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In vitro chemosensitivity and resistance assays (CSRAs) test primary tumor cells’ response to chemotherapeutic drugs. The goal of CSRAs is to potentially direct therapy based upon in vitro response. This is in contrast to empiric therapy of cancer patients where therapy is based on clinical trial evidence and the likelihood of patient response. However, most previous CSRAs have been unsuccessful; this may be because they employ tumor cells in mono-culture. increasing evidence indicates that heterogeneity in tumor cell biology and microenvironment and the interaction between the two play essential roles in regulating drug response. In addition, many of these assay and culture systems require more tumor cells per condition than can be obtained from every patient. Furthermore, coculture systems in the past often cultured tumor and non-tumor cells from different patients (trans-coculture). Therefore, a CSRA capable of being performed for all patients while incorporating both tumor cells and non-tumor microenvironmental cells from the same patient is needed.

Despite new therapies, patients with MM continue to relapse. Resistant MM cells are known to possess enhanced NF-?B activity which can be partially overcome in vitro by the proteosome inhibitor bortezomib. Bortezomib can also ameliorate topoisomerase IIainhibition and restore sensitivity to agents such as doxorubicin. We designed a phase II trial of bortezomib combined with doxorubicin and dexamethasone to exploit this interaction. We also studied NF-?B activation induced by stromal cells and in primary MM cells derived from subjects. Eligibility: Progressive or refractory secretory MM, = 1 previous treatments, total previous doxorubicin = 220 mg/m2; previous bortezomib, transplantation, renal insufficiency with creatinine <5 mg/dl, were allowed. Methods: Patients received bortezomib 1.3 mg/m2 IV days 1,4,8, 11; doxorubicin 15 mg/m2 IV d 1,8 and dexamethasone 20 mg orally on d 1,4,8,11. Cycles were repeated every 21 days up to a maximum of 8 cycles. NF-?B activity was assayed by electrophoretic mobility shift assay using a P32labelled DNA probe on whole cell extracts derived from CD-138 positive cells isolated from pretreatment bone marrow aspirates. For stromal cell studies, CD-138 negative cells were plated, incubated for 14 days and washed. Residual adherent fibroblast like cells were then cocultured with RPMI 8226 MM cells for 24 hours and NF-?B activation in these cells analyzed as described above. Results: 9 patients have been enrolled; 7 patients are assessable for response. Previous treatments included 2 lenalidomide failures, 2 bortezomib failures, 2 transplant failures 1 subject with plasma cell leukemia. Median number of previous regimens: two (range 1–5), age range 40–88. Six of seven (85%) responded with 1 CR, 2 near CR, and 3 partial responses, including both lenalidomide failures. One patient, who had failed all previous therapy, rapidly progressed after one cycle. Of note, MM cells isolated from this resistant patient showed the highest levels of constitutive NF-?B activity whereas MM cells from responding patients showed far less constitutive activity. Stromal cells isolated from other patients demonstrated the ability to induce NF-?B activation in RPMI 8226 cells. Toxicities: no grade 3 hematologic toxicity was observed; 1 patient experienced grade 3 neuropathy, 2 patients grade 2 neuropathy after 6 cycles requiring dose reductions of bortezomib. Cost of doxorubicin $184/cycle (versus $8884/cycle if liposomal doxorubicin substituted). Conclusions: Bortezomib/doxorubicin/dexamethasone is an active, cost effective salvage regimen even in patients failing lenalidomide and single agent bortezomib. Neuropathy was the dose limiting toxicity of this regimen. High levels of constitutive NF-?B may predict for resistance to this regimen and may imply a proteosome independent pathway for NF-?B activation. Further analysis of constitutive NF-?B activity of primary MM and stromal cells in comparison to response is planned.