Low Levels Of Residual Disease Research Articles

MM-329: Evaluation of Immunophenotype of the Non-Neoplastic Plasma Cell Compartment and Post-Therapy Immunomodulation to Understand Nuances in MRD Assessment of Multiple Myeloma Using Next-Generation Flow Cytometry

Context A deeper understanding of the normal plasma cell (PC) compartment and changes in the immunophenotypic profile post-treatment is required to assess low levels of residual disease in multiple myeloma (MM) using multiparametric flow cytometry. Objective The expression profile of selective antigens on normal and aberrant plasma cells prior to and following anti-myeloma therapy was assessed to ascertain its relevance in MRD assessment. Design This is a prospective analysis of 702 patients with the diagnosis of MM (January 2015 to June 2020). Setting The study was conducted at a tertiary care cancer center. Patients A total of 702 cases (321 treatment naive; 321 post-therapy; 60 control samples) were evaluated. Sixty samples from non-MM staging marrow were assessed to study antigenic profiles of normal PC. Interventions Bone marrow samples were collected in EDTA and processed within 6 hours of collection per the consensus recommendation from the Euro-flow protocol using two tube 8-color/single tube 10-color panel. An aberrant PC cluster was defined by presence of >30 plasma cells with light chain restriction and/or aberrant immunophenotypes. Main Outcomes Measures 1. Expression profile of antigens on the polyclonal PC compartment; 2. Post-therapy immunomodulation in MM. Results Polyclonal PC were observed in 65.42% of treatment naive samples compared to 88.16% post-treatment samples (p=0.001). Frequency of aberrant expression of markers in polyclonal PC was higher at follow-up time point: CD45 (33% vs 38%; p=0.6), CD19 (19% vs 41%; p=0.001), CD56 (14% vs 41%; p Conclusions Flow cytometry-based MRD assessment in MM is challenged by wide immunophenotypic variation in non-neoplastic PCs and changes in immunophenotype after chemotherapy; thus, simultaneous analysis of multiple immunophenotypic markers, including light chain expression, is required for reliable MRD assessment.

A Dose-Finding Study of Lenalidomide As Post-Remission Therapy for Older Adults with Acute Myeloid Leukemia (AML)

Introduction : Optimal post-remission therapy for older adults with AML is unclear. Lenalidomide (LEN) is an oral immunomodulatory agent that reconstitutes T- and NK-cell immunodeficiency in patients with other malignancies. These immunomodulatory properties could benefit patients with Acute Myeloid Leukemia (AML) in remission. LEN, alone and in combination, has led to complete remissions in older patients with AML treated with doses above those approved for other malignancies. We sought the optimal dose of LEN in older AML patients who are not candidates for intensive post-remission therapy.Methods: Subjects were: age ≥ 60 with AML; had 1-2 prior courses of induction chemotherapy; in CR, CRi, PR or had residual AML with ≤ 1 x 109/L circulating blasts; poor candidates for cytotoxic consolidation therapy as deemed by their physician or patient preference. A Bayesian dose-finding design was used to assign subjects to 3 cohorts (25 mg, 35 mg and 50 mg daily days 1-28 for one cycle). Subjects without progressive AML after cycle 1 then received up to 12 months of LEN 10 mg daily. Dose-limiting toxicities (DLTs) were CTCAEv4 grade ≥3 non-hematologic toxicity during cycle 1 and grade ≥3 hematologic toxicity beyond day 35 of cycle 1. AML response and relapse were measured by International Working Group criteria. Geriatric assessments were performed before and after cycle 1. Measures of T- and NK-cell immunity performed before and after months 1 and 4 included flow cytometry to quantify circulating T- and NK-cell subsets as well as NK-cell cytotoxicity using subject peripheral blood mononuclear cells as effector cells and K562 cell lines as targets. Effector cell-to-K562 ratios (E:T) were titrated between 1:100 and 1:1, and the percentage of dead K562 cells was plotted against E:T ratio. Cytotoxicity was reported as the slope of the curve of the percentage of dead K562 cells versus E:T ratio.Results: 19 subjects were treated with at least one 28-day course of LEN. Baseline variables are shown in the table. DLTs occurred in 2/9 subjects (hemolytic anemia, rash) at 25 mg, 0/7 subjects at 35 mg, and 1/3 subjects (elevated bilirubin) at 50 mg. No subject in complete remission required hospital admission for adverse events. Among 7 subjects who began LEN with measureable AML, two experienced improvement in AML response category (table). Median relapse-free survival for subjects starting in or entering CR was 4.3 months (range: 1.9-26.9). There was no change in proportion of T- and NK-cell subsets before and after 1 month of LEN. However, a significant decline in NK cell cytotoxicity was observed after month 1, compared with baseline cytotoxicity (p=0.04, paired t test, figure). Among three subjects with NK cell cytotoxicity measured after 4 months of LEN, there was a non-significant trend toward higher cytotoxicity than at baseline and 1-month (figure). Serial geriatric assessments did not demonstrate changes in subject physical, cognitive, psychological or social function.Conclusions: High dose LEN can be safely administered to older patients with AML after induction. Expansion of the 35 mg dose cohort is ongoing. Unexpected declines in NK-cell cytotoxicity may be due to delayed reconstitution of NK- and T-cell numbers in older patients who have been recently treated with induction therapy. Further investigation should explore responses to LEN in patients with low levels of residual disease, the impact of LEN on geriatric assessment domains, and on the T- and NK-cell repertoire after >1 month of LEN. [Display omitted] DisclosuresFoster:Celator Pharmaceuticals: Research Funding; Pfizer, Inc.: Research Funding; Macrogenics, Inc.: Research Funding; Amgen, Inc.: Honoraria; Shire: Honoraria; Celgene Corporation: Research Funding; Incyte: Honoraria. Vincent:Merck: Research Funding; Pharmacyclics: Research Funding; Nanostring: Membership on an entity's Board of Directors or advisory committees. Zeidner:Tolero Pharmaceuticals: Research Funding; Takeda: Research Funding; Tolero Pharmaceuticals: Honoraria; Celgene: Honoraria; Agios: Honoraria; Merck: Research Funding.

Clinical Significance and Biomarkers to Predict Unsustained Complete Remission in Transplant-Eligible Multiple Myeloma

Clinical Significance and Biomarkers to Predict Unsustained Complete Remission in Transplant-Eligible Multiple Myeloma

Minimal Residual Disease Assessment Within the Bone Marrow of Multiple Myeloma: A Review of Caveats, Clinical Significance and Future Perspectives.

There is an increasing clinical interest in the measure and achievement of minimal residual disease (MRD) negativity in the bone marrow of Multiple Myeloma (MM) patients, as defined equally either by Multicolor Flow Cytometry (MFC) or by Next Generation Sequencing (NGS) technologies. At present, modern technologies allow to detect up to one on 104 or on 105 or even on 106 cells, depending on their throughput. MFC approaches, which have been progressively improved up to the so-called Next Generation Flow (NGF), and NGS, which proved clear advantages over ASO-PCR, can detect very low levels of residual disease in the BM. These methods are actually almost superimposable, in terms of MRD detection power, supporting the lack of unanimous preference for either technique on basis of local availability. However, some technical issues are still open: the optimal assay to use to detect either phenotype (e.g., next generation multidimensional flow cytometry, imaging) or genotype aberrations (e.g., ASO-RQ PCR, digital droplet PCR, NGS) and their standardization, the sample source (BM or peripheral blood, PB) and its pre-processing (red-cell lysis vs. Ficoll, fresh vs. frozen samples, requirement of CD138+ cells enrichment). Overall, MRD negativity is considered as the most powerful predictor of favorable long-term outcomes in MM and is likely to represent the major driver of treatment strategies in the near future. In this manuscript, we reviewed the main pitfalls and caveats of MRD detection within bone marrow in MM patients after front-line therapy, highlighting the improving of the currently employed technology and describing alternative methods for MRD testing in MM, such as liquid biopsy.

PS1172 DDPCR AT THE TIME OF TKI DISCONTINUATION IN CHRONIC PHASE CHRONIC MYELOID PATIENTS IS PREDICTIVE OF TREATMENT‐FREE REMISSION OUTCOME

Background:Only few prognostic factors ‐ the median duration of treatment or of deep molecular response (DMR) ‐ have so far been associated to a positive outcome after TKI discontinuation in chronic myeloid leukemia (CML) patients. Thus, the identification of candidates that may impact on this primary treatment endpoint remains elusive. Digital droplet PCR (ddPCR) is an accurate molecular tool that could prove advantageous for the quantification of the BCR‐ABL1 fusion transcript copy number compared to classic RQ‐PCR and could help in better identify patients candidate for TKI discontinuation.Aims:To evaluate the predictive role of ddPCR in CML patients in sustained DMR tested at the time of TKI treatment discontinuation.Methods:We studied 50 RNA samples of CML patients who discontinued treatment while in RQ‐PCR undetectable DMR (MR4–4.5) according to IS for more than 2 years. The ddPCR assay was performed in triplicate using the Biorad‐QX200 platform. To test the BCR‐ABL fusion transcript and ABL control gene, we used the same primers and probes used in the standard RQ‐PCR reaction. Each reaction mixture was partitioned into approximately 20,000 droplets and then amplified. Cycled droplets were read in the QX200 droplet‐reader and analysis of the ddPCR data was performed using the QuantaSoft analysis software (Biorad Laboratories GmbH, Munich, Germany). We collected the clinical follow‐up data from the patients’ medical history and correlated them with ddPCR positivity or negativity at the time of discontinuation using the Wilcoxon signed‐rank test.Results:Fifty patients discontinued treatment in DMR after a median duration of response of 2.5 years (range 1.5–7.2). The median age was 59 years (range 32–87.4), 33 were female. According to the Sokal risk at baseline, 25 patients were classified as low risk, 21 as intermediate and 4 as high risk. Forty‐two patients received front‐line imatinib and 8 were treated with second generation TKIs. The median duration of treatment before discontinuation was 11.9 years (range 3.3–17.9). At the time of discontinuation, 28 patients (56%) were ddPCR‐negative and 24 of them (86%) remained in treatment‐free remission (TFR), while 4 patients (14%) relapsed after a median time of 5.5 months (range 4.0–27.0). Twenty‐two patients (44%) were ddPCR positive at the time of discontinuation: 12 of them (55%) remained in TFR after a median time of observation of 13.2 months, while 10 patients (45%) relapsed after a median of 3.5 months (range 1.0–16.0). The difference in TFR rate between the two groups is statistically significant (p = 0.026, Figure 1). We compared the characteristics of ddPCR‐negative versus ddPCR‐positive patients and failed to detect differences in the type of transcript (p = 0.155), Sokal risk score (p = 0.684), previous IFN treatment (p = 0.70), type of TKI received (p = 0.250), median duration of TKI treatment (10.9 years in positive versus 13.1 years in negative patients, p = 0.639) and median duration of DMR (2.4 years in positive versus 2.8 years in negative patients, p = 0.134).Summary/Conclusion:These results suggest that by ddPCR it is possible to predict the TFR outcome of CML patients in DMR (MR4–4.5) at the time of TKI discontinuation. This method, compared to RQ‐PCR, appears to be superior in terms of quantification of low levels of residual disease in patients who have already achieved a DMR and may successfully identify candidates to treatment discontinuation.image

Central pathology review with two-stage quality assurance for pathological response after neoadjuvant chemotherapy in the ARTemis Trial.

The ARTemis Trial tested standard neoadjuvant chemotherapy±bevacizumab in the treatment of HER2-negative early breast cancer. We compare data from central pathology review with report review and also the reporting behavior of the two central pathologists. Eight hundred women with HER2-negative early invasive breast cancer were recruited. Response to chemotherapy was assessed from local pathology reports for pathological complete response in breast and axillary lymph nodes. Sections from the original core biopsy and surgical excision were centrally reviewed by one of two trial pathologists blinded to the local pathology reports. Pathologists recorded response to chemotherapy descriptively and also calculated residual cancer burden. 10% of cases were double-reported to compare the central pathologists' reporting behavior. Full sample retrieval was obtained for 681 of the 781 patients (87%) who underwent surgery within the trial and were evaluable for pathological complete response. Four hundred and eighty-three (71%) were assessed by JSJT, and 198 (29%) were assessed by EP. Residual cancer burden calculations were possible in 587/681 (86%) of the centrally reviewed patients, as 94/681 (14%) had positive sentinel nodes removed before neoadjuvant chemotherapy invalidating residual cancer burden scoring. Good concordance was found between the two pathologists for residual cancer burden classes within the 65-patient quality assurance exercise (kappa 0.63 (95% CI: 0.57-0.69)). Similar results were obtained for the between-treatment arm comparison both from the report review and the central pathology review. For pathological complete response, report review was as good as central pathology review but for minimal residual disease, report review overestimated the extent of residual disease. In the ARTemis Trial central pathology review added little in the determination of pathological complete response but had a role in evaluating low levels of residual disease. Calculation of residual cancer burden was a simple and reproducible method of quantifying response to neoadjuvant chemotherapy as demonstrated by performance comparison of the two pathologists.

A New Computational Method to Predict Long-Term Minimal Residual Disease and Molecular Relapse after TKI-Cessation in CML

▪Predicting minimal residual disease (MRD) levels in tyrosine kinase inhibitor (TKI)-treated chronic myeloid leukemia (CML) patients is of major clinical relevance. The reason is that residual leukemic (stem) cells are the source for both, potential relapses of the leukemicclone but also for its clonal evolution and, therefore, for the occurrence of resistance.The state-of-the art method for monitoring MRD in TKI-treated CML is the quantification of BCR-ABL levels in the peripheral blood (PB) by PCR. However, the question is whether BCR-ABL levels in the PB can be used as a reliable estimate for residual leukemic cells at the level of hematopoietic stem cells in the bone marrow (BM). Moreover, once the BCR-ABL levels have been reduced to undetectable levels, information on treatment kinetics is censored by the PCR detection limit. Clearly, BCR-ABL negativity in the PB suggests very low levels of residual disease also in the BM, but whether the MRD level remains at a constant level or decreases further cannot be read from the BCR-ABL negativity itself. Thus, also the prediction of a suitable time point for treatment cessation based on residual disease levels cannot be obtained from PCR monitoring in the PB and currently remains a heuristic decision.To overcome the current lack of a suitable biomarker for residual disease levels in the BM, we propose the application of a computational approach to quantitatively describe and predict long-term BCR-ABL levels. The underlying mathematical model has previously been validated by the comparison to more than 500 long-term BCR-ABL kinetics in the PB from different clinical trials under continuous TKI-treatment [1,2,3].Here, we present results that show how this computational approach can be used to estimate MRD levels in the BM based on the measurements in the PB. Our results demonstrate that the mathematical model can quantitatively reproduce the cumulative incidence of the loss of deep and major molecular response in a population of patients, as published by Mahon et al. [4] and Rousselot et al. [5]. Furthermore, to demonstrate how the model can be used to predict the BCR-ABL levels and to estimate the molecular relapse probability of individual patients, we compare simulation results with more than 70 individual BCR-ABL-kinetics. For this analysis we use patient data from different clinical studies (e.g. EURO-SKI: NCT01596114, STIM(s): NCT00478985, NCT01343173) where TKI-treatment had been stopped after prolonged deep molecular response periods. Specifically, we propose to combine statistical (non-linear regression) and mechanistic (agent-based) modelling techniques, which allows us to quantify the reliability of model predictions by confidence regions based on the quality (i.e. number and variance) of the clinical measurements and on the particular kinetic response characteristics of individual patients.The proposed approach has the potential to support clinical decision making because it provides quantitative, patient-specific predictions of the treatment response together with a confidence measure, which allows to judge the amount of information that is provided by the theoretical prediction.

Next-Generation Sequencing-Assisted DNA-Based Digital PCR for a Personalized Approach to the Detection and Quantification of Residual Disease in Chronic Myeloid Leukemia Patients

Recent studies indicate that 40% of chronic myeloid leukemia patients who achieve sustained undetectable BCR-ABL1 transcripts on tyrosine kinase inhibitor therapy remain disease-free after drug discontinuation. In contrast, 60% experience return of detectable disease and have to restart treatment, thus highlighting the need for an improved method of identifying patients with the lowest likelihood of relapse. Here we describe the validation of a personalized DNA-based digital PCR (dPCR) approach for quantifying very low levels of residual disease, which involves the rapid identification of t(9;22) fusion junctions using targeted next-generation sequencing coupled with the use of a dPCR platform. t(9;22) genomic breakpoints were successfully mapped in samples from 32 of 32 patients with early stage disease. Disease quantification by DNA-based dPCR was performed using the Fluidigm BioMark platform on 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission. dPCR detected persistent disease in 81% of molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%). We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual-disease detection in chronic myeloid leukemia and may prove useful in the management of tyrosine kinase inhibitor withdrawal.

Conversion, correction, and International Scale standardization: results From a Multicenter External Quality Assessment Study for BCR-ABL1 testing.

Monitoring BCR-ABL1 expression levels relative to clinically validated response criteria on the International Scale (IS) is vital in the optimal management of patients with chronic myeloid leukemia, yet significant variability remains across laboratories worldwide. To assess method performance, interlaboratory precision, and different IS standardization modalities in representative laboratories performing routine BCR-ABL1 testing. Fifteen blinded test specimens with 5-level nominal BCR-ABL1 to ABL1 IS percentage ratios ranging from 5% to 0.0005% and 4-level secondary IS reference panels, the ARQ IS Calibrator Panels, were tested by relative quantitative polymerase chain reaction in 15 laboratories in 5 countries. Both raw and IS percentage ratios calculated by using local conversion factors (CFs) or analytic correction parameters (CPs) were collected and analyzed. A total of 670 valid positive results were generated. BCR-ABL1 detection was associated with variable ABL1 quality metric passing rates (P < .001) and reached at least 0.01% in 13 laboratories. Intralaboratory precision was within 2.5-fold for all sample levels combined with a relative mean difference greater than 5-fold across laboratories. International Scale accuracy was increased by using both the CF and CP standardization methods. Classification agreement for major molecular response status was 90% after CF conversion and 93% after CP correction, with precision improved by 3-fold for the CP method. Despite preanalytic and analytic differences between laboratories, conversion and correction are effective IS standardization methods. Validated secondary reference materials can facilitate global diffusion of the IS without the need to perform sample exchange and improve the accuracy and precision of BCR-ABL1 quantitative measurements, including at low levels of residual disease.

Nelarabine Is Safe and Effective In Adult Relapsed or Refractory T Cell Acute Lymphoblastic Leukemia (T-ALL) and Lymphoblastic Lymphoma (T-LBL): The Bologna Experience

Abstract 4250 Background.Nelarabine (N) is approved for the treatment of T-ALL and T-LBL that have not responded to or has relapsed after treatment with at least 2 chemotherapy regimens.Aim. To evaluate safety profile and efficacy of N as savage therapy in 16 adult relapsed or refractory T-ALL or T-LBL. Methods.After obtaining an informed consent, 16 patients (median age 33 years, range 19–45, M/F= 13/3) affected by T-ALL (n=10) and T-LBL (n=6) received savage therapy with N (median cycle=1, range 1–3), administered at standard adult dosage (1500 mg/sqm on days 1, 3 and 5, every 21). Four patients were primary resistant to induction treatment, 7 patients were relapsed after two previous chemotherapy regimens (including allogeneic BMT in 4 cases and autologous SCT in 1 case); the remaining 6 patients had a molecular relapsed disease (MRD positive). Molecular characterization was performed, including NOTCH and WT-1 genes mutational status. GEP analysis, according to Ferrando A. stratification (Cancer Cell 2002), is still ongoing. Results.Currently, 12 out of 16 patients are evaluable, due to a too short follow up in the other 4 cases. Seven out of 12 patients obtained a complete remission (CR) (5 T-ALL 2 T-LBL);a partial remission (PR) was documented in 2 cases, with an overall response rate (ORR) of 75%. Median duration of CR was 10 weeks (range 2.8–54+). Among these, 2 out of 4 patients in molecular relapse reached a molecular CR and underwent an allogenic BMT (currently in CR after a median follow up of 12 months). Extra- hematological toxicity, not clearly related to the drug, occurred in 3 cases, determining, a complete and irreversible paraplegia, a condition of mental confusion, and a peripheral neuropathy, respectively. Conclusions.N showed a strong efficacy also in cases with low levels of residual disease, in addition to a good safety profile. Neurological toxicity needs to be strictly monitored. Acknowledgments.European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, PRIN 2009, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures:Baccarani:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Abstract 2237: Nelarabine is safe and effective in adult relapsed or refractory T cellacute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LBL): The Bologna experience

Abstract Background. Nelarabine (N) is approved for the treatment of T-ALL and T-LBL that have not responded to or has relapsed after treatment with at least 2 chemotherapy regimens. Aim. To evaluate safety profile and efficacy of N as savage therapy in 16 adult relapsed or refractory T-ALL or T-LBL. Methods. After obtaining an informed consent, 16 patients (median age 33 years, range 19-45, M/F = 13/3) affected by T-ALL (n=10) and T-LBL (n=6) received savage therapy with N (median cycle=1, range 1-3), administered at standard adult dosage (1500 mg/sqm on days 1, 3 and 5, every 21). Four patients were primary resistant to induction treatment, 7 patients were relapsed after two previous chemotherapy regimens (including allogeneic BMT in 4 cases and autologous SCT in 1 case); the remaining 6 patients had a molecular relapsed disease (MRD positive). Molecular characterization was performed, including NOTCH and WT-1 genes mutational status. GEP analysis, according to Ferrando A. stratification (Cancer Cell 2002), is still ongoing. Results. Currently, 12 out of 16 patients are evaluable, due to a too short follow up in the other 4 cases. Seven out of 12 patients obtained a complete remission (CR) (5 T-ALL 2 T-LBL);a partial remission (PR) was documented in 2 cases, with an overall response rate (ORR) of 75%. Median duration of CR was 10 weeks (range 2.8-54+). Among these, 2 out of 4 patients in molecular relapse reached a molecular CR and underwent an allogenic BMT (currently in CR after a median follow up of 12 months). Extra- hematological toxicity, not clearly related to the drug, occurred in 3 cases, determining, a complete and irreversible paraplegia, a condition of mental confusion, and a peripheral neuropathy, respectively. Conclusions. N showed a strong efficacy also in cases with low levels of residual disease, in addition to a good safety profile. Neurological toxicity needs to be strictly monitored. Acknowledgments. European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2237. doi:10.1158/1538-7445.AM2011-2237

Molecular genetic analysis of haematological malignancies II: mature lymphoid neoplasms

Molecular genetic techniques have become an integral part of the diagnostic assessment for many lymphomas and other chronic lymphoid neoplasms. The demonstration of a clonal immunoglobulin or T cell receptor gene rearrangement offers a useful diagnostic tool in cases where the diagnosis is equivocal. Molecular genetic detection of other genomic rearrangements may not only assist with the diagnosis but can also provide important prognostic information. Many of these rearrangements can act as molecular markers for the detection of low levels of residual disease. In this review, we discuss the applications of molecular genetic analysis to the chronic lymphoid malignancies. The review concentrates on those disorders for which molecular genetic analysis can offer diagnostic and/or prognostic information.

Molecular genetic analysis of haematological malignancies: I. Acute leukaemias and myeloproliferative disorders

Molecular genetic techniques are now routinely applied to haematological malignancies within a clinical laboratory setting. The detection of genetic rearrangements not only assists with diagnosis and treatment decisions, but also adds important prognostic information. In addition, genetic rearrangements associated with leukaemia can be used as molecular markers allowing the detection of low levels of residual disease. This review will concentrate on the application of molecular genetic techniques to the acute leukaemias and myeloprolferative disorders.

Clinical significance of HOX11L2 expression linked to t(5;14)(q35;q32), of HOX11 expression, and of SIL-TAL fusion in childhood T-cell malignancies: results of EORTC studies 58881 and 58951

AbstractIn a series of 153 children with T-cell malignancies enrolled in 2 consecutive European Organization for Research and Treatment of Cancer (EORTC) trials, we assessed the HOX11L2 expression and/or the presence of a t(5;14)(q35;q32). Additionally, in 138 of these patients, HOX11 expression and SIL-TAL rearrangement were also assessed. These alterations were mutually exclusive, and their frequency was 23% (n = 35), 7% (n = 10), and 12% (n = 17), respectively. HOX11L2/t(5;14) positivity was more frequent in acute lymphoblastic leukemia (ALL) with cortical T immunophenotype and in children aged between 6 and 9 years. In contrast with previously reported data, patients positive and negative for HOX11L2/t(5;14) were comparable with regard to clinical outcome as well as to the response to a 7-day prephase treatment or to residual disease at completion of induction therapy. The 3-year event-free survival (EFS) rate (± SE percentage) for patients positive and negative for HOX11L2/t(5;14) was 75.5% (± 8.1%) and 68.3% (± 5.0%), respectively; the hazard ratio was 0.84 (95% confidence interval, 0.40-1.80). Patients with HOX11-high expression and those with SIL-TAL fusion had low levels of residual disease at the end of induction and a favorable prognosis: the 3-year EFS rate was 83.3% (± 8.5%) and 75.3% (± 12.6%), respectively. The results obtained in HOX11L2/t(5;14) patients in this study do not confirm the unfavorable prognosis reported in previous studies.

Circulating plasma cells in multiple myeloma: characterization and correlation with disease stage.

The aim of this study was to develop a flow cytometric test to quantitate low levels of circulating myeloma plasma cells, and to determine the relationship of these cells with disease stage. Cells were characterized using five-parameter flow cytometric analysis with a panel of antibodies, and results were evaluated by comparison with fluorescent consensus-primer IgH-PCR. Bone marrow myeloma plasma cells, defined by high CD38 and Syndecan-1 expression, did not express CD10, 23, 30, 34 or 45RO, and demonstrated weak expression of CD37 and CD45. 65% of patients had CD19- 56+ plasma cells, 30% CD19- 56(low), and 5% CD19+ 56+, and these two antigens discriminated myeloma from normal plasma cells, which were all CD19+ 56(low). Peripheral blood myeloma plasma cells had the same composite phenotype, but expressed significantly lower levels of CD56 and Syndecan-1, and were detected in 75% (38/51) of patients at presentation, 92% (11/12) of patients in relapse, and 40% (4/10) of stem cell harvests. Circulating plasma cells were not detectable in patients in CR (n = 9) or normals (n = 10), at a sensitivity of up to 1 in 10,000 cells. There was good correlation between the flow cytometric test and IgH-PCR results: myeloma plasma cells were detectable by flow cytometry in all PCR positive samples, and samples with no detectable myeloma plasma cells were PCR negative. Absolute numbers decreased in patients responding to treatment, remained elevated in patients with refractory disease, and increased in patients undergoing relapse. We conclude that flow cytometry can provide an effective aternative to IgH-PCR that will allow quantitative assessment of low levels of residual disease.