A phase II study of UCN-01 in combination with irinotecan in patients with metastatic triple negative breast cancer

Between January 2007 and November 2010, 25 patients with metastatic TNBC were enrolled. Patient characteristics are summarized in Table 1. Patients had prior treatment with an anthracycline and a taxane and the median number of prior regimens was 3 (ranges 1–4).

Twenty of 25 patients completed at least one cycle of treatment (range 1–6). All 25 patients were evaluated for treatment-related adverse events (Table 2). The most common hematological toxicities were neutropenia and anemia. Only 1 patient developed febrile neutropenia. The most common non-hematological toxicities were gastrointestinal toxicities including nausea/vomiting and diarrhea. Two patients developed colitis while on study (1 due to Clostridium difficile). Laboratory abnormalities were observed and were most often associated with gastrointestinal toxicities. Two treatment-related elevations in AST/ALT were observed. One patient with baseline grade 1 AST/ALT and liver metastases developed a grade 2 fever, grade 3 hyperglycemia, and transient grade 3 ALT/AST elevation within 24 h following cycle 1, day 2 UCN-01 administration. The patient’s serum levels of ALT/AST decreased to baseline level (grade 1) within 2 days and she completed the cycle without further transaminase elevations. The other patient developed a grade 3 ALT following cycle 1 week 1 and grade 4 ALT/grade 3 AST during cycle 2 week 4. As per protocol, this patient was removed from study due to grade 4 ALT. This patient’s transaminase elevations resolved to grade 1 2 weeks after the last treatment. Several patients had toxicities related to UCN-01. Two patients experienced hypoxia (grade 2 and grade 3, respectively), and five patients experienced grade 1 or 2 hypotension during the UCN-01 infusion that responded to intravenous fluid. As per protocol, the patient with grade 3 hypoxia, associated with grade 2 hypotension, was removed from study. Hyperglycemia occurred in over half of the patients, with grade 3 or 4 in five patients.

Among the 25 patients enrolled, only 22 were evaluated for response as one withdrew consent and two went off study during cycle 1 due to toxicities, one due to elevated transaminases and the other as a result of hypoxia during the UCN-01 infusion (Table 3). No complete responses were observed. One PR lasting 24 weeks occurred in a patient (patient 15) with liver and lung metastasis. This patient was previously treated with neoadjuvant FEC-T (5-Fluorouracil, epirubicin, cyclophosphamide followed by docetaxel) at initial diagnosis and, at disease progression, with capecitabine followed by paclitaxel and bevacizumab. Tumor from this patient was found to be basal-like, and carry a TP53 mutation (Table 4). The overall response rate (CR + PR) was 1 in 25 [4, 95 % CI (0, 20 %)]. Two patients had prolonged disease stabilization that lasted for 37 and 28 weeks, respectively. The clinical benefit rate (CR + PR + SD > 6 months) was 3 in 25 [12, 95 % CI (3, 31 %)]. The median TTF and OS were 1.7 months, 95 % CI (1.2, 2.6 months) and 11.3 months, 95 % CI (3.8, 19.0 months), respectively, as shown in Fig. 1a, b. The study did not meet the pre-defined efficacy criteria for further evaluation.

PBMCs were collected to evaluate the bioavailability of UCN-01 over time. Since UCN-01 inhibits PDK1, treatment with UCN-01 was expected to reduce levels of pS6, pAKT, and pGSK3β [32, 33]. The levels of the phosphorylated S6, AKT, and GSK3β were decreased 24 h after UCN-01 on day 3, but returned to baseline on day 8 in most patients (n = 24, data not shown). Representative western blots pre- and post-therapy are shown for two patients (Fig. 2a). The ratio of pS6 to S6 was quantified at baseline (n = 24), day 1 (n = 24), day 3 (n = 22), and day 8 (n = 20) (Fig. 2b). Compared to baseline, the ratio of pS6 to S6 was decreased significantly (p < 0.001) 24 h after UCN-01 on day 3, but not significantly different on day 8, indicating that UCN-01 activity was not present by day 8.

To evaluate the molecular effects of UCN-01, serial tumor biopsies were collected at three time points including baseline, day 2 (24 h post irinotecan), and day 3 (24 h post UCN-01) in five patients (1, 3, 4, 6, and 11) with chest wall recurrences. Tumors from patients 1 and 11 were mutant for TP53, while those from patients 3, 4, and 6 were wild type for TP53 (Table 4). Tumor biopsies from patient 3 were not submitted for further analysis because there were no tumor cells in the baseline sample. Immunostaining of γH2AX (marker for double strand DNA breaks), pChk1^S296 (marker of Chk1 activation), phistone H3 (marker for mitosis), cleaved caspase 3 (CC3) (marker for apoptosis), and pS6 were performed. As expected from treatment with irinotecan, there was an increase in double strand DNA breaks (↑ γH2AX) (Fig. 2c, d) and activation of Chk1 (↑ pChk1^S296) (Fig. 2c, e) in all tumors on day 2 biopsy (representative staining from patient 11’s tumor). phistone H3 staining was low at baseline (1–2 %); therefore, the anticipated further reduction of phistone H3 due to cell cycle arrest from irinotecan was difficult to demonstrate (Fig. 2c, f). Despite the anticipated pharmacodynamic effects of irinotecan, there was little induction of apoptosis on day 2 (Fig. 2c, g). Following UCN-01 therapy, pS6 was reduced in all tumors (Fig. 2c, h). However, loss of Chk1^S296 was only observed in tumors from patients 6 (TP53 WT (wild type)) and 11 (TP53 deletion) (Fig. 2c, e). Tumor from patient 11, which had a deletion in TP53, demonstrated the most dramatic increase in apoptosis with close to 10 % of tumor cells stained positive for cleaved caspase 3 on day 3 (Fig. 2g). The same tumor demonstrated a slight increase in phistone H3 (Fig 2f) and an increase in γH2AX (Fig. 2d) following combination therapy, suggesting cell cycle abrogation and enhanced DNA damage from the addition of UCN-01. The patient had a best response of stable disease and a TTF of 2.6 months. Interestingly, tumor from patient 6, which was WT (wild type) for TP53, showed the most dramatic increase in phistone H3 (Fig. 2f) following UCN-01, suggesting cell cycle abrogation, but this patient had a best response of PD and a TTF of 1.6 months on study, indicating ineffective therapy.

In summary, data on pChk1^S296 indicated an inconsistent effect of UCN-01 on Chk1, with one (patient 11) of two TP53 mutant tumors demonstrating the anticipated cell cycle pharmacodynamic effect (loss of pS296) of UCN-01. However, given that active Chk1 (p296-phosphorylated form) is targeted for ubiquitin-mediated proteolysis, there may have been less total Chk1 in this tumor sample [34‐36].

To investigate the molecular characteristics of the TNBC and their potential predictive value in prognosis and therapeutic response, tumor specimens for TP53 mutation and molecular subtype by PAM50 were analyzed (Table 4). Among the 25 patients enrolled, one withdrew consent and a specimen was not requested, three were without available archival tumor specimens, and five had archival tumor specimens with limited cellularity, and one each had poor quality DNA and RNA, respectively. Among the 15 patients with sufficient specimens for PAM50 analysis, there were ten basal-like, one basal/HER2-enriched, two HER2-enriched, and two luminal B. Among the 15 patients with sufficient tumors for analysis, eight (53 %) carried mutations in TP53. In this limited dataset, there was no significant difference in TTF between the two populations (Fig. 1c), but a significantly worse OS was observed in patients with mutations in TP53. The median OS was 5.5 (95 % CI: 2, 11) months and 20.3 (95 % CI: 3, 24) months in patients with and without TP53 mutation (p = 0.004), respectively (Fig. 1d).