we present a Phase II clinical trial of veliparib plus temozolomide in mCRC patients. The primary endpoint was DCR, and we have analyzed patient tumor samples for MMR enzyme expression, PTEN expression, and MGMT expression to identify a subgroup of patients who are more likely to benefit from this therapeutic combination.
PATIENTS AND METHODS
Patients
Patients with mCRC whose disease has progressed on, or who were intolerant of or ineligible for all standard therapies (including regimens containing fluoropyrimidine, oxaliplatin, irinotecan, bevacizumab, and an anti-EGFR antibody (where appropriate)) were eligible. (Prior treatment on the since approved regorafenib and TAS-102 was not a requirement). Patients were aged ≥18 years, had an Eastern Cooperative Oncology Group performance status score of ≤2, and had adequate organ and bone marrow function (hemoglobin ≥9.0 g/dL, absolute neutrophil count ≥1.5 × 109/L, platelet count ≥75 × 109/L, serum creatinine level <1.5 mg/dL, non-fasting direct bilirubin level ≤2.5 × upper limit of normal (ULN), and ALT/AST levels ≤3 × ULN in patients without liver metastases, and ≤5 × ULN in patients with liver metastases). The study protocol, amendments, the informed consent forms were approved by the Institutional Review Board at Georgetown University. Investigators obtained informed consent from each participant or participant’s guardian prior to screening.
Study Design and Treatment Schedule
This was a single center, Phase II, open label study of veliparib plus temozolomide. Veliparib was given at 40mg twice a day (BID) days 1-7 of each 28-day cycle. For patients enrolled in the original protocol cohort, temozolomide was given at 150mg/m2 orally daily days 1-5 of each 28-day cycle. The protocol was amended to enroll an additional 20 patients in a high-dose temozolomide cohort (200mg/m2 orally daily days 1-5 of each 28-day cycle (high-dose cohort)). Finally, a third patient cohort prospectively identified as being deficient in mismatch repair enzyme expression (dMMR) was enrolled (dMMR cohort). This cohort was closed for slow accrual after 5 patients. Safety assessments were performed weekly for the first two cycles, then biweekly thereafter. Tumor response was assessed radiographically every 2 cycles (unless clinically indicated to be done sooner) using Response Evaluation Criteria in Solid Tumors criteria. Study treatment was continued without interruption in the absence of unacceptable toxicity or progressive disease (PD). The primary endpoint was disease control rate (DCR), defined as the percent of patients with complete or partial response, or stable disease after 2 cycles. Secondary clinical endpoints included response rate, progression-free survival (PFS), and overall survival (OS).
Correlative Markers of Response to Therapy
Archived tumor samples were obtained and freshly cut slides were prepared to perform immunohistochemical (IHC) analysis. Additionally, patients in the high-dose and dMMR cohorts had fresh, pre-treatment tumor biopsies performed. IHC analysis of the MMR enzymes MLH1, MSH2, MSH6, and PMS2 was performed and scored by clinical pathologists in CLIA-certified hospital pathology laboratories. Immunohistochemistry of PTEN and MGMT protein expression was performed using standard assays with the following antibodies: anti-PTEN (138G6, 1:100 dilution; Cell Signaling, MA), and MGMT (sc-56432, 1:50 dilution; Santa Cruz, TX). MGMT staining was defined as “positive” when more than 10% of the tumor cells showed nuclear staining(36,37). PTEN “loss” was defined as >50% of cells showing loss of cytoplasmic staining, as previously described(38-40). Patients were grouped into clinical benefit (those with stable disease [SD] after 2 cycles or a partial response [PR]) versus no clinical benefit (those with PD).
Statistical Analysis
The initial 50 patients were enrolled following a Simon’s 2-stage design(41) to differentiate a 25% vs. 10% DCR, with 90% power and one-sided 10% significance level. DCR was defined as the percentage of patients with CR or PR any time or SD at the end of the 2nd cycle. This design continued if 3 or more patients of the first 21 disease control, and concluded successful treatment if 8 or more patients have disease control. The protocol was later amended to add 2 pilot cohorts of 20 patients each, 1 for high-dose treatment and one for dMMR patients. The selection of 20 patients for these pilot cohorts was to collect initial data for description only. Patient characteristics, medical features at study entry, and adverse events at least possibly related to study therapy were tabulated overall and by study cohort. Differences in DCR among subgroups were tested with chi-square tests, using exact calculations as needed for small sample sizes. OS was defined as the number of months from enrollment until death or last contact. Patients who were alive at the time of analysis were censored at their last contact. PFS was defined as the number of months from enrollment to progression or death, whichever occurred first. Patients who were alive and progression-free at the time of analysis were censored at their last tumor assessment. Kaplan-Meier curves were presented for OS and PFS. Analyses were performed in SAS 9.3 [SAS Institute Inc., Cary, NC, USA.] and figures were created using STATA 12.1 [StatCorp LP, College Station, TX, USA].
RESULTS
Patient Characteristics and Treatment Cohorts
Between September, 2009 and May, 2012, 75 patients were enrolled (Figure 1) to the 3 cohorts. The first stage of Simon’s 2-stage design continued with 3 patients having SD at the second cycle. After observing the low rate of toxicities and passing the Simon’s design success rate in the original 50 patients, the protocol was amended to allow for the enrollment of an additional 20 patients at a higher dose of temozolomide, in which patients received temozolomide at 200mg/m2 daily, days 1-5 of each 28 day cycle. Finally, based on pre-clinical evidence suggesting that patients with defects in mismatch repair gene expression (dMMR) would be more likely to respond to this combination (10,42), a third cohort of prospectively identified dMMR patients was enrolled. The dMMR cohort was meant to enroll 20 patients, but enrollment was stopped after 5 patients due to slow accrual.
Figure 1. Cohort Flowchart.
Patients were enrolled in three cohorts. There were 10 screen failures, with the reasons listed in the flowchart.
Patient characteristics are listed in Table 1. The median age was 56 years (range 35-72 years), most patients had an ECOG score of 0 or 1 (97%), and 56% of patients were male. All patients had received standard treatments including 5-fluorouracil (and/or capecitabine), oxaliplatin, irinotecan, and bevacizumab, with a median of 3 lines of prior therapy (range, 2-7). Of 67 patients with KRAS testing, 43% had KRAS wild-type tumors, all of whom received prior anti-EGFR therapy. Three patients were withdrawn prior to first restaging - 1 each for non-compliance, patient choice, and toxicity, all in the original cohort. All other patients were taken off study for PD. Figure 1 depicts the patient cohorts, screen failures, and reasons for withdrawal.
Table 1.
Patient Characteristics
| Cohort
|
|||||
|---|---|---|---|---|---|
| All | Original | High-Dose | dMMR | ||
| N (%) | N (%) | N (%) | N (%) | ||
|
| |||||
| All | 75 (100%) | 50 (100%) | 20 (100%) | 5 (100%) | |
| Age- median (min,max) | N=75 | 56 (35,72) | 54 (35,72) | 58.5 (40,68) | 52 (45,64) |
| Gender | |||||
| Female | 33 (44%) | 22 (44%) | 9 (45%) | 2 (40%) | |
| Male | 42 (56%) | 28 (56%) | 11 (55%) | 3 (60%) | |
|
| |||||
| Race/Ethnicity | |||||
| White/Non-Hispanic | 45 (60%) | 30 (60%) | 14 (70%) | 1 (20%) | |
| Black/Non-Hispanic | 17 (23%) | 12 (24%) | 3 (15%) | 2 (40%) | |
| Asian-PI/Non-Hispanic | 2 (3%) | 2 (4%) | 0 (0%) | 0 (0%) | |
| Any/Hispanic | 3 (4%) | 2 (4%) | 1 (5%) | 0 (0%) | |
| Unknown | 8 (11%) | 4 (9%) | 2 (10%) | 2 (22%) | |
|
| |||||
| ECOG | |||||
| 0 | 22 (29%) | 15 (30%) | 4 (20%) | 3 (60%) | |
| 1 | 51 (68%) | 33 (66%) | 16 (80%) | 2 (40%) | |
| 2 | 2 (3%) | 2 (4%) | 0 (0%) | 0 (0%) | |
|
| |||||
| Number of Primary Therapies -median (min,max) | N=75 | 3 (2,7) | 3 (2,7) | 3 (2,5) | 3 (2,5) |
|
| |||||
| Therapies* | |||||
| Cetuximab/Panitumumab | 37 (49%) | 26 (52%) | 9 (45%) | 2 (40%) | |
| Other Therapy | 13 (17%) | 10 (20%) | 3 (15%) | 0 (0%) | |
|
| |||||
| MMR | |||||
| Stable | 49 (65%) | 29 (58%) | 20 (100%) | 0 (0%) | |
| dMMR | 6 (8%) | 1 (2%) | 0 (0%) | 5 (100%) | |
| Indeterminate | 2 (3%) | 2 (4%) | 0 (0%) | 0 (0%) | |
| Missing | 18 (24%) | 18 (36%) | 0 (0%) | 0 (0%) | |
|
| |||||
| PTEN | |||||
| LOSS | 30 (40%) | 23 (46%) | 7 (35%) | 0 (0%) | |
| NO LOSS | 19 (25%) | 16 (32%) | 3 (15%) | 0 (0%) | |
| Missing | 26 (35%) | 11 (22%) | 10 (50%) | 5 (100%) | |
|
| |||||
| MGMT | |||||
| NEG | 26 (35%) | 23 (46%) | 3 (15%) | 0 (0%) | |
| POS | 22 (29%) | 13 (26%) | 9 (45%) | 0 (0%) | |
| Missing | 27 (36%) | 14 (28%) | 8 (40%) | 5 (100%) | |
|
| |||||
| KRAS | |||||
| Mutant | 38 (51%) | 26 (52%) | 11 (55%) | 1 (20%) | |
| WT | 29 (39%) | 18 (36%) | 8 (40%) | 3 (60%) | |
| Missing | 8 (11%) | 6 (12%) | 1 (5%) | 1 (20%) | |
All patients received prior 5-fluorouracil, oxaliplatin, irinotecan, and bevacizumab. These are additional prior therapies
Suspected Drug Related Adverse Events
All 75 patients who received treatment were evaluable for adverse events. No grade 5 events occurred. Overall, the combination of veliparib and temozolomide was well tolerated. Most patients experienced mild fatigue, but it was attributed to study therapy in only 25%. Almost half had nausea, but none were grade 3 or 4. There were very few other Grade 3 or 4 non-hematologic adverse events, which included one patient each with dysphagia, vaginal hemorrhage, and febrile neutropenia in the high-dose cohort and urinary hemorrhage in the dMMR cohort. The primary toxicity of concern was myelosuppression. In the original cohort, the rate of Grade 3 or 4 myelosuppression was very low, and only 10% of patients experienced treatment changes with 4 patients delayed due to myelosuppression and 1 patient stopped. One patient in the original cohort had severe pancytopenia and was taken off study. However, in the high dose cohort, and the MSI cohort the rate of significant myelosuppression was higher, with 36% of patients having Grade 3 or 4 anemia, thrombocytopenia, or neutropenia and associated treatment delays. None of the patients out of 25 in the high-dose and dMMR cohorts were taken off study due to myelosuppression. In all cases, the myelosuppresion did resolve within one to 1-4 weeks. Table 2 provides the number of patients experiencing adverse events by category and cohort that are possibly, probably, or definitely related to study drug.
Table 2.
Numbers (Percentages) of Patients Experiencing Adverse Events at Least Possibly Related to Study Drug
| Cohort
|
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| All N=75 Grade |
Original N=50 Grade |
High-dose N=20 Grade |
dMMR N=5 Grade |
||||||||||
|
|
|||||||||||||
| All | 1,2 | 3,4 | All | 1,2 | 3,4 | All | 1,2 | 3,4 | All | 1,2 | 3,4 | ||
| BLOOD/BONE MARROW | |||||||||||||
| Platelets | 40 (53) | 29 (39) | 11 (15) | 24 (48) | 20 (40) | 4 (8) | 14 (70) | 8 (40) | 6 (30) | 2 (40) | 1 (20) | 1 (20) | |
| Hemoglobin | 35 (47) | 29 (39) | 6 (8) | 18 (36) | 17 (34) | 1 (2) | 14 (70) | 9 (45) | 5 (25) | 3 (60) | 3 (60) | 0 (0) | |
| Leukocytes | 15 (20) | 9 (12) | 6 (8) | 2 (4) | 1 (2) | 1 (2) | 11 (55) | 7 (35) | 4 (20) | 2 (40) | 1 (20) | 1 (20) | |
| Neutrophils | 13 (17) | 5 (7) | 8 (11) | 5 (10) | 3 (6) | 2 (4) | 6 (30) | 1 (5) | 5 (25) | 2 (40) | 1 (20) | 1 (20) | |
| Lymphopenia | 2 (3) | 2 (3) | 0 (0) | 2 (4) | 2 (4) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
|
| |||||||||||||
| CONSTITUTIONAL SYMPTOMS | |||||||||||||
| Fatigue | 19 (25) | 19 (25) | 0 (0) | 16 (32) | 16 (32) | 0 (0) | 2 (10) | 2 (10) | 0 (0) | 1 (20) | 1 (20) | 0 (0) | |
| Fever | 2 (3) | 2 (3) | 0 (0) | 1 (2) | 1 (2) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Weight loss | 1 (1) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
|
| |||||||||||||
| DERMATOLOGY/SKIN | |||||||||||||
| Alopecia | 1 (1) | 1 (1) | 0 (0) | 1 (2) | 1 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Peeling on feet | 1 (1) | 1 (1) | 0 (0) | 1 (2) | 1 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Rash | 1 (1) | 1 (1) | 0 (0) | 1 (2) | 1 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
|
| |||||||||||||
| GASTROINTESTINAL | |||||||||||||
| Nausea | 36 (48) | 36 (48) | 0 (0) | 24 (48) | 24 (48) | 0 (0) | 10 (50) | 10 (50) | 0 (0) | 2 (40) | 2 (40) | 0 (0) | |
| Vomiting | 14 (19) | 14 (19) | 0 (0) | 7 (14) | 7 (14) | 0 (0) | 6 (30) | 6 (30) | 0 (0) | 1 (20) | 1 (20) | 0 (0) | |
| Anorexia | 9 (12) | 9 (12) | 0 (0) | 5 (10) | 5 (10) | 0 (0) | 3 (15) | 3 (15) | 0 (0) | 1 (20) | 1 (20) | 0 (0) | |
| Diarrhea | 5 (7) | 5 (7) | 0 (0) | 4 (8) | 4 (8) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Constipation | 2 (3) | 2 (3) | 0 (0) | 2 (4) | 2 (4) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Ascites | 1 (1) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Dehydration | 1 (1) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (20) | 1 (20) | 0 (0) | |
| Dysphagia | 1 (1) | 0 (0) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 1 (5) | 0 (0) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | |
| Dysgeusia | 1 (1) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (20) | 1 (20) | 0 (0) | |
|
| |||||||||||||
| HEMORRHAGE/BLEEDING | |||||||||||||
| Urinary Hemorrhage | 1 (1) | 0 (0) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (20) | 0 (0) | 1 (20) | |
| Vaginal Hemorrhage | 1 (1) | 0 (0) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 1 (5) | 0 (0) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | |
| Petechiae/purpura | 1 (1) | 1 (1) | 0 (0) | 1 (2) | 1 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
|
| |||||||||||||
| INFECTION | |||||||||||||
| Febrile Neutropenia | 1 (1) | 0 (0) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 1 (5) | 0 (0) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | |
|
| |||||||||||||
| LYMPHATICS | |||||||||||||
|
| |||||||||||||
| Edema: limb | 3 (4) | 3 (4) | 0 (0) | 2 (4) | 2 (4) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
|
| |||||||||||||
| METABOLIC/LABORATORY | |||||||||||||
| Hypoalbuminemia | 1 (1) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Creatinine | 1 (1) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
|
| |||||||||||||
| NEUROLOGY | |||||||||||||
| Neuropathy: sensory | 3 (4) | 3 (4) | 0 (0) | 3 (6) | 3 (6) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Dizziness | 2 (3) | 2 (3) | 0 (0) | 1 (2) | 1 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (20) | 1 (20) | 0 (0) | |
|
| |||||||||||||
| PAIN | |||||||||||||
| Pain NOS | 3 (4) | 3 (4) | 0 (0) | 2 (4) | 2 (4) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Headache | 1 (1) | 1 (1) | 0 (0) | 1 (2) | 1 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
|
| |||||||||||||
| PULMONARY/UPPER RESPIRATORY | |||||||||||||
| Shortness of Breath | 2 (3) | 2 (3) | 0 (0) | 1 (2) | 1 (2) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Cough | 1 (1) | 1 (1) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (5) | 1 (5) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
Clinical Responses
The primary endpoint was DCR. All patients were evaluable for response, on an intention to treat basis (Table 3). Two of the 50 original cohort patients had a PR, and 9 more had SD after 2 cycles (DCR=22%), defined as a success by Simon’s 2-stage design. In the high-dose cohort, 7 patients had SD after 2 cycles (DCR=35%). The DCR in the high-dose vs. original protocol cohort revealed no statistically significant increase in the high-dose group (p=0.26). None of the patients in the dMMR cohort had SD or PR.
Table 3.
Clinical Outcomes for Cohorts and Correlative Markers
| DCR | OS | PFS | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
|
||||||||||
| N | N (%) | P-Value* | Events | Median (95% CI) |
P-Value | Events | Median (95% CI) | P-Value | ||
| All** | 75 | 18 (24.0%) | 70 | 6.6 (5.6,7.4) | 74 | 1.8 (1.6,1.9) | ||||
| Cohort | NC | NC | NC | |||||||
| Original** | 50 | 11 (22%) | 46 | 6.7 (5.2,7.7) | 49 | 1.8 (1.6,1.9) | ||||
| High-dose** | 20 | 7 (35%) | 19 | 6.2 (4.9,10.2) | 20 | 2.0 (1.6,2.7) | ||||
| dMMR | 5 | 0 (0%) | 5 | 6.5 (1.8,17.8) | 5 | 1.4 (1.0,1.8) | ||||
| KRAS | 0.78 | 0.62 | 0.83 | |||||||
| Mutant | 38 | 8 (21.1%) | 33 | 6.6 (4.9,9.7) | 38 | 1.8 (1.6,1.9) | ||||
| WT | 29 | 7 (24.1%) | 29 | 6.3 (5.2,7.2) | 28 | 1.8 (1.5,2.1) | ||||
| Missing | 8 | 3 (37.5%) | ||||||||
| MGMT | >0.99 | 0.15 | 0.88 | |||||||
| NEG | 26 | 6 (23.1%) | 25 | 6.2 (4.8,7.0) | 26 | 1.7 (1.6,2.1) | ||||
| POS | 22 | 5 (22.7%) | 19 | 7.6 (5.2,12.7) | 21 | 1.8 (1.5,2.0) | ||||
| Missing | 27 | 7 (25.9%) | ||||||||
| MMR | 0.21 | 0.38 | 0.001 | |||||||
| Indeterminate | 2 | 0 (0.0%) | 1 | NR (5.1,NR) | 2 | 1.0 (0.5,1.5) | ||||
| Stable | 49 | 14 (28.6%) | 45 | 6.7 (5.4,7.9) | 49 | 1.8 (1.6,2.1) | ||||
| dMMR | 6 | 0 (0.0%) | 6 | 5.6 (0.7,17.8) | 6 | 1.3 (0.2,1.8) | ||||
| Missing | 18 | 4 (22.2%) | ||||||||
| PTEN | 0.69 | 0.76 | 0.40 | |||||||
| LOSS | 30 | 4 (13.3%) | 29 | 6.2 (5.1,7.9) | 29 | 1.7 (1.5,1.8) | ||||
| NO LOSS | 19 | 4 (21.1%) | 16 | 6.3 (4.2,12.7) | 19 | 1.8 (1.6,2.0) | ||||
| Missing | 26 | 10 (38.5%) | ||||||||
DCR=Disease Control rate; OS=Overall Survival; PFS=Progression-Free Survival; CI=Confidence Interval; NC=Not Calculated; NR=Not Reached.
p-value calculations included patients with non-missing marker information.
All responses were SD except 2 PRs in the original cohort
The median (95% CI) OS and PFS for all patients was 6.6 months (5.6,7.4 months) and 1.8 months (1.6,1.9 months), respectively (Table 3, and Figures 2A and 2B). Broken down by cohort, the OS and PFS of the original cohort was 6.7 and 1.8 months, respectively; for the high-dose cohort was 6.2 and 2.0 months, respectively; and for the dMMR cohort was 6.5 and 1.4 months, respectively (Table 3). Figures 2C and 2D show no differences among the cohorts for OS (p=0.81) or PFS (p=0.14), respectively.
Figure 2. Survival Curves.
Figure 2A. Overall Survival. Figure 2B. Progression-Free Survival. Figure 2C. Overall Survival by Cohort. Figure 2D. Progression-Free Survival by Cohort
Correlative Markers of Response to Therapy
There was no association between DCR and age or gender. Since KRAS status has been implemented as a predictive marker for CRC therapy(43), we analyzed whether KRAS genotype status correlated with DCR and found no association (Table 3). Archived tumor samples were obtained for all patients. In addition, the 20 patients in the high-dose cohort, and the 5 patients in the dMMR cohorts underwent fresh tumor biopsies. MMR analysis was available for 76% (57/75) of patients and was determined for 55 patients. Patients with MMR-deficient (dMMR) vs. MMR-PROficient (MS Stable) tumors had worse PFS with medians of 1.8 vs. 1.3 months, respectively (p=0.01). The trend held true but was not statistically significant for OS (p=0.27) and DCR (p=0.32). Given reports that tumors lacking PTEN exhibit a defect in homologous recombination and are hypersensitive to PARP-inhibitors(44), we examined the association between PTEN expression and the DCR. In our patient samples, using a previously established scoring system(38-40), PTEN protein expression was detected mainly at the cytoplasmic level, although occasional nuclear positivity was present. PTEN expression was lost in 39% (19/49) of available samples. However, no clear association was evident between a loss of PTEN expression and DCR (Table 3). Finally, given the previously published association between MGMT expression and response to temozolomide (14-16), we examined MGMT protein expression in our samples. Surprisingly, no association was found between MGMT status and DCR, OS, or PFS (Table 3).
DISCUSSION
Patients with advanced-refractory mCRC are in desperate need of additional treatment options. PARP inhibitor-based treatments have demonstrated great promise in pre-clinical models and in several clinical trials, particularly in DNA repair deficient subtype of cancers (i.e., predominantly ovarian, breast, prostate, and pancreatic cancers). However, pre-clinical studies and the logical concept that chromosomally instable or MSI-high cancer cells may benefit from PARP-inhibitor therapies launched us into designing this trial. Previously, we demonstrated that by transfecting cancer cells with a dominant negative PARP-1 construct (i.e., inhibiting PARP-1), we sensitized cancer cells to various DNA damaging agents(6). From this preclinical work, we hypothesized that combining a PARP inhibitor with a potent DNA-alkylating agent could force a synthetic lethal setting that provides a therapeutic window targeting genetically unstable CRC cells.
Here we have demonstrated that veliparib plus temozolomide can be safely administered to patients with refractory mCRC. Patients tolerated the combination well, with very little need for treatment delays or dose modification. Given the higher DCR in the high-dose group, we recommend starting future studies at this higher dose, with pre-planned dose reductions for myelosuppression. This surprisingly contrasts a similar trial with veliparib plus temozolomide for patients with refractory breast and ovarian cancers, in which the veliparib dose had to be reduced to 30mg BID(45). We speculate that the breast and ovarian cancer patients, who have more “standard” treatment options, were more heavily pre-treated. Another possible explanation may be that more breast and ovarian cancer patients harbored underlying BRCA1 or -2 mutations. In a patient with an actionable synthetically lethal gene mutation that may render the tumor more sensitive to treatment, the mutation may also cause a greater degree of toxicity to other normal cells that harbor haploinsufficiency with rapid turnover, such as the bone marrow stem cells.
We did meet our protocol defined primary endpoint, with a DCR of 24%, and while only a small subgroup of patients, the DCR in the high-dose group of 35% is even more promising. Moreover, our median OS of 6.6 months is certainly promising, and worthy of further investigation.
Future clinical trials should ideally identify a patient population most likely to benefit from this combination. We were able to assess the impact of potential predictive biomarkers of response from patient tumors. While not all patient samples could be tested, there were enough samples in each subgroup to undergo a reasonable preliminary (i.e. hypothesis-generating) statistical assessment (Table 3). It has been shown that cells with dMMR are significantly more sensitive to the combination of veliparib and cisplatin, irinotecan, and temozolomide than MMR-proficient cells(10,33). Therefore, we were surprised to find no benefit to treatment in patients with dMMR tumors. In fact, there appeared to be a worse outcome. We are confident that determination of the MMR status by IHC was not a limiting factor, and the sensitivity for IHC detection of MMR enzyme (protein) loss has been shown to be 95% predictive of true microsatellite instability, as classically tested by PRC fragment analysis(31,46). Instead, the lack of benefit may have been an unexpected consequence of choosing temozolomide, as it has also been shown to be less effective in dMMR cells(15,47). In fact, Hochhauser, et al, demonstrated no clinical activity of temozolomide patients with MMR deficient cancers, despite the pre-selection of MGMT promoter methylation(15). Our results are consistent with Hochhauser’s conclusion that MMR enzymatic activity is necessary for temozolomide activity. However, enhanced efficacy in dMMR patients may be seen in patients treated with a PARP inhibitor and another alkylating agent, such as oxaliplatin.
We were also optimistic that patients with PTEN-deficient tumors would be more likely to respond to treatment(23,24,42,44,48). Our hope was based on pre-clinical evidence that tumors lacking PTEN exhibit a defect in homologous recombination(28,49). Additionally, PTEN-deficiency appeared to be a marker for PARP inhibitor therapy in endometrioid endometrial cancers both in pre-clinical models and in a case report(23,24). Unfortunately, our patient population did not show an association between PTEN loss and clinical response to PARP inhibitor therapy. It is possible that the mode of assessing PTEN status may be critical. In some cases, the pre-clinical data was demonstrated in cell lines that lack the PTEN gene(28. 47); but in at least one example, PTEN loss by IHC was sufficient to predict for benefit with a PARP inhibitor(23). Several reasons could account for our findings: 1) PTEN-deficiency association with PARP inhibitor efficacy may depend on the model (in vitro and/or cell type) utilized; 2) CRC cells are able to thrive even while harboring mutator phenotypes (CIN and MSI). Thus, these cells may be able to compensate and survive without PTEN function, leaving them less likely to be sensitive to DNA-damaging therapies.
Similarly, we analyzed whether MGMT expression correlated with response to therapy(50). This concept is based on the mechanism that MGMT extracts O6-methyl adducts from DNA guanine (06 position). We hypothesized that MGMT status could be a marker for response. Again, we did not find an association between MGMT expression and response. In this case, the lack of benefit may have been due to the choice to test for MGMT expression with IHC, rather than evaluating MGMT promotor methylation and gene silencing.
Notably, we do not know how these heavily pre-treated mCRC cells adjust their biology and sensitivity to this combination therapy or alter the predictability of the biomarkers evaluated herein. Nevertheless, the combination of veliparib plus temozolomide has demonstrated promising efficacy in a highly refractory mCRC patient population. In fact, the DCR is comparable to agents that have been since approved for refractory colorectal cancer, such as regorafenib or TAS-102(51,52). It is possible that PARP inhibitors such as veliparib may be better used for CRC in combination with other DNA damaging agents, such as oxaliplatin or irinotecan. Alternatively, additional biomarker testing may help refine the patients that are most likely to benefit. In an effort to identify patients that would respond to this targeted approach, future clinical studies should incorporate “omic profiling” of “naïvely treated” tumor cells along with complementary ex vivo modeling(53). This therapeutic strategy warrants further study, either in a broad based refractory population, or preferably in a prospectively defined patient subgroup.
Condensed Abstract.
Here we present the results of a Phase II trial of veliparib plus temozolomide for patients with metastatic colon cancer, wherein the combination was well tolerated, and achieved disease control in 24% of patients, with two confirmed PRs. There was a suggestion of a worse outcome in patients with mismatch repair deficiency, and there was no correlation between PTEN or MGMT protein expression and disease control.
Acknowledgments
Research Support: This work was funded by the Otto J. Ruesch Center for the Cure of GI Cancers, Lombardi Comprehensive Cancer Center.
Abbvie, Inc. has provided the Veliparib, Temozolomide, and partial research funding for the correlative science
We would like to thank Meeta Jaiswal, PhD for scientific guidance throughout protocol development and implementation.
MJP and JRB are supported by 1R01CA212600-01 (NCI, NIH) and a 2015 Pancreatic Cancer Action Network American Association for Cancer Research Acceleration Network Grant (15-90-25-BROD).
Footnotes
ClinicalTrials.gov identifier: NCT01051596; A Study of ABT-888 in Combination with Temozolomide for Colorectal Cancer
Presented in part at the 47th Annual Meeting of the American Society of Clinical Oncology, Chicago, IL, June 3-7, 2011.
Authors’ Contributions:
Michael J. Pishvaian: Planning, conduct, and reporting of the work described in the article; responsible for the overall content as guarantor.
Rebecca Slack: Planning, conduct, and reporting of the work described in the article
Wei Jiang: Conduct, and reporting of the work described in the article
Aiwu Ruth He: Conduct, and reporting of the work described in the article
Jimmy J. Hwang: Conduct, and reporting of the work described in the article
Amy Hankin: Conduct, and reporting of the work described in the article
Karen Dorsch-Vogel: Conduct, and reporting of the work described in the article
Divyesh Kukadiya: Conduct, and reporting of the work described in the article
Louis M. Weiner: Conduct, and reporting of the work described in the article
John L Marshall: Conduct, and reporting of the work described in the article
Jonathan Brody: Planning, conduct, and reporting of the work described in the article
Authors’ disclosures of potential conflicts of interest:
Michael J. Pishvaian, None
Rebecca Slack, None
Wei Jiang, None
Aiwu Ruth He, None
Jimmy J. Hwang, None
Amy Hankin, None
Karen Dorsch-Vogel, None
Divyesh Kukadiya, None
Louis M. Weiner, Consultant for Abbvie, Inc.
John L Marshall, None
Jonathan Brody, none
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