• bortezomib;
  • autologous transplantation;
  • multiple myeloma;
  • Fanconi anaemia;
  • DNA repair pathway


  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure of conflicts of interest
  8. References

We conducted a Phase 1/2 study of bortezomib administered in combination with high-dose melphalan followed by tandem autologous transplants in patients with primary resistant multiple myeloma. Thirty patients received two cycles of salvage bortezomib followed by stem cell mobilization with granulocyte colony-stimulating factor and harvest. Melphalan 100 mg/m2 per day on two consecutive days was administered, immediately followed by one dose of bortezomib (dose escalation) and stem cell infusion. The median beta 2-microglobulin was 4·35 mg/l (range: 1·8–11·4); albumin was 37 g/l (range: 3·1–4·9); high-risk karyotypes were noted in 45% of patients. The maximum planned dose of bortezomib at 1·3 mg/m2 was well tolerated and a formal maximum tolerated dose was not determined. The peak of best overall response (≥partial response) and complete response rates after tandem transplants were 84% and 36%, respectively. With a median follow-up of 48 months, the median progression-free survival was 15 [95% confidence interval (CI): 11–21] months and the median overall survival was 35 (95% CI: 22–43) months. Correlative studies demonstrated decreased expression of BRCA2 (= 0·0072) and FANCF (= 0·0458) mRNA following bortezomib treatment. Bortezomib combined with high-dose melphalan is a well-tolerated conditioning with some activity in patients with resistant myeloma.

Multiple myeloma (MM) represents approximately 10% of all haematological malignancies and the median overall survival (OS) of those diagnosed in the last decade has approached 4 years (Kumar et al, 2008; Jemal et al, 2011). The role of high-dose chemotherapy followed by autologous haematopoietic cell transplantation (HCT) has been established (Attal et al, 1996; Child et al, 2003; Koreth et al, 2007; Kumar et al, 2009). High-dose melphalan at 200 mg/m2 remains an integral modality of myeloma treatment and tandem transplants continue to be offered to a fraction of patients (Attal et al, 1996).

Historically, approximately 20–30% of patients with newly diagnosed MM fail to respond to induction treatment regimens such as vincristine, adriamycin and dexamethasone (VAD) or thalidomide and dexamethasone (Kumar et al, 2004), hence these patients were thought to have poorly responsive disease. Their median progression-free survival (PFS) ranges from 4 to 8 months with the median OS was approximately 12 months (Alexanian & Dimopoulos, 1994). In recent years, these primary refractory patients were shown to benefit from high-dose chemotherapy with complete response (CR) rates of 16–40% (Singhal et al, 2002; Alexanian et al, 2004; Kumar et al, 2004). However, the results are still inferior to patients with responsive disease prior to transplant. With the introduction of novel agents, including lenalidomide and bortezomib, the overall response rates (ORR) with newer induction regimens have increased dramatically to between 80% and 90% (Harousseau et al, 2010; Rajkumar et al, 2010; Richardson et al, 2010). Plasma cell leukaemia (PCL) represents, perhaps, the most aggressive form of MM (Sher et al, 2010). The European Group for Blood and Marrow Transplantation (EBMT) reported an OS of 25·7 months in 272 patients with PCL who underwent autologous HCT in a retrospective analysis (Drake et al, 2010), albeit limited by a selection bias. Currently there are no standard therapeutic strategies for PCL. To this end, there is an unmet need to improve and develop better treatment regimens for patients with primary resistant MM and PCL.

Bortezomib has demonstrated significant clinical anti-myeloma activity when used as a single agent (Richardson et al, 2003, 2005) and also in combination with melphalan (Berenson et al, 2006; San Miguel et al, 2008). Bortezomib targets both acquired and de novo mechanisms of drug resistance and sensitizes melphalan-sensitive and melphalan-resistant MM cell lines to melphalan via the down regulation of cellular responses to genotoxic stress (Ma et al, 2003; Mitsiades et al, 2003). Inhibition of nuclear factor (NF)-κB by bortezomib significantly reduces Fanconi anaemia (FA)/BRCA gene expression and FANCD2 protein expression in MM cells with a resultant reduction in DNA damage repair and enhanced melphalan sensitivity (Yarde et al, 2009). These studies suggested the utilization of bortezomib in combination with high-dose melphalan may be synergistic and provide significant clinical benefit.

Based on both preclinical and clinical data, we hypothesized that bortezomib would sensitize patients with primary resistant MM (defined in the method section) to high-dose melphalan, overcome both acquired and de novo drug resistance, and ultimately result in increased efficacy of autologous HCTs, and that these effects may result from the down regulation of the FA/BRCA gene expression. We designed this study to (i) determine the maximum tolerated dose (MTD) and to (ii) investigate the safety, tolerability, and response rates of bortezomib given in combination with high-dose melphalan, as a conditioning regimen for tandem autologous HCTs in patients with primary resistant MM, and to (iii) examine the effects of bortezomib on the FA/BRCA gene expression in vivo.


  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure of conflicts of interest
  8. References

Patient eligibility

Patients with primary resistant MM or PCL aged 18 years or older were eligible. Primary resistant disease was defined as failure to achieve at least a partial response (PR), based on International Uniform Response Criteria (Durie et al, 2006), after induction therapy in a bortezomib-naïve myeloma population, as bortezomib was only available for patients who failed two prior therapies at the time of study initiation (Bross et al, 2004). All patients were required to have histologically confirmed diagnosis by pathology review at the H. Lee Moffitt Cancer Center and Research Institute (MCC). Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki on the protocol approved by the Institutional Review Board of University of South Florida. The trial was registered at as NCT 00307086. A total of 34 patients were enrolled onto this study between June 2005 and February 2009. Three patients were excluded due to insurance denial for the participation in the study and one patient withdrew their informed consent.

Patients with inadequate haematological (a platelet count of <30 × 109/l and an absolute neutrophil count of <1·0 × 109/l), renal (serum creatinine >176·8 μmol/l or creatinine clearance <40 ml/min, requiring haemodialysis or peritoneal dialysis), endocrine, cardiopulmonary and hepatic functions were ineligible. Other notable criteria for exclusion at the time of enrollment were >grade 2 peripheral neuropathy, hypersensitivity to bortezomib, boron, or mannitol, active central nervous system (CNS) involvement, previous malignancy other than non-melanoma skin cancer, radiation therapy within 14 d prior to enrollment, previous bortezomib therapy, and prior stem cell transplantation.

Initial diagnostic and staging evaluations, baseline cytogenetics and fluorescent in situ hybridization (FISH), initial therapies and myeloma status at diagnosis were documented for all patients. High risk group by cytogenetic changes was defined as the presence of hypodiploidy, t(4;14), t(14;16), partial or whole deletion of chromosome 13q, or loss of 17p13 by conventional cytogenetics and 17p deletion (TP53), t(4;14) (IGH@, FGFR3), or deletion 13q14 (RB1) by FISH. Standard risk group by cytogenetic changes was defined as hyperdiploidy, or normal karyotype by conventional cytogenetics and other abnormalities not included in the high-risk group by FISH. Patients were staged by both the Durie-Salmon staging system and the International Staging System (ISS) (Woodruff et al, 1979; Greipp et al, 2005).

Study design

Patients were initially treated with bortezomib at a dose of 1·3 mg/m2 intravenously on days 1, 4, 8, and 11, every 3 weeks for two cycles. After the completion of bortezomib therapy, patients underwent peripheral-blood stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) mobilization at a dose of 20 μg/kg per day for four consecutive days, followed by stem cell collection. The goal of peripheral blood stem cell collection was to achieve CD34+ cell dose of 4–10 × 106/kg of body weight (BW) to perform tandem transplantation. The study schema is depicted in Fig 1.


Figure 1. Study schema. HCT1, first haematopoietic cell transplantation; HCT2, second haematopoietic cell transplantation. Melphalan 200: Melphalan 200 mg/m2. *Bortezomib given on day −3 (dose escalation: 0·7, 1·0, and 1·3 mg/m2).

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For the first transplant, melphalan (100 mg/m2) was administered intravenously via central catheter over 30 min once daily on days −4 and −3, with bortezomib dosed according to a Phase I dose escalation schema and infused by intravenous push over 3–5 s on day −3, immediately after the melphalan. A minimum of 2 × 106 autologous CD34+ cells/kg of BW was infused 72 h after the conditioning regimen was completed. The day of peripheral blood stem cell infusion was designated as day 0. At 90 (±15) days after the first transplant, patients underwent a second transplant using the same regimen and schedule as in the first transplant (Fig 1).

For bortezomib in combination with high-dose melphalan, a Phase I dose-escalation scheme with three patients in each cohort (standard 3 + 3 design) was employed. Escalated doses of bortezomib were administered in sequential cohorts of patients: 0·7 mg/m2 (Cohort 1), 1·0 mg/m2 (Cohort 2) and 1·3 mg/m2 (Cohort 3). As specified in the protocol, Cohort 2 was expanded as the last patient in Cohort 3 was <30 d after first HCT and the entire cohort had not completed the toxicity evaluation at the time of enrollment. Cohort 3 was subsequently expanded as a Phase 2 component of the study. We estimated that with 30 evaluable patients the width of a 95% confidence interval (CI) for the response rate would not exceed 37% using the exact Binomial distribution.

Post-transplantation supportive care

Patients received supportive care according to institutional clinical guidelines for autologous stem cell transplantation. G-CSF was administered at a dose of 5 μg/kg per day from day + 7 until a self-sustained absolute neutrophil count (ANC) recovery of ≥0·5 × 109/l was achieved. No maintenance therapy post-tandem autologous HCTs were allowed in this protocol. Herpes simplex and herpes zoster virus prophylaxis consisted of acyclovir 400–800 mg orally twice daily for 1 year post-transplant.

Assessment of response and toxicity

Treatment response and relapse definitions were based on International Uniform Response Criteria (Durie et al, 2006). Responses were scored separately after two cycles of bortezomib and 3 months post-tandem autologous HCT. After the 90-d evaluation, myeloma responses were recorded approximately every 3 months until progression or relapse was documented. Toxicity assessment was performed using the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 ( Dose-limiting toxicities (DLTs) were defined as delayed engraftment and/or the development of grade 3 or 4 toxicities. The toxicities expected during stem cell transplantation, including leucopenia, neutropenia, thrombocytopenia, mucositis, nausea, vomiting, and alopecia, were not considered dose-limiting.

Isolation of multiple myeloma cells

Serial bone marrow aspirations were collected (i) at baseline, (ii) after one dose of bortezomib, and (iii) after two cycles of bortezomib. Plasma cells were isolated via negative selection protocol (RosetteSep; StemCell Technologies Inc., Vancouver, BC, Canada), aiming for >85% purity. First, 1 ml of bone marrow aspirate was centrifuged in a Ficoll-Paque PLUS gradient (Amersham Biosciences and GE Healthcare, Little Chalfont Buckinghamshire, UK), and a cytospin of the isolated cells was used to determine the initial purity of the plasma cell population. In parallel, 10 ml of the bone marrow sample was incubated with the Millennium Rosette Antibody Cocktail (Millennium Pharmaceuticals Inc., Cambridge, MA, USA) for 20 min followed by Ficoll extraction. A new cytospin was prepared from these cells to assess the efficiency of selection (Yarde et al, 2009).

FA/BRCA pathway gene expression

RNA was extracted from CD 138-enriched fresh myeloma cells using RNeasy Micro kit (Qiagen Inc., Hilden, Germany) and cDNA was synthesized using SuperScript First Stand Synthesis kit (Life Technologies, Carlsbad, CA, USA). FA/BRCA DNA repair pathway mRNA expression was assessed by quantitative-PCR (q-PRC) with customized microfluidic cards (Life Technologies) designed to simultaneously analyze the expression of 11 FA/BRCA-related genes, BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51, AND RAD51C as previously described (Yarde et al, 2009). Gene expression was normalized to the internal standard of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous control, and externally standardized against the screening sample. For longitudinal intra-patient analysis of the FA/BRCA pathway, mRNA expression was reported as fold-change from screening bone marrow. The inter-patient comparison of FA/BRCA pathway expression involved an additional step. To control for comparison of values from different microfluidic cards, an additional myeloma cell line control sample was used on each card. All FA/BRCA pathway values from initial bone marrow were normalized to this, allowing for an accurate inter-patient comparison of pretreatment FA/BRCA pathway expression taking into account the card-to-card variability.

Study endpoints and statistical analysis

The primary objectives of this study were (i) to determine the MTD and (ii) to investigate the safety, tolerability, and response rates of bortezomib given in combination with high-dose melphalan in primary resistant MM or plasma cell leukaemia. Primary efficacy endpoints were analysed on those patients who received at least one transplant. Following treatment with two cycles of standard dose bortezomib, sequential cohorts of patients were given escalating doses of bortezomib in a standard 3 + 3 design combined with high dose melphalan. Neutrophil engraftment was defined as the first day the ANC was ≥0·5 × 109/l for three consecutive days. Platelet engraftment was defined as the first day the platelet count was ≥20 × 109/l for seven consecutive days independent of transfusions. Engraftment was considered successful (not delayed) if the time to achieve the pre-defined ANC occurred at or prior to 14 d, and the time to achieve the pre-defined platelet count occurred at or prior to 30 d.

Those patients who received at least one transplant at the MTD or maximum planned dose (MPD) were deemed as evaluable patients for efficacy analysis. The 95% confidence interval of the response rate was computed using the exact Binominal distribution. OS and PFS were calculated using the methods of Kaplan and Meier with standard errors computed using Greenwood's formula with log-log transformation, and were compared using log-rank test.

Statistical methods, including t-test, Spearman correlation, and Cox Proportional-Hazards model, were used to evaluate clinical association of FA/BRCA gene expression. Specifically, the t-test was used to examine if the gene expression changed from baseline to after one dose of bortezomib (and after two cycles). Spearman correlation was used to compare the FA/BRCA gene expression to the monoclonal spike ratios and disease response categories. Cox Proportional-Hazards model was used to test the relationship between the FA/BRCA gene expression to OS and time to progression. Prior to data analysis, the FA/BRCA gene expression was first normalized to baseline using the ratio and then transformed by logarithm to make the data less skewed. P values <0·05 were considered statistically significant unless otherwise specified.


  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure of conflicts of interest
  8. References

Patient characteristics and response to salvage bortezomib

A total of 30 patients received at least one dose of salvage bortezomib. Clinical characteristics of these patients are summarized in Table 1. Fifteen were males (50%) and the median age was 54·5 years (range, 36–70 years). Information on conventional cytogenetics was available in 28 patients and FISH analysis was available in 29 patients. Thirteen patients (45%) had high-risk chromosomal abnormality. Induction therapy had consisted of combination chemotherapy including dexamethasone, thalidomide, lenalidomide, or doxorubicin formulation. Twenty-three patients received one line of therapy and seven received two or more lines of therapy. Of those 29 patients with MM, 16 (55%) had disease progression either while on induction therapy or within 60 d of discontinuing the therapy prior to enrollment to the study.

Table 1. Patient characteristics.
N = 30n%
  1. a

    One patient with plasma cell leukaemia was not included in the chromosomal risk classification.

  2. b

    Disease progression during induction therapy or within 60 d of discontinuing induction therapy (prior to enrollment).

  3. c

    One patient was diagnosed as asymptomatic myeloma 6 years prior to study enrollment and only required systemic therapy 4 months before the enrollment.

  4. d

    Three patients received liposomal doxorubicin-containing regimen.

  5. e

    One patient with plasma cell leukaemia was excluded.

Age at transplant, years: median, year (range)54·5 (36–70)
Multiple myeloma subtype
 Light chain517
 Plasma cell leukaemia13
Chromosomal abnormalitya
 High risk1345
13q deletion1138
17p deletion27
 Standard risk1655
β2 microglobulin, median, mg/l (range)4·35 (1·8–11·4)
Refractory diseaseb1655
Albumin, median, g/l (range)37 (31–49)
Time from diagnosis to wenrollment, months: median (range)7 (3–80)c
Prior chemotherapy
 Lenalidomide-based regimend1137
 Vincristine/adriamycin/dexamethasone (VAD)517
 Liposomal doxorubicin-based regimen310
Number of prior regimens
Durie-Salmon staging systeme
International Staging System (ISS)

After two cycles of bortezomib (n = 30), the overall response [CR + very good partial response (VGPR) + PR] and CR rates were 37% [95% confidence interval (CI): 19·9–56·1%] and 3%, respectively. Two patients had progressive disease after the first cycle of bortezomib. One patient with obesity hypoventilation syndrome was not a candidate for high-dose chemotherapy. Adverse events included 10 patients (33%) with grade 1–2 sensory peripheral neuropathy and one patient (3%) with grade 1–2 cranial neuropathy. Grade 3 adverse events include anaemia (10%), thrombocytopenia (13%), neutropenia (10%), and pneumonia (3%). One patient developed significant cytokine release syndrome and pulmonary dysfunction after G-CSF mobilization and was taken off study. One patient experienced diffuse alveolar haemorrhage after the first cycle of bortezomib, which resolved, but prompted removal from the study. Thus, 25 patients proceeded to transplant conditioning with melphalan plus bortezomib.

Stem cell collections and engraftments

All 29 patients receiving mobilization therapy collected sufficient autologous stem cells for transplantation. Patients received a median of 2·59 × 106 (range: 2·07–11·2) CD34+ cell/kg for the first autologous HCT (HCT1) and a median of 2·71 × 106 (range: 2·0–11·2) CD34+ cell/kg for the second autologous HCT (HCT2). Neutrophils (ANC ≥ 0·5 × 109/l for 3 d) recovered at medians of 12 (range: 10–23) days after autologous HCT1 and 12 (range: 11–16) d after autologous HCT2. Platelets (≥20 × 109/l for 7 d without transfusions) recovered in medians of 13 (range: 11–20) d after HCT1 and 12 (range: 9–14) d after HCT2, respectively.

Bortezomib dose escalation and conditioning regimen safety

Bortezomib was administered at 0·7 mg/m2 to six patients in Cohort 1, at 1·0 mg/m2 to six patients in Cohort 2, and successfully escalated to 1·3 mg/m2 and administered to a total of 18 patients in Cohort 3, which was the MPD. One patient in Cohort 1 experienced delayed neutrophil engraftment and this patient did not receive G-CSF support post-transplant. Cohort 1 was expanded and there were no further DLTs. Cohort 2 was expanded while three initial patients on Cohort 3 were still <30 d post tandem transplants as specified in the protocol. No DLTs were observed in either Cohort 2 or 3. Thus, no formal MTD was determined. Three patients did not proceed with second HCT because of disease progression (n = 1) and delayed recovery from the first HCT (n = 2), hence a total of 22 patients received tandem transplants. Observed grade 3–4 adverse events are summarized in Table 2. There was no treatment-related mortality.

Table 2. Grades 3–4 adverse events after autologous haematopoietic cell transplantation (= 25 and 22).a
 Grade 3, n (%)Grade 4, n (%)
  1. HCT1, first haematopoietic cell transplantation; HCT2, second haematopoietic cell transplantation.

  2. a

    There were no grade 5 toxicities.

Febrile neutropenia12 (48)13 (59)0 (0)1 (5)
Hypokalaemia7 (28)5 (23)0 (0)0 (0)
Bacteraemia6 (24)2 (9)0 (0)1 (5)
Mucositis3 (12)4 (18)1 (4)0 (0)
Hypophosphataemia2 (8)3 (14)0 (0)0 (0)
Pneumonia2 (8)0 (0)0 (0)0 (0)
Diarrhoea1 (4)1 (5)0 (0)0 (0)
Nausea1 (4)0 (0)0 (0)0 (0)
Vomiting1 (4)0 (0)0 (0)0 (0)
Clostridium difficile infection1 (4)0 (0)0 (0)0 (0)
Systolic dysfunction1 (4)0 (0)0 (0)0 (0)
Diastolic dysfunction1 (4)0 (0)0 (0)0 (0)
Fatigue1 (4)0 (0)0 (0)0 (0)
Hypoxia1 (4)0 (0)0 (0)0 (0)
Dyspnea1 (4)0 (0)0 (0)0 (0)
Hypotension0 (0)0 (0)0 (0)1 (5)

Myeloma responses after transplants

The responses at 3 months after completing at least one transplant (n = 25) were as follows: CR 20%, VGPR 24%, PR 40%, stable disease (SD) 4%, progressive disease (PD) 4%, and 8% were not evaluable with an ORR of 84% (95% CI: 63·9–95·5%). The CR rate increased to 36% at best response (median of 8·1 months: range 4·3 – 12·3) and the overall response was at 84% (95% CI: 70·8–97·1%). Figure 2 shows the distribution of disease responses before and after the two cycles of bortezomib, at 3 months after the completion of transplant, and at their best responses.


Figure 2. Responses after treatment. The overall response and complete response rates increased after autologous haematopoiectic cell transplantation. HCT, haematopoietic cell transplantation; CR, complete response; PR, partial response; VGPR, very good partial response.

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Progression-free and overall survivals

The median OS from the first dose of bortezomib was 35 (95% CI: 22–43) months, with a median follow-up of 48 (range, 3–62) months (Fig 3A). The median PFS from the first dose of bortezomib was 15 (95% CI: 11–21) months (Fig 3B). For patients receiving a transplant, the median OS from the time of first transplant was 40 (95% CI: 32–40) months and the median PFS from the first transplant was 15 (95% CI: 10–20) months, respectively. Of those 25 transplanted patients, 19 had disease progression. Patients with standard risk by cytogenetics had a significantly higher OS (= 0·02: Fig 3C) and PFS (= 0·03: Fig 3D) than high-risk patients. There were no significant differences in the OS between the group with initial response (≥PR) to salvage bortezomib and those without the response (= 0·60). However, there was a suggestion of longer PFS in patients responsive to salvage bortezomib compared to non-responders (= 0·05: Fig 3E).


Figure 3. (A) Overall survival (OS). Kaplan–Meier probability of overall survival. Vertical tick marks indicate censored patients. (B) Progression-free survival (PFS). Kaplan–Meier probability of progression-free survival. Vertical tick marks indicate censored patients. (C) Overall survival according to risk groups. Kaplan–Meier probability of overall survival according to risk group. Solid line, high risk group; dashed line, standard risk group; vertical tick marks indicate censored patients. (D) Progression-free survival according to risk groups. Kaplan–Meier probability of progression-free survival according to risk group. Solid line, high risk group; dashed line, standard risk group; vertical tick marks indicate censored patients. (E) Progression-free survival according to response to salvage bortezomib. Kaplan–Meier probability of progression-free survival according to response to salvage bortezomib. Solid line, non-responders; dashed line, responders; vertical tick marks indicate censored patients.

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FA/BRCA pathway gene expression after bortezomib and correlation with time to progression and survival

A total of 12 patients had both baseline bone marrow aspirates and samples after first dose of bortezomib for FA/BRCA pathway gene expression analysis. Eleven patients had samples for both baseline and after two cycles of salvage bortezomib. There was a statistically significant lower BRCA2 and FANCF mRNA expression after the first dose of bortezomib (= 0·007 and = 0·046, respectively; Fig 4A) and there was a trend toward lower expression of FANCC, FANCD2, RAD51, and BRCA1 after the first dose of bortezomib (Fig 4A). There was also a statistically significant lower BRCA2 mRNA expression after two cycles of bortezomib (= 0·0055; Fig 4B). There was no statistically significant association between baseline expression of the FA/BRCA pathway genes and bortezomib-induced down-regulation of gene expression with either time to progression or OS when examined individually or when combined as a pathway.


Figure 4. (A) FA/BRCA gene expression by RT-PCR after first dose of bortezomib. BRCA2, also known as FANCD1, and FANCF expressions were significantly lower after first dose of bortezomib (= 0·0072 and = 0·0458, respectively). There was a trend toward decreased expression of four other genes including FANCC,FANCD2,RAD51 and BRCA1. (B) FA/BRCA gene expression by RT-PCR after two cycles of bortezomib. BRCA2 expression remained significantly lower after two cycles of bortezomib (= 0·0055).

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  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure of conflicts of interest
  8. References

Our trial offers several distinctive features in comparison to previously reported studies. First, the patient population studied was specifically primary resistant MM, defined as failure to achieve at least a partial response after induction therapy, and bortezomib-naïve. Second, we evaluated the ability of single agent bortezomib to salvage this population. Third, bortezomib was added to a standard high-dose melphalan conditioning regimen in tandem transplants and demonstrated its safety and unaffected engraftment kinetics. Additionally, we explored the effect on FA/BRCA DNA damage repair pathway and showed significantly decreased mRNA expression after bortezomib.

The use of high-dose chemotherapy in patients with refractory MM has been proven to be feasible and efficacious, as was demonstrated by the Intergroup Trial (Vesole et al, 1994). Acknowledging the methodological differences in documenting disease response utilizing more sensitive free light chain assay or immunofixation, previous high-dose therapy studies have shown ORR of 70–92% and CR rates of 16–40% with single autologous transplant in patients with primary refractory MM (Singhal et al, 2002; Alexanian et al, 2004; Kumar et al, 2004). However, median PFS and OS were 18–30 months and <5 years, respectively, therefore supporting the need for better treatment modalities for this patient population.

The patients enrolled to our trial had primary resistant MM or PCL. More specifically, 55% of MM patients had disease progression either while on induction therapy or within 60 d of discontinuing the therapy prior to enrollment to the study, albeit they were bortezomib-naïve. This poor response to initial therapy occurred despite the use of lenalidomide in more than one-third of our patients. OS from first transplant was longer than OS from the time of first bortezomib, reflecting the aggressive disease biology and progression prior to transplant. The ORR of 84% was seen after tandem transplants with novel combination of bortezomib plus melphalan. A CR rate of 20% at 3 months after the transplants increased to 36% at the peak of best response. This suggests that the addition of bortezomib to high-dose therapy may enhance the CR rate for a group of bortezomib-naïve patients with primary resistant disease. However, OS of 35 months with PFS of 15 months is in line with previously reported results (Gertz et al, 2010) (OS of 30·4 months and PFS of 13·1 months) for patients who did not achieve PR after thalidomide and/or lenalidomide Taken together, the evidence suggests the durable role of autologous HCT as salvage therapy in refractory patient population, albeit with a short response duration. The combination of bortezomib plus high-dose melphalan would need to be examined in a larger controlled trial before more definitive conclusions are drawn.

In a similar concept, others have combined bortezomib with high-dose melphalan in different cohorts. In a multicentre Phase II trial, Roussel et al (2010) reported at a minimum 70% VGPR rate including CR rate of 32% after autologous HCT in newly diagnosed MM patients with melphalan plus bortezomib conditioning. A matched cohort comparison with Intergroupe Francophone du Myélome (IFM) 2005-01 showed higher CR rate with combination of bortezomib and high-dose melphalan than standard regimen (35% vs. 11%, = 0·001) (Roussel et al, 2010). Bortezomib was administered intravenously at 1 mg/m2 on days −6, −3, +1, and +4. Lonial et al (2010) performed a dose- and schedule-finding randomized Phase I/II study by administering bortezomib either before or after high-dose melphalan in patients who did not achieve VGPR after induction. Pharmacodynamic studies showed greater plasma cell apoptosis among patients who received bortezomib following melphalan (Lonial et al, 2010). The data were only available only after we completed accrual to our current study, however, the result was in line with our trial and it underscores the importance of administration sequence for the synergism enhancement (Mitsiades et al, 2003).

Although the pathogenesis of primary resistance is poorly understood, it probably involves factors intrinsic to the myeloma cells, as well as those posed by the bone marrow microenvironment (Damiano et al, 1999, 2001; Mudry et al, 2000; Dalton et al, 2004). Increasing activity of NF-κB in MM correlates with an increase in tumour cell survival (Palombella et al, 1994; Andela et al, 2000; Huang et al, 2000; Kim et al, 2000). Regulation of anti-apoptotic products via NF-κB activation is one example of cell adhesion-mediated drug resistance (CAM-DR) (Meads et al, 2009). Bortezomib has been shown to reduce binding interactions between myeloma cells and bone marrow stromal cells (Hideshima et al, 2001), and sensitize highly resistant myeloma cell lines to chemotherapy (Ma et al, 2003). Sensitization of myeloma cells to melphalan by bortezomib, which may overcome chemoresistance, has been shown in vitro (Hideshima et al, 2001; Mitsiades et al, 2003).

The mechanisms by which bortezomib enhances melphalan response in myeloma cells are still not fully understood. The FA/BRCA DNA damage repair pathway has been reported to be a critical determinant of melphalan response and resistance (Hazlehurst et al, 2003; Chen et al, 2005). Measurement of FA/BRCA gene expression was incorporated to functionally evaluate the effect of bortezomib on FA/BRCA DNA damage repair pathway expression. Despite the small sample size, we demonstrated statistically significant lower BRCA2 and FANCF expression as well as a trend toward lower expression of four other FA/BRCA genes after the dose of bortezomib. Reduced BRCA2 and FANCF expression following bortezomib treatment would be sufficient to afford a drug sensitive phenotype, as in Fanconi anaemia, where loss of function mutations in individual FA/BRCA pathway complementation groups results in decreased DNA repair capacity and predisposition to cancer(Howlett et al, 2002). FAND1 is a critically important downstream effector of FA complex in homologous recombination, unlike other FA genes' products where the altered function may result in only mildly reduced DNA repair capacity (Nakanishi et al, 2005; Sung & Klein, 2006). These findings suggest the significant impact of decreased BRCA2 and/or FANCF expression in FA/BRCA DNA repair pathway, which would result in melphalan sensitization with bortezomib. No statistically significant associations among the FA/BRCA expression, disease response, and time to progression were observed which may be in part due to limited sample size and the timing of assessment. The effect of bortezomib on FA/BRCA pathway expression appears transient and decreases within 24–48 h (Yarde et al, 2009). To this end, it may be more effective to examine FA/BRCA expression within 24 h of the combined dosing and compare with clinical endpoints. Nonetheless, our data suggest that targeting the FA/BRCA DNA damage repair pathway may potentially enhance chemotherapeutic response in myeloma patients and may provide important insights into the mechanisms associated with the observed synergy between bortezomib and high-dose melphalan.

This report demonstrates the feasibility and safety of adding sequential treatment with bortezomib followed by a combination of bortezomib and high-dose melphalan as a conditioning regimen for tandem autologous HCTs in patients with primary resistant MM. As bortezomib in combination with high-dose melphalan was safely administered in these resistant MM patients and FA/BRCA gene expression was decreased after bortezomib, further exploration to discern the true efficacy of this novel combination is warranted in the context of randomized controlled trials with chemosensitive MM. Further evaluation of FA/BRCA DNA damage repair pathway gene analyses is also required to dissect the mechanisms of bortezomib synergy with high-dose melphalan.


  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure of conflicts of interest
  8. References

We thank the patients, physicians, nurses and research staff at MCC. We thank Mrs. Beth Maddox and Christine M. Simonelli for their excellent work in data collection. This work was supported in part by the NIH/NCI grant number P30 CA 76292-11 (W.S.D.) and by Research Support from Millennium Pharmaceuticals Inc. Correlative studies were performed at the Translational Core at MCC (PI: Christopher Cubitt, PhD). D.S., R.B., L.P., M.A.KD., J.L.OB., H.F.F., J.R., and M.A. performed the research, T.J.A., C.A., and M.A. designed the research study, K.S., D.N.Y., and V.O. contributed to the DNA repair pathway analysis, T.N., K.S., W.F. G.H., J.K., DT.C., C.A., and M.A. analysed the data and T.N., J.P., C.A., and M.A. wrote the paper.

Disclosure of conflicts of interest

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure of conflicts of interest
  8. References

Declared conflicts of interest include: MA Consultant and Research support from Millennium Pharmaceuticals Inc., Cambridge, MA, USA.


  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure of conflicts of interest
  8. References
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