Peripheral Blood Stem Cell Autografts Research Articles

Is Quantification of Measurable Clonal Plasma Cells in Stem Cell Grafts (gMRD) Clinically Meaningful?

With the introduction of more effective novel therapies, the prognosis of multiple myeloma (MM) has improved significantly over the past decade, resulting with a significant proportion of patients achieving durable remissions that may reach even more than 10 years. Several studies demonstrated that the real prognostic value of complete remission (CR) relies on sustained undetectable minimal residual disease (MRD). Additionally, advances in MRD detection methods used for the detection of clonal plasma cells (cPC) inside or outside the bone marrow have also improved the value of MRD. The use of peripheral blood for MRD detection could be an effective method that overcomes the spatial heterogeneity and invasive intervention with recurrent bone marrow aspirations. During the last two decades, many groups have investigated the role of circulating plasma cells (CPCs) at diagnosis. As also presented by multiple groups during the recent ASH 2021 annual meeting, CPCs are becoming recognized as an independent prognostic factor. In addition, measurement of post-induction residual plasma cells in the stem cell graft is identified as another option for MRD assessment. Earlier studies in the era of less intensive induction regimens attempts to analyze the level of CPC contamination in the graft was shown to contribute to myeloma relapse and progression. According to these recent results, higher graft purity has been found to be in concordance with deeper responses. As expected, graft minimal residual disease (gMRD) may reflect the efficacy of induction as an additional response assessment tool. Although gMRD is a non-invasive approach, it has not gained sufficient support for routine use. In view of the hurdles related to monoclonal protein assessments, high-sensitivity cellular component measurement continues to possess its value as an end point for therapeutic efficacy. In this review, we will present a structural framework for MRD testing in peripheral blood stem cell autografts in MM and review the clinical integration into MM management.

Aberrant Plasma Cell Contamination of Peripheral Blood Stem Cell Autografts, Assessed by Next-Generation Flow Cytometry, Is a Negative Predictor for Deep Response Post Autologous Transplantation in Multiple Myeloma; A Prospective Study in 199 Patients.

Simple SummaryAutologous stem cell transplantation (ASCT) remains the standard of care for transplant-eligible newly diagnosed Multiple Myeloma (NDMM) patients. However, despite its overall benefit, patients undergo various clinical outcomes post ASCT. Therefore, the identification of biomarkers that could explain, at least partially, this heterogeneity is of utmost clinical significance. The aim of this study was to assess clonal plasma cell contamination of the stem cell grafts of NDMM and evaluate its potency as a predictive/prognostic marker. Our results showed that worse responses to induction therapy correlate with higher levels of graft contamination. Importantly, our data revealed the significantly higher risk of delaying or not achieving complete remission and minimal residual disease (MRD)-negative responses among patients with graft contamination. Graft contamination is emerging as a promising predictive biomarker of clinical relevance that could be used to stratify patients post ASCT.High-dose chemotherapy with autologous stem cell support (ASCT) is the standard of care for eligible newly diagnosed Multiple Myeloma (MM) patients. Stem cell graft contamination by aberrant plasma cells (APCs) has been considered a possible predictive marker of subsequent clinical outcome, but the limited reports to date present unclear conclusions. We prospectively estimated the frequency of graft contamination using highly sensitive next-generation flow cytometry and evaluated its clinical impact in 199 myeloma patients who underwent an ASCT. Contamination (con+) was detected in 79/199 patients at a median level 2 × 10−5. Its presence and levels were correlated with response to induction treatment, with 94%, 71% and 43% achieving CR, VGPR and PR, respectively. Importantly, con+ grafts conferred 2-fold and 2.8-fold higher patient-risk of not achieving or delaying reaching CR (4 vs. 11 months) and MRD negativity (5 vs. 18 months) post ASCT, respectively. Our data also provide evidence of a potentially skewed bone marrow (BM) reconstitution due to unpurged grafts, since con+ derived BM had significantly higher prevalence of memory B cells. These data, together with the absence of significant associations with baseline clinical features, highlight graft contamination as a potential biomarker with independent prognostic value for deeper responses, including MRD negativity. Longer follow-up will reveal if this corresponds to PFS or OS advantage.

Bortezomib Maintenance (BM) or Consolidation (BC) Following Aggressive Immunochemotherapy and Autologous Stem Cell Transplant (ASCT) for Untreated Mantle Cell Lymphoma (MCL): 8 Year Follow up of CALGB 50403 (Alliance)

Bortezomib Maintenance (BM) or Consolidation (BC) Following Aggressive Immunochemotherapy and Autologous Stem Cell Transplant (ASCT) for Untreated Mantle Cell Lymphoma (MCL): 8 Year Follow up of CALGB 50403 (Alliance)

Influence of Clonal Plasma Cell Contamination of Peripheral Blood Stem Cell Autografts on Progression and Survival in Multiple Myeloma Patients After Autologous Peripheral Blood Stem Cell Transplantation in Long-term Observation

BackgroundHigh-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) remains the mainstay of treatment of eligible patients diagnosed multiple myeloma. The role of clonal plasma cell (CPC) contamination was found as a reason for relapse, but results in terms of survival, progression, and purging were ambiguous. Therefore, the aim of the study was to explore the influence of CPC contamination in the autograft on survival and progression after auto-PBSCT. Study DesignThe study included 59 patients diagnosed and treated for multiple myeloma in 1998–2004. Cells with coexpression of CD38+++CD138++CD56+ and lacking the expression of CD45, CD19, CD10, CD20, and CD23 were considered CPC in flow cytometry. ResultsThe risk of death and progression after auto-PBSCT increased significantly by 10% (P < .021) and 8% (P < .034) per 1 × 106/kg of the CPC number, respectively. For CPC number above 2.96 × 106/kg overall survival achieved clinical significance. Two years after auto-PBSCT, the risk of death was independent of CPC number among the patients who survived (P = .70). Analogous conclusions concerned results of progression-free survival at 1 year after auto-PBSCT. ConclusionsHigh clonal plasma cell contamination (>2.96 ×1 06/kg; 90th percentile of CPC number) is associated with the worst progression-free survival and overall survival. Therefore purging in vitro might be considered for the patients with the highest CPC contamination. Negative consequences of CPC contamination on the risk of death are observed for only 2 years after auto-PBSCT. Thereafter only those patients who had lower CPC contamination survived.

Acute Phase Proteins for Monitoring Hematopoietic Recovery After Early Engrafting of CD34+ Peripheral-Blood-Stem-Cells for Autografting or Allografting in Patients with Malignant Diseases.

BACKGROUND Neutropenic fever (NF) is associated with delayed engraftment after peripheral blood stem cell transplantation (PBSCT). MATERIAL AND METHODS We followed the levels of acute-phase proteins (APPs) serially in 60 patients after peripheral blood stem-cell autograft (n=39) or peripheral blood stem-cell allograft (n=21) for hematologic malignancies and germinal tumors; we then examined the correlation of those levels with the presence of fever and with markers of engraftment. RESULTS Fever (present in 60% of patients) was associated with a highly statistically significant delay in reaching conventional engraftment targets (ANC >500/μL [0.5×10^9/L]; platelets >20,000/μL [20×10^9/L]; reticulocytes >20,000/μL [20×10^9/L]) (for all associations, p<0.001). Every 4th day for 24 days, we measured the APPs levels and the number of neutrophils (ANC), platelets (PL), and reticulocytes (RET) to reach the reference values of >0.5 G/L or >1.0 G/L for ANC, >20 G/L or >50 G/L for PL, and >20 G/L for RET, respectively. The presence of NF resulted in longer time to engraft hematopoietic stem cells with ANC, PL, and PET counts statistically significant (range 0.001-0.004). The median day range for NF patients was 21.22-26.89 versus 13.88-19.13 for no NF patients. CONCLUSIONS Our results provide additional information for monitoring hematopoietic engraftment in patients following PBSCT; the presence of NF can be tracked by serial measurements in serum of three investigated APPs throughout an early phase of hematopoietic recovery.

Bortezomib Maintenance (BM) Versus Consolidation (BC) Following Aggressive Immunochemotherapy and Autologous Stem Cell Transplant (ASCT) for Untreated Mantle Cell Lymphoma (MCL): CALGB (Alliance) 50403

Introduction: Aggressive chemo-immunotherapy followed by peripheral blood stem cell autografting (ASCT) in CALGB 59909 achieved a median progression-free survival (PFS) in MCL of 5 years (Damon et al JCO, 2009), but late recurrences occurred. Bortezomib has a 33% response rate in relapsed/refractory MCL. Using the CALGB 59909 treatment backbone, we evaluated tolerability and efÞcacy of adding post-transplant BC or BM in a randomized phase II trial.Methods: The primary endpoint was PFS estimated from study entry for each treatment arm. Induction therapy was with 2-3 cycles of augmented R-CHOP (2000 mg/m2 cyclophosphamide) and methotrexate (300 mg/m2) followed by high-dose cytarabine/etoposide/rituximab(R)/Þlgrastim (EAR) stem cell mobilization and cyclophosphamide/carmustine/etoposide (CBV) ASCT. After 2 doses of post-transplant R, patients were randomized to BC (1.3 mg/ m2 days 1, 4, 8, 11 of a 3 week cycle for 4 cycles) or BM (1.6 mg/m2 weekly 4 of 8 weeks for 18 months) beginning at approximately day 90. Minimal residual disease (MRD) was analyzed using patient-specific PCR probes for the bcl-1 / IgH junction or the IgH CDR3 region.Results: 151 patients were enrolled at 14 sites and 147 received treatment. Median age was 59 (29-69); stage II (2.7%), III (12%), IV (86%); MIPI low (52.4%), int. (30.6%), high (17%); blastoid histology (14%); bone marrow involvement (81%). 118 (88%) underwent ASCT and 102 (68%) were randomized. Most withdrawals (45) were for progression (10) or adverse events (AEs) (19) including 4 treatment-related deaths. Following randomization, 34 (65%) completed BM and 33 (66%) completed BC. Withdrawal for AEs occurred in 14 (28%) of BC and 7 (13%) of BM patients (p = 0.088), most for cytopenias or peripheral neuropathy. Median follow-up was 5.5 years from registration. Median PFS was significantly greater than the null hypothesis (4 years) for both BM and BC (1-sided test of exponential parameter p < 0.001). The 5-year PFS estimates from study entry in the BM and BC arms were 70% (55-81%) and 69% (54-80%), respectively. Progression occurred in 17 BM (12 post-treatment) and 19 BC patients (all post-treatment). Five-year PFS from time of transplantation in CALGB studies 50403 (n=118) and 59909 (n=66) was 72.7% (63-80%) and 51.5% (36.7-62%), respectively (log rank p=0 0006) favoring the 50403 trial which differed from 59909 only by the addition of post-transplant bortezomib. MRD results were available in 47 patients. Five-year PFS from study entry was 93% if MRD-negative (n=15) and 51% if MRD-positive (n=32) following induction chemo-immunotherapy (log rank p=.003) (See figure).Conclusions: Induction chemotherapy followed by ASCT and either BC or BM was efficacious and tolerable, although BC was associated with more withdrawals for toxicity. The comparison between studies 50403 and 59909 suggests a PFS benefit from the addition of BC or BM. MRD-negativity following induction chemo-immunotherapy is highly associated with improved PFS and could provide an important tool for designing future trials. [Display omitted] DisclosuresOff Label Use: Post-autotransplant use of bortezomib . Bartlett:Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy; Janssen: Research Funding; Pharmacyclics: Research Funding; Astra Zeneca: Research Funding; ImaginAB: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Medimmune: Research Funding; Millenium: Research Funding; Celgene: Research Funding. Byrd:Acerta Pharma BV: Research Funding. Blum:cephalon: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding. Hurd:Procter and Gamble: Equity Ownership; Medtronic: Equity Ownership; Pfizer: Equity Ownership; Merck: Equity Ownership; Bristol Myers Squib: Equity Ownership. Czuczman:MorphoSys: Consultancy; Cellgene: Employment; Immunogen: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees. Leonard:Weill Cornell Medical College: Employment; Genentech: Consultancy; Medimmune: Consultancy; AstraZeneca: Consultancy; Spectrum: Consultancy; Boehringer Ingelheim: Consultancy; Vertex: Consultancy; ProNAI: Consultancy; Biotest: Consultancy; Seattle Genetics: Consultancy; Pfizer: Consultancy; Mirati Therapeutics: Consultancy; Gilead: Consultancy; Novartis: Consultancy. Cheson:AstraZeneca: Consultancy; Astellas: Consultancy; Ascenta: Research Funding; Spectrum: Consultancy; Teva: Research Funding; MedImmune: Research Funding; Pharmacyclics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Roche/Genentech: Consultancy, Research Funding; Celgene: Consultancy, Research Funding.

Content of Endothelial Progenitor Cells in Autologous Stem Cell Grafts Predict Survival after Transplantation for Multiple Myeloma

Multiple myeloma (MM) is considered an incurable B cell malignancy, although many patients can benefit from high-dose therapy with autologous stem cell transplantation (ASCT) as a first-line treatment. In non-Hodgkin lymphoma (NHL), ASCT is usually performed after relapse with curative intent. Disease progression is often associated with increased angiogenesis, in which endothelial progenitor cells (EPC) may have a central role. Here, we investigated the clinical impact of EPC levels in peripheral blood stem cell (PBSC) autografts for MM and NHL patients who received ASCT. EPC were identified by flow cytometry as aldehyde dehydrogenasehi CD34+ vascular endothelial growth factor receptor 2+ CD133+ cells in both MM and NHL autografts. In MM, there was a positive correlation between EPC percentage and serum (s)-β2-microglobulin levels (r2 = .371, P = .002). Unlike for NHL patients, MM patients with high numbers of infused EPC (EPC cells per kilogram) during ASCT had significant shorter progression-free survival (PFS) (P = .035), overall survival (P = .044) and time to next treatment (P = .009). In multivariate analysis, EPC cells per kilogram was a significant independent negative prognostic indicator of PFS (P = .03). In conclusion, the presence of high number of EPC in PBSC grafts is associated with adverse prognosis after ASCT in MM.

A Comparison Between Next-Generation Sequencing and ASO-qPCR For Minimal Residual Disease Detection In Multiple Myeloma: The Clinical Value In ASCT Setting

BackgroundAlthough molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We utilized a sequencing method, termed the LymphoSIGHT™ platform, which employs consensus primers and high-throughput sequencing to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone. The sequencing method is quantitative at frequencies above 10-5 and the lower limit of detection is below 10-6. Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR. MethodsWe compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting. Because myeloma cells exist patchily in bone marrow (BM), myeloma cells in PBSC autografts may reflect the whole amount of tumor in vivo. Thirty-six Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or complete response (CR) after ASCT. BM slides from 28 MM patients and fresh BM cells from 8 MM patients at diagnosis as well as autografts were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using next-generation sequencing (NGS). Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in follow-up samples (Faham et al, Blood 2012). ResultsMRD in autografts could be assessed 36 of 36 (100%) by NGS and 30 of 36 (83%) by ASO-qPCR. MRD in autografts was detected in 27 of 36 (75%) by NGS and 11 of 30 (37%) by ASO-qPCR (Figure 1A). Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, ASO-qPCR could not detect myeloma cells at MRD levels of 10-5 or lower. Two cases where MRD was not detected by NGS (MRDNGS(-)) and 14 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide while 7 MRDNGS(-) cases and 13 MRDNGS(+) cases were followed without post-ASCT therapy. The best post-ASCT responses were as follows: 6 (67%) mCR, 1 (11%) sCR and 2 (22%) VGPR in 9 MRDNGS(-) cases; 2 (14%) mCR, 2 (14%) sCR, 2 (14%) CR, 8 (58%) VGPR in 14 MRDNGS(+) cases with post-ASCT therapy; 2 (15%) sCR, 10 (78%) VGPR and 1 (7%) PR in 13 MRDNGS(+) cases without post-ASCT therapy. The MRDNGS(-) cases tended to show a better PFS than the MRDNGS(+) cases with post-ASCT therapy (P = 0.400) and showed a significantly better PFS than those without post-ASCT therapy (P = 0.032) (Figure 1B) although overall survival rates were comparable among the three groups. To investigate the value of sensitive detection by NGS, we compared PFS in 7 MRDNGS(-) cases (Group 1) with the 6 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 tended to show a better PFS than Group 2 (P = 0.091) (Figure 1C). This underscores the value of sensitive detection of MRD in MM. [Display omitted] ConclusionsA high correlation between NGS and PCR MRD results was observed, and MRD-negativity in PBSC autografts revealed by NGS may be more closely associated with durable remission of MM than that revealed by ASO-qPCR. Disclosures:Zheng:Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment.

Detection of minimal residual disease in patients with multiple myeloma using clonotype-specific PCR primers designed from DNA extracted from archival bone marrow slides

Polymerase chain reaction (PCR)-negative molecular complete remission (mCR) can be induced by stem cell transplantation in some patients with multiple myeloma (MM) and is associated with long-term progression-free survival (PFS). The detection of molecular minimal residual disease (MRD), however, requires fresh or frozen materials for designing clone-specific primers, which are not always readily available. In this study, we used DNA extracted from archival bone marrow (BM) slides for PCR to detect MRD in 50 patients with MM who received various induction therapies and autologous peripheral blood stem cell transplantation (ASCT). Clonotype-specific immunoglobulin (Ig) H PCR primers were prepared for 32 of 50 cases (64%) using BM slides, and for 9 of 14 cases (64%) using fresh BM cells. DNA in peripheral blood stem cell autografts of the 22 patients who achieved at least a partial response after ASCT was subjected to PCR to amplify clonotype-specific rearranged IgH gene sequences. The median PFS of the eight patients with MRD-positive autografts was 18 months, whereas that of 14 patients with MRD-negative autografts was not reached at a median follow-up of 27 months (p = 0.012). Post-ASCT PFS of the four patients who achieved mCR was 100% at a median follow-up of 47 months. These results indicate that archival BM slides can serve as a source of DNA for preparing clonotype-specific primers for MRD monitoring in patients with MM whose cryopreserved myeloma cells are not available for DNA preparation. Our results also suggest that patients with MM who received MRD-negative autografts and achieved mCR have a long PFS.

Nrf2 Deficiency Leads to Reduced Hematopoietic Stem Cell Self-Renewal and Increased Sensitivity to Genotoxic Stressors Through Impaired P53 Function

Abstract 397Nrf2 is a basic region-leucine-zipper transcription factor that regulates expression of numerous genes required for oxidative stress response, drug and toxin detoxification, protein ubiquitination, and proteasomal degradation of damaged proteins. Therefore Nrf2 represents a major cellular defense mechanism against oxidative and toxic stresses. We have shown that CD34+ hematopoietic stem/progenitor cells (HSPC) drawn from peripheral blood stem cell (PBSC) autografts of lymphoma patients who develop therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) after autologous hematopoietic cell transplantation (aHCT) demonstrate significantly reduced expression of Nrf2-mediated stress response pathway genes compared to cells from patients who do not develop t-MDS/AML (Cancer Cell 20, 591–605). We have extended these findings to study the role of Nrf2 in HSPC regulation and the response to genotoxic chemotherapeutic agents by using an Nrf2 knockout (KO) mouse model. Nrf2 KO mice demonstrated impaired bone marrow (BM) long-term hematopoietic stem cell (LT-HSC) function, manifested by significantly reduced competitive engraftment of BM cells in recipient mice at 16 weeks compared to wild-type (WT) controls (42.5±2.8% vs. 57.2±3.3% peripheral blood engraftment for KO and WT donors respectively, p<0.01). An even more significant defect in engraftment of KO cells was seen after transplantation to secondary recipients (16.4±1.7% vs. 61.9±1.5% peripheral blood engraftment at 8 weeks for KO and WT donors respectively, p<0.01). Nrf2 KO mice also demonstrated increased sensitivity to ENU treatment compared to WT mice. ENU (100mg/kg) treatment resulted in severe anemia at 7 months in 100% of KO mice compared with 36% of WT mice (p<0.001). Furthermore, anemia developed at a median of 5 months in Nrf2 KO mice compared to 10 months in WT controls. Pathological examination of BM from anemic mice revealed reduced erythropoiesis with impaired erythroid maturation and expansion of myeloid cells, consistent with myeloid dysplasia. Q-RT-PCR analysis demonstrated significantly reduced expression of Nrf2 target genes, including Hmo-1, Nqo-1, Gclc and Gpx1, in BM c-kit+ HSPC from KO mice compared to WT mice (p<0.05). Interestingly, expression of several P53 target genes including Bax, Puma, Cdkn1a and Necdin were also significantly reduced in BM c-kit+ cells from Nrf2 KO compared to WT mice. We also observed reduced P53 target gene expression in Nrf2 KO c-Kit cells compared with WT cells 4 hours after treatment with ENU (p<0.05), indicating impaired P53 response in Nrf2 deficient HSPC. To examine the P53 response to DNA damaging agents in LT-HSC, we exposed Nrf2 KO and WT mice to 2Gy irradiation and selected BM LT-HSC (Lin-Sca-1+c-kit+Flt-3-CD150+CD48-) using flow cytometry 12 hours after irradiation. Q-RT-PCR analysis showed significantly reduced expression of P53 target genes Bax, Puma and Necdin (p<0.05), and a trend towards reduced expression of Cdkn1a (p=0.08), in Nrf2 KO compared to WT LT-HSC. Immunofluorescence microscopy showed significantly reduced nuclear and increased cytoplasmic localization of P53 in Nrf2 KO compared to WT LT-HSC. Western blotting analysis showed increased levels of ubquitinated proteins in BM c-kit+ HSPC from Nrf2 KO compared to WT mice, and immunoprecipitation followed by Western blotting for P53 revealed increased high molecular weight bands indicative of ubiquitinated P53 in Nrf2 KO HSPC. Nrf2 is a key regulator of protein ubiquitination and proteasomal degradation, and these results suggest that abnormal cytoplasmic accumulation of ubiquitinated P53 may contribute to altered P53 function in Nrf2 deficient HSPC. In conclusion, our results show that Nrf2 deficiency results in impaired HSC self-renewal capacity under physiological conditions, and significantly increased sensitivity to genotoxic stress, potentially explained by altered P53 protein modulation in Nrf2 deficient HSPC. These observations suggest that Nrf2 deficiency could contribute to development of t-MDS/AML following exposure to genotoxic agents, and support further evaluation of Nrf2 as a potential target of chemopreventive efforts. Disclosures:No relevant conflicts of interest to declare.

Thawed autologous peripheral blood stem cells require modified quantification methods for hematopoietic progenitor cell evaluation

The aim of this study was first, to analyze the post-thaw progenitor assays usually performed on peripheral blood stem cell autografts and second, to achieve standardization with improved flow cytometric and CFU-GM assays. In the first part of the study (n=79), recovery and Intraclass Correlation Coefficient (ICC) of total nucleated cells, CD34 and CFU-GM were analyzed before and after cryopreservation. In the second part (n=20), evaluation methods were modified : the washing step was suppressed in the flow cytometric method and 500 CD34 were plated compared to 4×10(4) total nucleated cells in the CFU-GM assay. The recovery rates were analyzed and the CFU-GM results were regarded as reliable when 30-100 colonies were observed, according to the manufacturer recommendation. The analysis of the first part showed an ICC that was perfect for total nucleated cells (0.93), substantial for CD34 (0.67) and fair for CFU-GM (0.25). Median CD34 recovery was 112.6% (29.9-222%). The CFU-GM median recovery was 31.7% (0.19-142%) leading to reliable results for 27 grafts. In the second part, the median CD34 recovery was 85.75% (54-99%). No recovery over 100% was observed. The CFU-GM assay led to 18 out of 20 evaluable autografts when 500 CD34 were seeded, compared to 10 out of 20 when total nucleated cell were seeded. Avoiding cell washing in the flow cytometric method limited the overestimate of the CD34 percentage. Plating 500 thawed CD34 improved reliability of the results and allowed a better standardization of the assay.

Detection of Minimal Residual Disease in Patients with Multiple Myeloma by PCR-Based IGH Gene Clonality Analysis Using DNA Extracted From Archival Bone Marrow Slides

Abstract 1879 Background:Multiple myeloma (MM) can be cured by allogeneic stem cell transplantation (SCT), which induces PCR-negative molecular remission (MR) in patients with MM. Autologous peripheral blood stem cell transplantation (ASCT) followed by maintenance therapy can also induce PCR-negative MR in MM patients, with a progression-free survival (PFS) rate of 100% at 42 months (JCO 2010). Accordingly, it is essential to assess the depth of remission using clonotype-specific PCR primers in the treatment of MM. To prepare primers for amplification of rearranged regions of the IgH gene specific to individual MM patients, fresh or frozen bone marrow (BM) or peripheral blood cells containing MM cells are required. However, these materials are not always available, particularly when patients are diagnosed and treated at local hospitals. Archival BM slides that had been prepared for diagnosis may serve as a source of DNA for PCR-based minimal residual disease (MRD) detection. Therefore, we examined whether DNA extracted from archival BM slides was useful for designing clonotype-specific IgH PCR primers for MRD detection in autografts as well as in the BM in the post-ASCT setting. Patients and Methods:Twenty-two Japanese patients (9 men and 13 women; median age, 56 years; age range, 48–68) with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a very good partial response (VGPR) or complete response (CR) after ASCT. BM slides from 19 MM patients, which had been stored in a steel box at room temperature for 19 to 90 months (median, 60 months), and fresh BM cells from 3 MM patients, obtained at first diagnosis, were subjected to DNA extraction using the QIAamp DNA Micro Kit (QIAGEN). The IGH Gene Clonality Assay Kit (InVivoScribe Technologies) was used to detect IgH gene rearrangements in the extracted DNA. The CDR III region of the IgH gene was sequenced to design clonotype-specific primers for amplification of the rearranged sequence in MM patients. BM slides prepared at 3 to 12 months post-ASCT were also subjected to DNA extraction, followed by MRD detection using clonotype-specific PCR primers. Results:IgH gene clonality was detected in 6 of 8 (75%) unstained slides and 4 of 5 (80%) Giemsa-stained slides that had been preserved for less than 5 years. For slides that had been preserved for more than 5 years, IgH gene clonality was detected in 5 of 11 (45%) unstained slides and 1 of 4 (25%) Giemsa-stained slides. Clonotype-specific IgH PCR primers were prepared in 9 of 19 cases (47%) using BM slides and in 3 of 3 cases (100%) using fresh BM cells. DNA in peripheral blood stem cell autografts of 12 patients who had achieved a VGPR or CR after ASCT was subjected to PCR to amplify clonotype-specific rearranged IgH gene sequences; rearranged sequences were amplified in four patients. Within 2 years post-ASCT, 3 of 4 (75%) patients with MRD-positive autografts (Group A) developed progressive disease, whereas all of the 8 patients with MRD-negative autografts (Group B) remained in remission. Group A showed a higher risk of progression than Group B (P = 0.023) (Figure 1A), although overall survival was comparable between the two groups (P =0.371). When BM slides (10 patients) and fresh BM cells (2 patients) obtained after ASCT were examined for the presence of MRD using clonotype-specific PCR primers, the rearranged IgH fragments were amplified in 5 of 12 patients. PFS of 5 MRD-positive patients tended to be shorter than that of 7 MRD-negative patients (P = 0.147) (Figure 1B). [Display omitted] Conclusions:Archival BM slides can be used as a source of DNA to prepare clonotype-specific primers for MRD detection in MM patients whose cyropreserved myeloma cells are not available for DNA preparation. Furthermore, MRD status determined by PCR-based clonality analysis was useful in predicting prognosis of patients with MM. This approach enables us to select appropriate consolidation/maintenance therapy in MM patients based on their MRD status. A large multi-institutional study is currently underway to verify the clinical relevance of MRD detection using this technique. Disclosures:No relevant conflicts of interest to declare.

Platelet-released supernatants enhance hematopoietic stem cell proliferation in vitro

Peripheral blood stem cell transplantation (PBSCT) is used to reconstitute normal hematopoiesis after myeloablative chemotherapy. The hematopoietic stem cells are collected from the blood by apheresis machines using density gradient centrifugation. Because of density similarities the grafts contain high levels of leukocytes and platelets that release various mediators into the grafts. The collected hematopoietic stem cells are therefore exposed to platelet released mediators including platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor and platelet factor-4. To investigate whether platelet activation and secretion can affect hematopoietic stem cells during PBSCT, we cultured (i) normal cord blood stem cells and (ii) mobilized peripheral blood stem cells from autografts together with the total secretion product of thrombin activated platelets (i.e. platelet released supernatants). Platelet released supernatants enhanced the cell proliferation of both peripheral blood stem cell (PBSC) autograft and normal cord blood CD34+ cells. Our study shows that platelet secretion in PBSCT autografts affect the hematopoietic stem cell function and possibly thereby the hematopoietic reconstitution after PBSCT.

Efficacy of High-Dose Sequential Chemotherapy and Autograft for Transformed Non Hodgkin's B Cell Lymphoma: Results of a Retrospective Analysis from GITIL (Gruppo Italiano Terapie Innovative nei Linfomi).

Background - Follicular lymphoma (FL) transforms in a more aggressive lymphoma in 25–60% of patients representing the outgrowth of a more malignant subclone. Transformation is usually associated to a rapidly progressive clinical course, refractoriness to treatment, and short survival. To define the impact of high dose sequential (HDS) therapy and peripheral blood stem cell (PBSC) autograft on outcome of transformed FL (TFL), we analyzed a consecutive series of 66 pts with a confirmed diagnosis of TFL registered at the GITIL centers from March 1988 to September 2004 and treated with the HDS regimen.Methods - Biopsy-proven histological transformation (HT) in diffuse large B cell lymphoma (DLBCL) was observed at diagnosis (n=24; 36%) or at relapse after a treatment for FL (n= 42; 64%). Main patient characteristics were as follows: male/female 36/30; median age 51 yrs (range 33–66), stage I-II/III-IV 8/58, IPI score 0–1/≥2 28/38. HDS regimen included: i. 3 APO or 3 DHAP courses (for patients relapsed after anthracycline-containing regimens); ii. sequential administration of hd-CTX (7g/mq), hd-Ara-C, (2g/mq q12h for 6 days) hd-Etoposide (2.4 g/mq), with PBSC harvests following hd-CY and hd-Ara-C; iii. myeloablative regimen with hd-Mitoxantrone/L-Pam (n=28) or BEAM (n=28 pts who could not receive additional anthracycline), or TBI-PAM (n=3); iv. PBSC autograft; v. consolidation radiotherapy on bulky disease. From January 1999, hd-CTX and hd-Ara-C has been supplemented with Rituximab (RHDS; n=34) with in-vivo purging intent.Results - Overall 59 patients achieved a complete remission (CR; 89%), 1 patient responded partially and underwent allogeniec bone marrow transplantation, 6 patients died for progressive disease while on therapy (PD; 9%). With a median follow-up of 67 months (range 23–170), 42 patients are alive (63.6%), 24 patients relapsed and died for progressive disease (n= 23) or toxicity (n= 1). Five-year event free survival (EFS) and overall survival (OS) are 53.0% and 63.6%, respectively. No significant differences in OS and EFS were observed between patients with HT at diagnosis or at relapse, with IPI O-1 vs IPI >2. Of note, pts treated with R-HDS showed an improved clinical outcome (OS: 76% vs. 50%; EFS: 67.6 vs. 37.5 respectively), with a large difference that did not reach statistical significance because of the limited number of patients.Conclusion - Our data strongly suggest that HDS regimen, in particular when supplemented with rituximab (R-HDS), is a very effective regimen in transformed B cell lymphoma.

Gene Expression Profiles of CD34+ Cells from PBSC Grafts Predict for Development of t-MDS Following Autologous Transplantation for HL/NHL.

Therapy-related myelodysplasia (t-MDS) is a lethal complication of autologous hematopoietic cell transplant (HCT) for Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). The development of t-MDS after HCT appears related to pre-HCT genotoxic exposures. Here we investigated whether alterations in gene expression patterns in hematopoietic stem cells (HSC) from peripheral blood stem cell (PBSC) autografts was associated with subsequent development of t-MDS after HCT. We analyzed pre-HCT PBSC samples from 18 patients who developed t-MDS and 38 controls (matched for primary diagnosis, age at HCT, race/ ethnicity, and length of follow-up) that did not develop t-MDS after HCT for HL/NHL. CD34+ cells were selected using flow cytometry. RNA was extracted from 1000 cells, processed using the Affymetrix 2-Cycle Target labeling kit and hybridized on Affymetrix U133 Plus 2.0 microarrays. Following QC assessment, 16 t-MDS and 29 matched controls were selected for further analysis. Raw data were normalized using a RMA algorithm. 35042 transcripts were selected for analysis based on ≥4 arrays having intensity >16. Limma package was used to identify genes differentially expressed between t-MDS and control samples (FDR ≤ 0.01; and > 1.5-fold up or down-regulation) while controlling for matched groups. 877 differentially expressed transcripts representing 781 unique genes were identified. Of several classification algorithms tested, the K nearest neighbor (KNN) and Naïve Bayes (NB) were found to best predict t-MDS. To select the best genes for classification, a step forward method was used for prediction analysis. First, redundant transcripts were removed with only the most significant one of the redundant transcripts being kept and genes were ranked based on p-value. Prediction analyses were performed by starting with top 2 genes and incrementing one more gene at a time. Using this approach the top 5 genes best predicted the class labels using leave-one-out (LOO) cross validation with a prediction error of 22%. To determine the statistical significance of the prediction error rate, a permutation based approach was implemented to establish the null distribution of the error rate. This determines the probability of obtaining a cross-validated classification error as small as would be achieved if there were no difference between t-MDS and control. Sample labels were randomly permuted 10,000 times and cross-validated error rates were recalculated using LOO cross validation. Based on the null distribution of the permutated error rates, the original error rate (ER0=0.22) had a P value of 0.005, indicating that the likelihood of obtaining such a small prediction error for a cross-validated classifier by chance is very low. We are currently analyzing the differentially expressed genes to investigate for altered response to genotoxic stress and stem cell regulation in HSC from patients who subsequently develop t-MDS. In conclusion, we have shown that gene expression profiles of HSC from PBSC autografts from HL/NHL patients can differentiate patients who develop t-MDS after autologous HCT from those who do not develop t-MDS. The prediction power of these gene sets will be further verified using different sets of t-MDS case and control samples.

Ideal body weight correlates better with engraftment after PBSC autograft than actual body weight, but is under-estimated in myeloma patients possibly due to disease-related height loss

Stem cell dose is important in determining rate of engraftment following autograft. We show closer correlation with haematopoietic reconstitution when the CD34+ cell dose is calculated using ideal (IBW) rather than actual (ABW) body weight in 218 patients receiving peripheral blood stem cell (PBSC) autograft for haematological malignancy. ABW was 21% greater than IBW thus the median CD34+ dose of 5.0 x 10(6)/kg (ABW) rose to 6.1 x 10(6)/kg when calculated by IBW. Neutrophils reached 0.5 x 10(9)/l in 11 days (range 8-21), while platelets reached 20 x 10(9)/l unsupported in 12 days (range 7-38). For both neutrophil and platelet engraftment, a greater inverse correlation was seen when IBW was used to calculate stem cell dose (r2=0.082 vs r2=0.104 for neutrophils and r2=0.085 vs r2=0.135 for platelets). Those non-myeloma patients who failed to achieve a CD34+ dose of 4 x 10(6) cells/kg by ABW but did so by IBW achieved neutrophil and platelet engraftment not significantly different from those who achieved that stem cell dose by both methods. This was not confirmed in patients treated for myeloma, possibly owing to inaccurate IBW in patients with skeletal height loss. We confirm that calculation of CD34+ cell dose by IBW safely predicts engraftment for patients with haematological malignancies other than myeloma undergoing PBSC autograft.

Long-term lymphoma survivors following high-dose chemotherapy and autograft: Evidence of permanent telomere shortening in myeloid cells, associated with marked reduction of bone marrow hematopoietic stem cell reservoir

To investigate telomere length (TL) and hematopoietic progenitors in long-term survivors after high-dose chemotherapy and peripheral blood stem cell (PBSC) autograft. Peripheral blood (PB) and bone marrow (BM) samples were obtained from 31 subjects in continuous complete remission from a high-risk lymphoma, at a median of 5.8 years (range: 1-11 years) since autograft. Most of them were grafted with large PBSC quantities (median CD34(+ve) cells/kg: 7 x 10(6)). TL was determined by Southern blot analysis, BM progenitors by in vitro long-term culture-initiating cells (LTC-IC) and colony assays. TL of PB granulocytes was significantly shortened in autografted subjects compared with age-matched healthy subjects; a similar finding was observed in BM. The median TL reduction in granulocytes from autografted subjects compared with age-matched controls (Delta(TelShortening)) was then assessed according to time interval since autograft. Three subject subgroups were identified-at 1 to <3 years, 3 to <6 years, and 6 to 11 years since autograft-and their telomere loss was the same, with Delta(TelShortening) of 1132, 1379, and 1214 bp in the three subgroups, respectively. The longitudinal assessment of TL in five representative patients followed for up to 40 months since autograft confirmed that telomere shortening occurring during exposure to chemotherapy as well as postautograft is persistent at long term. BM LTC-IC and multipotent and committed progenitors were assessed in subjects at >3 years after autograft and found to be markedly reduced compared with normal controls. High-dose chemotherapy and PBSC autograft may result in myelopoietic cell abnormalities that appear to be irreversible.

Altered Gene Expression in CD34+ Cells from PBSC Grafts from Patients Developing t-MDS Following Autologous Transplantation for Lymphoma.

Therapy-related myelodysplasia (t-MDS) is a lethal complication of autologous hematopoietic cell transplant (HCT) for Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). The sequence of molecular abnormalities leading to t-MDS is unknown. To gain insights in pathogenesis of t-MDS, we have initiated a prospective study of a cohort of patients undergoing autologous HCT for HL and NHL. Patients are followed longitudinally from before HCT to five years post-HCT, with serial collection of samples. Here we investigated the association of gene expression patterns in hematopoietic stem cells (HSC) from peripheral blood stem cell (PBSC) autografts with development of t-MDS after HCT. We analyzed samples from 11 patients who developed t-MDS and 33 controls (matched for primary diagnosis, age at HCT, race/ethnicity, and length of follow-up) from within the cohort who did not develop t-MDS. RNA from 1000 CD34+ cells selected by flow cytometry sorting was processed using the Affymetrix 2-Cycle Target labeling kit and hybridized on Affymetrix U133 Plus 2.0 microarrays. Following quality control assessment, 7 informative t-MDS samples and their 18 matched controls were selected for further assessment. Raw data were normalized using a GCRMA algorithm. Limma package with paired test was used to identify genes differentially expressed between t-MDS and control samples (P Value < = 0.01; and > 2-fold up or down-regulation). 148 differentially expressed transcripts representing 136 unique genes were identified. The log2 intensity of these transcripts was mean-centered within each group and two-way clustering applied. Samples clustered into 2 major groups, with one sub-cluster containing all t-MDS samples. Supervised classification analysis using DLDA (diagonal linear discriminant analysis) provided the best classification model when using all 148 transcripts. To select the best genes for classification, redundant transcripts were removed. Staring with the top 2 genes and incrementing one gene at a time, the top 8 genes best classified case vs. control using leave-one-out validation and DLDA, with an 8% error rate. To select the best 2 or 3 gene combinations, we tested the error rate using all such possible combinations within the top 20 genes. The three best 3-gene combinations (of 1140 possible combinations) accurately classified cases and controls with a 0% error rate. Functional assessment of differentially expressed genes identified overexpression of immediate-early "stress" response genes (e.g. NR4A1-3, EGR-1-4, WT-1) and reduced expression of xenobiotic processing (e.g. GSTM2, GSTT1, and CYP1B1) and DNA repair genes (e.g. XPA, ERCC4, FANCF) in samples from patients developing t-MDS. We also observed altered expression of genes playing important roles in regulating HSC growth and associated with leukemogenesis (e.g. HOXA5, A6, B6, MEIS1, MLL5, TCF4, HES1, MCL1, SMAD5, and SMAD7). In conclusion our results suggest that additional studies in a test population are warranted to examine whether gene expression patterns in PBSC samples can predict for subsequent development of t-MDS. Importantly, perturbation in genes determining response to genotoxic stress and regulating stem cell growth are observed in CD34+ cells from patients who subsequently develop t-MDS.

Prolonged survival and low incidence of late toxic sequelae in advanced follicular lymphoma treated with a TBI-free autografting program: updated results of the multicenter consecutive GITMO trial

This study provides an updated report of the consecutive multicenter Gruppo Italiano Trapianto Midollo Osseo trial employing an intensified, purging-free, total body irradiation-free, high-dose sequential chemotherapy schedule with peripheral blood stem cell autograft (i-HDS) in advanced-stage follicular lymphoma (FL). Special interest has been devoted to late toxicities and outcome in terms of molecular status. Ninety-two untreated FL patients aged <or=60 were enrolled by 20 Italian centers and evaluated on an intention-to-treat basis. Main findings are as follows: (1) 5.5-years overall survival projection of 80% (median follow-up: 68 months), with no differences related to age-adjusted IPI score; (2) 46 (50%) of 92 patients presently in continuous complete remission; (3) projected long-term progression-free survival exceeding 80% for patients collecting PCR-negative stem cell harvests or achieving molecular remission within the first 2 years from the end of therapy; (4) actuarial 5-years risk of developing secondary myelodysplasia and acute myeloid leukemia of 3.7%, with most of these events occurring in patients re-treated for recurrent lymphoma. These results demonstrate that i-HDS is feasible, effective and safe even in terms of long-term outcome. As the HDS schedule can be easily supplemented with Rituximab, it is one of the best options for random comparison with Rituximab-supplemented conventional chemotherapy.

Long-term follow-up of patients with peripheral T-cell lymphomas treated up-front with high-dose chemotherapy followed by autologous stem cell transplantation

We report the results of two prospective phase II studies investigating the role of high-dose sequential chemotherapy, followed by autologous stem cell transplantation (ASCT) in 62 patients with advanced stage peripheral T-cell lymphomas (PTCLs) at diagnosis. Conditioning regimen consisted of mitoxantrone (60 mg/m2) and melphalan (180 mg/m2) or carmustine, etoposide, Ara-C and melphalan followed by peripheral blood stem cell autografting. In an intent-to-treat analysis, 46 out of 62 patients (74%) completed the whole programme, whereas 16 patients did not undergo ASCT, mainly because of disease progression. At a median follow-up of 76 months, the estimated 12-year overall (OS), disease-free and event-free survival (EFS) were 34, 55 and 30%, respectively. OS and EFS were significantly better in patients with anaplastic lymphoma-kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL), as compared with the remaining PTCL. Multivariate analysis showed that patients attaining complete remission (CR) before ASCT had a statistically significant benefit in terms of OS and EFS (P<0.0001). Overall treatment-related mortality rate was 4.8%. In conclusion, our findings indicate (1) up-front high-dose therapy and ASCT are feasible, but could induce a high rate of long-term CR only in patients with ALK-positive ALCL and (2) the achievement of CR before autografting is a strong predictor of better survival.