Phase II Trial of Bortezomib Plus Doxorubicin in Hepatocellular Carcinoma (E6202): A Trial of the Eastern Cooperative Oncology Group: Berlin J 1, Powell M 2, Su Y 1, Horton L 1, Short S 1, Richmond A 1, Kauh JSW 3, Staley C 3, Mulcahy M 4, Benson III AB 4. 1 Vanderbilt University/Vanderbilt-Ingram Cancer Center, 2 Dana Farber Cancer Institute, 3 Emory University, 4 Northwestern University

Abstract Background: Until ASCO 2007, the there was no proven systemic therapy for HCC, but dox was used often because of a response rate (RR) of up to 10%. We tested the effects of bor, dox and bor + dox in HCC cell lines and mouse models and found dox + bor to be at least additive compared to dox alone. Methods: Eligible pts had biopsy proven HCC with no prior chemotherapy or hormonal therapy, ECOG PS 0-2, with measurable and biopsiable disease. Pts had adequate bone marrow function (lower levels allowed for splenomegaly), bilirubin ≤ 2.0 mg/dl, Child’sPugh class A or B cirrhosis, INR ≤ 1.5, no peripheral neuropathy of grade 2 or higher and normal ejection fraction. Primary endpoint is RR. Null hypothesis = 10% RR. 40 pts were planned (37 eligible) to give 91% power to detect RR of 27% (7 responses) with 1-sided significance of Secondary endpoints were toxicity, survival (OS) and progression-free survival (PFS). Dox (20 mg/m2) was given days 1, 8 (day 1 dox was omitted in cycle 1) and Bor was given days 1, 4, 8 and 11 of a 21-day cycle. Serum was analyzed prior to treatment, cycle 1 day 8 and cycle 2 day 8 for changes in chemokines/cytokines (CKS). Results: 42 pts accrued, 39 confirmed eligible: 28 male, 11 female; 27 white, 9 African American, 3 other; PS = 0, 6, PS = 1, 28, PS =2, 5. 1 response (2.3%) was seen. Median OS was 5.7 months (4.4, 7.9) and PFS was 2.4 months (2.1, 3.0). 28/39 had grade 3 toxicity, 11 had grade 4. Most common toxicities were grad 3 leukocytes (11 pts), grade 3 platelets (8 pts) and grade 4 platelets (8 pts). including: CKS showed changes in serum levels of GROα, MIP-1α, IL-6 and IL-8 in several pts. Conclusions: dox + bor did not show significant activity in HCC. The regimen was generally well-tolerated with mostly hematologic toxicity and no toxic deaths. Bor caused changes in several CKS levels in pts with HCC. We will analyze the lab data to try to correlate with clinical effects (efficacy and toxicity) prior to the ASCO 2008 meeting. Supported by: R21 CA , Eastern Cooperative Oncology Group (Robert L. Comis), and supported in part by Public Health Service Grants CA23318, CA66636, CA21115, CA49957, CA17145 and from the National Cancer Institute, National Institutes of Health and the Department of Health and Human Services

Background In 2002, when this study was designed, doxorubicin was the de facto standard of care in HCC with a 0-10% Response Rate A single agent bortezomib trial reported at ASCO had 7/20 patients with stable disease At Vanderbilt-Ingram Cancer Center, pre-clinical studies showed in HCC models –Activity of bortezomib > activity of doxorubicin –Additive activity for bortezomib + doxorubicin

Methods Phase II trial, single-stage design –Early stopping rule designed for excessive toxicity Primary Endpoint –27% Response Rate considered positive –10% Response Rate is null hypothesis –40 patients needed for 37 eligible 91% power to detect alternative hypothesis with 1-sided significance level of 0.07 Secondary Endpoints –4.5 month PFS (21% increase from 50% to 71%) 84% power to detect alternative hypothesis with 1-sided significance of 0.07 –Overall Survival –Time to Tumor Progression

Key Eligibility Unresectable HCC Measurable Disease by RECIST, potentially amenable to biopsy ECOG PS 0-2 Adequate organ function –Creatinine < 2.0 mg/dl –Child Pugh A or B only –Platelet ≥ 100K or ≥ 75 K if splenomegaly is present –ANC ≥ 1.5 or ≥ 1.0 if splenomegaly is present –Bili ≤ 2.0 mg/dl –AST and Alk phos ≤ 5x ULN Prior Liver Transplant allowed

Key Ineligibility No prior systemic hormonal therapy, chemotherapy or hepatic chemotherapy although bland embolization, ethanol injection, RFA were allowed EF < 50% Peripheral neuropathy > grade 1 INR > 1.5

Treatment Plan Bortezomib (supplied by CTEP/NCI) 1.3 mg/m2 days 1, 4, 8 and 11 of a 21-day cycle Doxorubicin 20mg/m2 days 1 and 8, except on cycle 1 when it is administered day 8 only (and if bili 1.3-2, then dose is 15 mg/m2 on days 1 and 8)—21-day cycle Maximum cycles = 10 Disease evaluation q 3 cycles and at end of treatment (Response assessed by RECIST) Routine ECHO/MUGA scans

Correlates Optional tumor biopsies on day 1 and day 8 were requested initially, but after biopsies had too little tissue on most early samples, this was discontinued Blood was drawn cycles 1, 2 and 9, 10 on days 1 and 8 for –Serum: IL-1, IL-8, VEGF and MIP-1 α –WBC’s: p21, p27, p53, BCL-2 and Bax

Results ParameterN (total = 39*)% Sex: Male Female Median Age: 58 Ethnicity: White African American Other ECOG PS: *42 patients enrolled, 39 eligible

Treatment Prior Treatment: –4 patients had prior liver transplants –2 patients had prior bland emoblizations On Study –Median of 3 cycles administered Range: patients (33%) did not complete the first 3 cycles, 5 of whom had only 1 cycle Off-study –22 patients (56.4%) stopped due to progression –10 patients (26.4%) stopped due to AE’s, side effects or complications –3 patients (7.7%) died on study –4 patients (10.3%) withdrew consent or refused further treatment without progressive disease

Toxicities I ToxicityGrade 1,2Grade 3Grade 4 Hemoglobin3111 Platelets1288 Leukocytes1311 Neutrophils1162 Alkaline Phos151 AST (SGOT)222 Bilirubin10 Creatinine9 INR2

Toxicities II ToxicityGrade 1,2Grade 3Grade 4 Fatigue157 Anorexia14 Diarrhea95 Nausea131 Vomiting51 Constipation9 Taste Disturbance7 Dehydration12 Sensory Neuropathy10

Response N=39% PR12.6 Stable Progression Unevaluable Reasons for Unevaluable Pts: 4 patients died, not related to therapy 5 patients without available follow-up scans 2 patients switched type of scan performed (MRI vs CT)

Med Survival 5.7 months (90% CI = 4.4, 7.9 months)

Median PFS = 2.4 months (90% CI months)

Change of RelAp65 EMSA Value for Cycle 1 Individual Patient Increase or Decrease in Nuclear RelA Detected In EMSA. Each diamond represents one patient’s change during cycle 1.

Change of RelAp65 EMSA Value for Cycle 2 Individual Patient Increase or Decrease in Nuclear RelA Detected In EMSA. Each diamond represents one patient’s change during cycle 2

Change in Chemokine Levels Between Treatment Cycles

Conclusions The combination of of doxorubicin and bortezomib was tolerable in a relatively healthy HCC population The combination of doxorubicin and bortezomib is not sufficiently promising to investigate it further in HCC Laboratory correlatives suggest that some patients experienced changes suggestive of bortezomib affecting its target –Clinical correlation with changes in laboratory parameters is pending