Predicting Minimal Residual Disease in Multiple Myeloma: Allelic-Specific Oligonucleotide Real-Time Quantitative PCR or Multi Parametric Flow Cytometry

17552 Background: At present, the prognostic value of the amount of residual tumor cells in PB, BM or stem cell harvests and its changes over time is stil not clear. Also the advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by using flow cytometry. The aim of the present study was to evaluate a real time PCR test for the IgH gene using alellespecific molecular beacons as fluorescence probes to quantify residual disease and also correlate flow cytometric detection of plasma cells in MM patients during followup after treatment with high dose of chemotherapy or standart chemotherapy. Methods: After clinical diagnosis of 17 MM patients, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify its clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. We have also examined the co-expression of CD19, CD38, CD45, CD56, and CD138 molecules in cells of bone marrow aspirates in patients with multiple myeloma by flow cytometry. Results: The active disease had been accepted of whom plasma cell infiltration ratio was over 10% in bone marrow and also of whom labeled by CD38 and CD138 by FCM. The detection of the MRD was positive in 13 patients by RT-PCR, respectively. The infiltration ratio was correlated with CD138 expression (p = 0.009) and RT-PCR detection of plasma cells (p = 0.006) and also significant correlation had been found between RT-PCR detection and CD138 expression respectively. No any correlaton was found between other surface antigens (CD38, CD45, CD56). Conclusion: Our results indicated that real time PCR with specific molecular beacons provides a feasible, accurate and reproducible method for the determination of minimal residual disease in MM. By FCM only CD138 expression may have been used as disease marker in addition of the RT-PCR detection. No significant financial relationships to disclose.

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Prognostic Value of Minimal Residual Disease by Real-Time Quantitative PCR in AML with CBFB-MYH11 Rearrangement: The French Experience.

Blood ◽

10.1182/blood.v110.11.3496.3496 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3496-3496

Author(s):

Romain Guièze ◽

Aline Renneville ◽

Jean-Michel Cayuela ◽

Raouf Ben Abdelali ◽

Nicolas Boissel ◽

...

Keyword(s):

Real Time ◽

Minimal Residual Disease ◽

Quantitative Pcr ◽

Residual Disease ◽

Transcript Level ◽

Prognostic Impact ◽

Consolidation Therapy ◽

Consolidation Treatment ◽

Real Time Quantitative Pcr ◽

Minimal Residual

Abstract Background. Despite the favorable prognosis of AML patients with inv(16)/t(16;16) leading to CBFB-MYH11 rearrangement, relapses still occur in about 40% of the cases. With the possible exception of receptor tyrosine kinase mutations, no pretreatment-factor can strongly predict risk of relapse. Methods. To explore minimal residual disease (MRD) prognostic impact, we prospectively monitored CBFB-MYH11 rearrangement by real-time quantitative PCR (RQ-PCR) in patients with inv(16)/t(16;16) treated in French cooperative trials; most patients were treated in Acute Leukemia French Association (ALFA) trials and Leucémies Aiguës Myéloblastiques de l’Enfant (LAME) Cooperative Groups trials. RQ-PCR was performed in three molecular French labs (CHU of Lille, Necker Hospital and St-Louis Hospital) according to the EAC procedure and results are expressed as CBFB-MYH11/100 ABL copies(%). Results. 61 AML patients with inv(16)/t(16;16) who had reached CR with one of the anthracyclin-aracytin regimens were analyzed. Median age was 37.5 years (range 2.1–76.5) and median baseline WBC 46 G/L (range 1.8–246). With a median follow-up of 23.3 months, median overall DFS was 23 months and median OS not reached (93.5% of patients alive). No initial clinical or hematological characteristic including age, gender and initial WBC was predictive of relapse in adults, but children had poorer DFS than adults (17.6 months vs not reached, p=0.03). Regarding chromosomal abnormalities in addition to inv(16)/t(16;16), trisomy 22 was the most frequent but no significant prognostic impact was observed. Of the 61 patients, MRD monitoring could be performed sequentially in 45. Pretreatment CBFB-MYH11 transcript level had no impact on DFS. However, after induction therapy, transcript levels >0.5% were significant predictors of poorer DFS (median 16.4 months vs not reached if <0.5%, p=0.002). Likewise, after first consolidation therapy course, transcript level >0.001% was also predictive of poorer DFS (median 22 months when >0.001% vs not reached when <0.001%; p=0.03). 34 paired analyses of bone marrow (BM) and peripheral blood (PB) samples revealed only moderate correlation (R2=0.86); especially for low MRD levels, CBFB-MYH11 transcript expression being generally higher (up to 78-fold) in BM samples than in PB samples. Conclusion. MRD monitoring by RQ-PCR appears to be a major prognostic factor of DFS in AML with CBFB-MYH11 rearrangement and could be a powerful tool to early identify good and bad responders which could have an impact on therapeutic decisions for consolidation therapy. DFS according to MRD level after first consolidation treatment DFS according to MRD level after first consolidation treatment

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PHOX2B Is a Novel and Specific Marker for Minimal Residual Disease Testing in Neuroblastoma

Journal of Clinical Oncology ◽

10.1200/jco.2007.13.6531 ◽

2008 ◽

Vol 26 (33) ◽

pp. 5443-5449 ◽

Cited By ~ 51

Author(s):

Janine Stutterheim ◽

Annemieke Gerritsen ◽

Lily Zappeij-Kannegieter ◽

Ilona Kleijn ◽

Rob Dee ◽

...

Keyword(s):

Real Time ◽

Minimal Residual Disease ◽

Quantitative Pcr ◽

Peripheral Blood ◽

Residual Disease ◽

Specific Marker ◽

Real Time Quantitative Pcr ◽

Normal Tissues ◽

Specificity And Sensitivity ◽

Minimal Residual

Purpose Polymerase chain reaction (PCR)–based detection of minimal residual disease (MRD) in neuroblastoma can be used to monitor therapy response and to evaluate stem cell harvests. Commonly used PCR markers, tyrosine hydroxylase (TH) and GD2 synthase, have expression in normal tissues, thus limiting MRD detection. To identify a more specific MRD marker, we tested PHOX2B. Patients and Methods To determine PHOX2B, TH, and GD2 synthase expression in normal tissues, it was measured by real-time quantitative PCR in samples of normal bone marrow (BM; n = 51), peripheral blood (PB; n = 37), and peripheral-blood stem cells (PBSCs; n = 24). Then, 289 samples of 101 Dutch patients and 47 samples of 43 German patients were tested for PHOX2B and TH; these samples included 52 tumor, 214 BM, 32 BM, and 38 PBSC harvests. Of the 214 BM samples, 167 were compared with cytology, and 47 BM samples were compared with immunocytology (IC). Results In contrast to TH and GD2 synthase, PHOX2B was not expressed in any of the normal samples. In patient samples, PHOX2B was detected in 32% cytology-negative and in 14% IC-negative samples and in 94% of cytology-positive and in 90% of IC-positive BM samples. Overall, PHOX2B was positive in 43% compared with 31% for TH. In 24% of all samples, TH expression was inconclusive, which is similar to expression found in normal tissues. In 42% of these samples, PHOX2B expression was positive. Conclusion PHOX2B is superior to TH and GD2 synthase in specificity and sensitivity for MRD detection of neuroblastoma by using real-time quantitative PCR. We propose to include PHOX2B in additional prospective MRD studies in neuroblastoma alongside TH and other MRD markers.

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