Patient-specific Primers Research Articles

In-Utero Origin of Childhood Acute Leukaemias.

While there is enough convincing evidence in childhood ALL, the data on the pre-natal origin in childhood AML are less comprehensive. Our study aimed to screen Guthrie cards (neonatal blood spots) of childhood acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) patients for the presence of their respective leukaemic markers. For the analysis we used PML/RARa, AML1/ETO and CBFb/MYH11 fusion genes, translocations of MLL gene and internal tandem duplication of Flt3 gene (Flt3/ITD) in patients with AML and TEL/AML1 fusion gene and immunoglobulin (Ig) and/or T-cell receptor (TCR) gene rearrangements in patients with ALL. These molecular markers on the DNA level present suitable candidates for backtracking of leukaemic cells in newborn material, because they are clonotypic and altogether their incidence is ~40% and >90% in AML and ALL, respectively. We screened Guthrie cards from 13 AML patients (4x PML/RARa, 3x CBFb/MYH11, 2x AML1/ETO, 2xFlt3/ITD, 3x MLL rearrangement - MLL/AF6, MLL/AF9 and MLL/AF10) and from 15 ALL patients (13x Ig/TCR rearrangements, 3x TEL/AML1). One AML patient from our group had PML/RARa fusion gene together with Flt3/ITD, in 1 ALL patient we screened the Guthrie card for both Ig/TCR and TEL/AML1. We designed patient-specific PCR primers and we established nested PCR assay for each clonotypic sequence prior to the analysis of the corresponding Guthrie card. Assay specificity was determined using serial dilutions of patient DNA into the DNA of a healthy donor. The sensitivity of PCR was >=10(−4) in all patients. Therefore, this approach allowed us to detect the pre-leukaemic clone provided 1–10 positive cells were present on the Guthrie card. In three patients with ALL we reproducibly detected identical rearrangements both in the presentation sample and on the Guthrie card. The first patient harboured two independent rearrangements: TCR-delta Vd2/Dd3 and IgH VH3/JH5 and both were detectable on Guthrie card. In the second patient two PCR systems were optimised with adequate identical sensitivity: Igkappa Vk1/Kde and TCRgamma VgI/Jg1.3-2.3. However, only the latter detected pre-leukaemic cells on Guthrie card. The third patient had TEL/AML1 and we found this translocation on her Guthrie card. We did not find patient-specific molecular markers in any patient with AML. In the present study we confirmed the prenatal origin in 20 % of ALL patients. The negative results in the AML group can not disprove the theory that some childhood AML cases are also initiated in-utero. The negative results might be caused also by the fact that the age at presentation was higher in our AML group compared to ALL patients (median 7 and 3 years, respectively), the size of pre-leukaemic clone in AML might be below the sensitivity threshold of our approach, and Flt3/ITD could be of post-natal origin as recently suggested. However, our present data based on the largest cohort examined so far show that there is much less evidence for the pre-natal origin of childhood AML. Support: grants #7436 (Czech Ministry of Health) and #0021620813 (Czech Ministry of Education).

Molecular Quantitation of Minimal Residual Disease in Multiple Myeloma Patients Achieving Complete Remission after Treatment with Revlimid or Velcade.

In multiple myeloma (MM), new therapeutic strategies utilize the proteasome inhibitor bortezomib (Velcade) and the thalidomide immunomodulatory analog lenalidomide (Revlimid). While promising, MM invariably recurs, necessitating a better understanding of the malignant cells that persist during periods of complete remission (CR). In MM, minimal residual disease (MRD) is a valuable prognostic indicator for predicting time to relapse. There are no published studies to quantify the MRD or malignant cells that resist Velcade or Revlimid. In MM, the uniquely rearranged IgH VDJ gene provides a molecular signature. PCR strategies incorporating amplification of genomic DNA using patient specific primers provide a means to quantify MRD in MM patients. To date, the patient cohort tested included three MM patients achieving CR as part of a randomized trial for Revlimid plus dexamethasone, one MM patient with CR in response to dexamethasone (dex) alone, and two MM patients achieving CR after Velcade. All of the CR patients had clonotypic VDJ mRNA transcripts in BM by RT-PCR. Analysis of genomic DNA (gDNA) quantifies the number of malignant cells, as each cell has only one copy of the MM IgH VDJ. Three strategies were employed to quantify the extent of minimal disease during CR to these agents: semi-quantitative PCR analysis of purified gDNA from BM aspirates, quantitative realtime PCR using SYBR Green, and PCR analysis of cells captured from BM aspirate slides (laser pressure catapulting). 1) The semi-quantitative method measures the amount of gDNA template in the PCR reaction required to attain a positive signal, independent of cell type or morphology. 2) The quantitative PCR confirms method 1 by plotting rate of amplification of gDNA for a control sequence as compared to the clonotypic sequence, to calculate the frequency of all clonotypic MM cells in BM. 3) Laser pressure catapulting of BM cells from slide preparations provides a measure that most closely approximates the in vivo situation because it does not introduce potential artifacts arising from purification of cells and DNA that could confound interpretation of the results, and enables morphological identification of the cell types harboring clonotypic IgH VDJ genes. BM aspirate slides were viewed and numerous small patches of cells (8–15 cells) were captured into individual tubes for PCR analysis. Overall, we found that CR patients treated with Revlimid+ Dex have a 100 fold lower frequency of clonotypic MM cells (requiring 1.59e5 cells to detect a positive signal) than did the MM patient in CR from dex (detectable in 1.01e3 cells), indicating that Revlimid substantially reduced, but did not eradicate the malignant clone. Furthermore, for one patient, for whom sequential BM samples were available, the clonal frequency continued to decrease over time. Patients in CR after Velcade treatment have MM cells detectable in 1.32e4 cells. Both therapies exert more depletion as compared to dex alone. Larger patient cohorts are being analyzed to further quantify levels of MRD achieved in response to these agents.

Analysis of Validated Clonotypic Igh VDJ Sequences from a Large Cohort of MM Patients.

Studies in B cell malignancies such as chronic lymphocytic leukemia have revealed associations between immunoglobulin V gene characteristics and clinical outcomes. Few studies of this kind have been conducted in multiple myeloma (MM), a B lineage cancer characterized by malignant plasma cells that display somatically hypermutated VDJ, clonal homogeneity, and post switch (IgG or IgA) clinical isotypes. In this study we analyzed the clonotypic VDJ sequences for a large cohort of 115 MM patients to define associations with clinical parameters such as staging at diagnosis (e.g. clinical isotype, percent PC, M protein, beta-2-microglobulin) and overall survival. A key feature of this analysis is the rigorous validation of the experimentally derived clonotypic VDJ sequence by confirming its presence in the majority of single MM PC, to exclude any VDJ sequences arising from expanded but unrelated clones of normal PC that are also present in MM bone marrow (BM) samples. This process involves the amplification of patient specific clonotypic VDJ using sets of VH family and constant region primers, direct sequencing of VDJ fragments, and CDR2 / CDR3 patient specific primer design. The proposed VDJ is sequence is confirmed by one of several single cell assays, preferably RT-PCR on CD138+ sorted PC, but also including in-situ RT-PCR and limiting dilution analysis. The set of confirmed clonotypic VDJ sequences were analyzed for several parameters including: VH, DH, and JH gene usage, VH and CDR3 length, percent mutation in the CDR1-CDR3 region, and CDR3 amino acid sequence. We found that V gene usage amongst MM patients was not significantly different from expected values based on the frequency of functional germline alleles: VH3 (52.2%), VH4 (19.1%), VH1 (17.4%), VH2 (7.8%), VH5(3.5%). Frequently used VH alleles included VH3-30 (10.4%), VH1-69 (9.6%), VH3-23 (7.8%). Amongst VH5 alleles, VH5-51 was observed exclusively. VH4-34, which has been associated with autoimmune diseases and is not present in normal plasma cells was observed in a single case. DH and JH usage from most to least frequent was as follows: JH4, JH6, JH3, JH5, JH1, and JH2; DH3, DH2, DH1, DH6, DH5, and DH4. Sequence analysis of the CDR1-CDR3 region revealed 1.8–18.8% mutation with a mean of 9.4%. Using mutation frequency as a guideline, we identified three groups, high (12–18.8%), medium (6–11.9%) and low (1.8–5.9%) frequencies of mutation, and compared their clinical characteristics. In a similar fashion, CDR3 length, selected VH, DH, and JH alleles were also evaluated in the context of clinical parameters. In a preliminary analysis, there were no detectable correlations between any of these V gene characteristics and overall survival. Future studies with this database will include CDR3 sequence analysis to determine if subsets of recurrent amino acid patterns, suggestive of specific antigen involvement are detectable. Unlike other B lineage malignancies, such as B-CLL or Waldenstrom's macroglobulinemia, the V gene usage of MM appears to be a largely random representation of the normal VH gene repertoire, with no selection for particular VH gene families or individual VH genes, and a broad range of mutation frequencies. This type of analysis has meaning only if the IgH VDJ sequences analyzed are derived from the MM clone itself, making validation of experimentally derived clonotypic sequences a critical priority. Overall, this is the largest analysis to date of rigorously validated IgH VDJ gene sequences for MM.

Specific detection of Flt3 point mutations by highly sensitive real-time polymerase chain reaction in acute myeloid leukemia

Among activating class III receptor tyrosine kinase (Flt3) mutations, internal tandem duplications of Flt3 (Flt3-ITD) are detected in about 25% of patients with acute myeloid leukemia (AML). In contrast, mutations within the tyrosine kinase domain of Flt3 (Flt3-TKD mutations) are less frequent (approximately 7%), and there are only limited data on the frequency of recently demonstrated activating Flt3 point mutation at codon 592 (Flt3-V592A mutation). We evaluated a new approach for rapid screening of Flt3-TKD and Flt3-V592A mutations using the fluorescence resonance energy transfer (FRET) principle in a group of 122 patients. Based on individual Flt3-TKD mutations, we designed patient-specific primers to perform a highly sensitive polymerase chain reaction (PCR) assay for rapid detection of minimal residual disease (MRD). We also used a model system with MonoMac-6 cells carrying the Flt3-V592A mutation to establish a mutation-specific real-time PCR approach also for this molecular aberration. We identified 9 cases (8%) of Flt3-TKD mutations (5 cases of mutation D835Y, 3 cases of mutation D835H, and 1 case of mutation Del836), and no cases of Flt3-V592A mutation. Screening for Flt3-TKD mutations with fluorescent probes is equivalent to conventional screening using standard PCR followed by EcoRV restriction. We present a real-time PCR protocol that can be used for MRD analyses based on individual Flt3-TKD mutations. Examples of MRD analyses are presented for all 3 subtypes of Flt3-TKD mutation identified in this study. In summary, we demonstrate new methodological approaches for rapid screening of Flt3 point mutations and for detection of MRD based on patient-specific Flt3-TKD mutations.

Detection of Central Nervous System Leukemia in Children with Acute Lymphoblastic Leukemia by Real-Time Polymerase Chain Reaction

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification.

Touch-Down Reverse Transcriptase-PCR Detection of IgVH Rearrangement and Sybr-Green-Based Real-Time RT-PCR Quantitation of Minimal Residual Disease in Patients with Chronic Lymphocytic Leukemia

Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific, sensitive, and economic alternative to the current quantitative methods.

Assessing Immunoglobulin Heavy Chain Rearrangements in Pediatric CD20-Positive and CD20-Negative Classic Hodgkin's Diseas

Approximately 15% of all cases of childhood classical Hodgkin's disease (HD) express CD20, a B-cell marker associated with immunoglobulin heavy chain rearrangements. Immunoglobulin heavy chain rearrangements in Reed-Sternberg cells could be used to assess minimal residual disease (MRD), as was shown with immunoglobulin heavy chain patient-specific primers (PSPs) in non- Hodgkin's lymphoma. The aim of this study was to analyze pediatric HD for future design of immunoglobulin heavy chain PSP for MRD detection. DNA was extracted from paraffin-embedded tissue from unstained slides of 8 pediatric CD20+ nodular sclerosis HD cases and 10 CD20− nodular sclerosis HD cases. Immunoglobulin heavy chain polymerase chain reaction and sequencing were performed on 16 of 18 cases, which had adequate DNA for further analysis. Sequence analysis from 3 cases (19% of HD cases) demonstrated unique V(D)J regions, which could potentially be used to design PSP. Unique PSPs could be used to assess MRD in advanced-stage HD specimens. Future studies should focus on improved detection and analysis of more cases to identify appropriate specimens in assessing clinical implications of MRD detection.

Eradication of Minimal Residual Disease during Treatment of Mantle Cell Lymphoma: CALGB 59909.

Recent studies suggest that detection of subclinical, or minimal residual disease (MRD) in apheresis products used for autografting correlates with poor disease-free survival following ASCT for mantle cell lymphoma (MCL). To validate this observation and to gain insights into the kinetics of MRD during treatment, we are performing a prospective analysis of MRD using quantitative real-time PCR (Q-PCR) in patients (pts) undergoing treatment for MCL on a CALGB study (59909). Q-PCR of sequential paired bone marrow (BM) and blood (B) samples and of apheresis products was performed using either a patient-specific immunoglobulin heavy chain (IgH) or BCL-1 gene rearrangement. All samples were analyzed in triplicate using LightCycler technology and reported as a normalized ratio of IgH or BCL-1 copy number to GAPDH copy number. The sensitivity of the assay ranged from 1 X 104–1 X 105. To date, a clonal IgH or BCL-1 gene rearrangement was detected in 36 of 41 (88%) pts entered on study. Patient-specific primers and consensus probes were used for Q-PCR monitoring of MRD following two courses of intensive induction therapy, during stem cell mobilization, and 3 and 12 months after ASCT and post-transplant immunotherapy with Rituximab (R). 27 pts have completed all protocol treatment with a median follow-up of 7 months (range: 0–28). 26 pts were evaluated for MRD following two courses of induction therapy with cyclophosphamide, methotrexate, doxorubicin, vincristine, prednisone, and R. 10 of 26 became MRD negative (−) following induction while 16 remained MRD positive (+). Following mobilization with high-dose cytarabine, etoposide and R, apheresis products from 9 of 10 MRD- pts were evaluated and all products were MRD- (1 pt not evaluable). Of the 16 pts who were MRD+ prior to mobilization therapy, MRD- apheresis products were collected in 8; 5 had MRD+ stem cell collections, and 3 were not evaluable. In total, apheresis products were evaluable in 22 of 26 pts; 17 (77%) had MRD- stem cells collected prior to ASCT. None of these MRD- pts has relapsed to date although 2 pts with MRD- products became weakly MRD+ 12 months following ASCT and R. Of the 5 pts with MRD+ apheresis collections, 4 have remained persistently MRD+ following ASCT and R; 1 has relapsed 19 months after completion of all treatment. Rising levels of MRD in BM and B samples were noted in this patient 3 and 12 months post-ASCT. Statistical comparison of MRD values in paired BM and B samples prior to, and post-ASCT demonstrated good agreement with an intraclass correlation coefficient of.814 and.777, respectively. In conclusion, our results demonstrate that prospective MRD monitoring using Q-PCR provides important insights into the kinetics of response during treatment of MCL. MRD- apheresis products were collected in the majority of pts following intensive induction and mobilization chemo-immunotherapy with R on CALGB 59909. To date, no pts with MRD- apheresis products have relapsed following ASCT. In contrast, it appears that pts with MRD+ apheresis products remain MRD+ following ASCT and R and may be more likely to relapse. Longer clinical follow-up is needed to clarify the significance of the persistence of MRD in apheresis products and following ASCT for MCL.

VDJ-Switch Region Analysis in Multiple Myeloma Patients Reveals Homogeneity and Long-Term Stability of Switch Junctions, and Ongoing Mutation Upstream of Switch Mu.

During class switch recombination (CSR) B cells change their immunoglobulin heavy chain (IgH) constant region genes by a deletional recombination mechanism involving well characterized switch (S) regions several Kb downstream of IgH variable region (VDJ) sequences. Unlike VDJ recombination, the junction formed by CSR between pre-switch S-mu (IgM) and post-switch S-gamma (IgG) is imprecise, and the key factors mediating this process are not fully understood. In multiple myeloma, a bone marrow cancer characterized by post-switch plasma cells bearing a unique clonotypic VDJ, switch regions have been shown to mediate translocations in a subset of MM patients, suggesting a role in transformation. In this study we cloned and sequenced the VDJ-hybrid immunoglobulin switch (VDJ-S) regions of post-switch multiple myeloma (MM) cells at diagnosis and a second time point to determine if the switch junction was normal, homogeneous within a time point, and stable over the course of malignancy.The VDJ-S of five stage III IgG MM patients were examined in this study. Each patient has a specific clonotypic VDJ region and downstream hybrid switch junction (S-mu/S-gamma) arising after class switch recombination (CSR) from IgM to IgG. For each patient two samples were tested: a diagnosis sample and a second sample taken between 1.6 and 4.2 years later. The 5–7 Kb VDJ-S was amplified by long distance PCR using bulk DNA samples from bone marrow and blood as template, CDR2 specific sense primers and S-gamma specific antisense primers. In each case a single DNA product was amplified, cloned, and sequenced with primers covering the VDJ-S region. Patient specific CDR2 primers were used to confirm that the fragments were clonotypic. Unique switch junctions for patients 1–5 were detected at positions 125, 251, 435, 590, and 750 of S-mu, respectively. This agrees with previous studies mapping MM switch junctions to the 5′ portion of S-mu. The sequence and mutation profile of the switch junctions, with more frequent mutations in the S-mu region upstream of the hybrid junction, suggests that these junctions arise from normal CSR. A single hybrid junction was detected in all patients, corresponding to the single VDJ-Switch fragment amplified by long-distance PCR. The switch junction sequences also remained constant between time point samples, suggesting switch junction stability. Interestingly, new mutations were observed in the 3.5 Kb region upstream of the switch region, in the second time point samples of 2/5 patients. Most notably, 19 new mutations were detected in one patient, including 3 bp and 64 bp deletions 135 and 223 bp downstream of the intronic enhancer, respectively. The significance of these mutations is unclear, considering previous studies showing mutations, insertions, and deletions in this region in normal CSR. Nevertheless, it seems this process continues in clonotypic cells of some patients throughout malignancy. This ongoing mutation may lead to ‘remodeling' of the VDJ-S region, which is significant in the context of immunoglobulin translocations where proximity with immunoglobulin enhancers may confer a selective advantage.

Molecular Analysis of T-Cell Receptor Gene Rearrangements in Children with T-Cell Acute Lymphoblast Leukemia on DFCI ALL Consortium Protocol 95-01.

During normal T-cell differentiation, T-cell receptor (TCR) genes undergo recombination in an ordered way. TCRd genes rearrange first followed by TCRg genes, which may result in TCRgd+ T lymphocytes. TCRab T lymphocytes develop via a separate differentiation lineage, with TCRb preceding TCRa gene rearrangements. Unlike in normal T-cells, TCRd, TCRg and TCRb genes rearrangements can be detected in T-cell acute lymphoblastic leukemia (T-ALL) and these rearranged gene sequences can be used as molecular targets to detect minimal residual disease (MRD). Knowledge of the association between rearrangement patterns and disease pathogenesis of pediatric T-ALL is relatively limited. We analyzed 45 sequences of the TCR gene rearrangements (17 TCRd and 28 TCRg) obtained at presentation from 37 children with T-lineage ALL and these sequences were used as molecular targets to perform real-time PCR (RQ-PCR). Seven children had both TCRd and TCRg gene rearrangements. Sequence analysis revealed a higher frequency of Vd1-Jd1 rearrangements (88%) compared to Vd2-Jd1 (6%) and Vd2-Dd3 (6%). Of the 28 TCRg sequences, Vg2 gene segment was identified in 7 sequences, followed by Vg4 (5), Vg3 (4), Vg8 (4), Vg1 (2), Vg7 (2), Vg10 (2), Vg5 (1), and Vg9 (1). In keeping with previous findings the median number of nucleotides in the junctional regions was higher in TCRd (28bp) than in TCRg sequences (7.5bp, p<0.0001) suggesting random nucleotide insertions and that Dd3 genes contribute a longer junctional region in TCRd gene rearrangement. Detailed analysis of junctional region of TCRg sequences revealed that this region is larger in patients who also have a TCRd rearrangement (n=7, median 11bp) compared to those patients for whom only a TCRg sequences was found (n=21, median 5bp, p=0.02), perhaps explained by a recent finding that lack of expression of TdT was associated with the absence or shorter N sequences which contributed to shorter CDR3 regions in immature lymphocytes. Sequence specific junctional region primers were designed and amplified with J gene consensus primers and RQ-PCR performed using J regionconsensus probes. 13 of 14 TCRd targets (93%) reached a high reproducible sensitivity of 10−5 and 10−6, whereas 10 of 18 TCRg targets (56%) showed such sensitivity (p=0.044, Fisher's exact test). The junctional features of the TCR rearrangements for cases with only TCRg sequences and short N-regions were identified suggest that leukemic transformation is initiated at an early stage of T-cell ontology . The high junctional region diversity in TCRd rearrangements provides a better target to design patient specific primers (about 20bp) providing a higher sensitivity to detect MRD. RQ-PCR analysis was performed on 31 patients in whom BM (31) or PB samples (26) at day 30 were available for analysis. At this time point18 patients (58%) had no PCR detectable disease, six had MRD levels between 10−6 and 10−3 and 7 has MRD levels > 10−3. However, there was no difference in EFS in these three groups, and only one patient relapsed. We conclude that TCRg/d rearrangements provide suitable markers for RQ-PCR MRD analysis, but that in this treatment protocol high levels of MRD at the end of induction are not necessarily predictive of relapse in T-lineage ALL as they are in B-lineage ALL

Applicability of FLT3-Length Mutations in Exon 14 as a Follow up Marker in AML: An Experience of 148 Cases and 517 Follow up Samples Using Conventional and Real Time PCR.

Quantification of minimal residual disease (MRD) is becoming increasingly important to guide therapy in patients with acute myeloid leukemia (AML). As only 25% of all AML have fusion genes that have been proven useful as MRD-markers, new targets are urgently needed to make high sensitive PCR studies possible in more AML cases. As length mutations in exon 14 of the FLT3 gene (FLT3-LM) are the most frequent genetic aberrations detected in AML and are predominantly found in AML with normal karyotype this is a highly promissing marker to assess prognosis predicted by MRD in this prognostically intermediate group. Recently, FLT3-LM as a follow up marker was discussed controversly because of possible instabillity. To further evaluate this issue we analyzed 62 paired samples at diagnosis and at relapse that were positive at both or only one of these two time points. 53 were positive at both time points. In these cases the mutation (mt) to wildtype (wt) ratio was increasing in 25 and decreasing in 6 cases. Five cases were mt- at diagnosis and gained the mutation at relapse. Four cases (6.5%) lost the mt at relapse. However, these cases also had a change in cytogenetics (n=2), or immunophenotype (n=2), and FAB subtype (n=4). Thus, these cases may be regarded as different leukemias. In addition, we analyzed 517 follow up samples (day 16 – day 1548) of 148 different patients with FLT3-LM at diagnosis by conventional RT-PCR (sensitivity: up to 10−3). First, all follow up samples were rated positive (n=281) or negative (n=236) for the FLT3-LM as determined at any time point. The negative group had a longer OS (median not reached vs 902 days; p=0.0008) and EFS (413 vs. 279 days, p=0.0004). In a next step, five different follow up checkpoints were defined: 1 (up to day 21 after start of therapy), 2 (day 22–60), 3 (day 61–120), 4 (day 121–365), and 5 (after day 365). Survival was best predicted by interval 4 for OS (median not reached vs. 580 days, p=0.0031) and EFS: (406 vs. 221 days; p=0.0001) in 79 negative and 57 positive samples analyzed at this checkpoint. Also prognostically relevant for OS were checkpoints 2 (31 negatives, 16 positivs, 614 vs. 325 days, p=0.0213), and checkpoint 3 (56 negatives and 23 positives, 614 vs. 325 days, p=0.0215). Thus, sole FLT3-LM status during follow up is a highly predictive parameter for prognosis. In 13 cases that were positive for a second marker 72 samples were analyzed by quantitative real time PCR in parallel (PML-RARA: n=5, MLL-PTD: n=5, DEK-CAN: n=2, MLL-AF6: n=1). A high correlation of the results was found (p=0.01). In addition, 10 cases were 8–15 available follow up samples including at least one relapse were selected for real time PCR. In these cases patient specific primers spanning parts of the individual mutation were designed. With these PCRs a high sensitivity of 10e5–10e7 was riched. The data obtained clearly indicate the relapses 3–4 months before clinical relapse. In conclusion, these data show that 1) FLT3-LM is a very powerful marker for the analysis of therapy response and early detection of relapse. 2) Instability is a minor problem especially because the instabilily in most cases is in the direction of accumulation of the mutation.

Detection of noroviruses in raspberries associated with a gastroenteritis outbreak

Following an acute foodborne gastroenteritis outbreak in southern Sweden, stool specimens from five of nine ill patients were found positive for norovirus using reverse transcriptase polymerase chain reaction. Epidemiological data pointed to raspberry cakes as the source of the outbreak. Using a combination of generic and patient-specific primers and novel food analysis methodology (with extraction efficiency control and inhibitor removal), norovirus strains from two different genogroups were directly identified in the contaminated raspberries.

AMPLIFICA TION OF HVR1 REGION OF HEPA TITIS C VIRUS WITH PA TIENT SPECIFIC NESTED PCR PRIMERS

The understanding of the function of the hyper variable region (HVR) at the NH2-terminus of the E2protein remains very limited. Comparison of published sequences of HCV has led to the identification of anumber of distinct virus types that may differ from each other by as much as 33% over the whole viralgenome allowing the virus to escape cellular and humoral immunity. This phenomenon is related to thecapacity of HCV to escape immune recognition, by frequent mutations HVR1 region. Several investigationshave observed that the antibodies present in a proportion of infected patients had significant cross reactivitywith apparently unrelated HVR1 sequences presented as synthetic peptides.To study the HVR1 cross reactivity and evolution in individuals with varying liver disease severity, nestedPCR was performed to obtain patient specific 150 bp long HVR1 genome. For this, previously amplifiedfirst round single molecule amplimers were reamplified using new patient specific sense and antisenseprimers. The patient specific sense primers did not work in the second round amplification. So, the targetDNA was amplified by three rounds of nested PCR, using patient specific primers on the third round.Key Words: HCV, patient specific HVR1, nested PCR

Assessment of minimal residual disease in childhood non-hodgkin lymphoma by polymerase chain reaction using patient-specific primers.

A feasibility study was undertaken to identify patient-specific primers (PSPs) from childhood non-Hodgkin lymphoma (NHL) specimens to detect minimal residual disease (MRD). Eleven tumor specimens were amplified using immunoglobulin heavy chain and T-cell receptor primers to identify PSPs, which were then used to evaluate staging/follow-up specimens. Disease was detected in 19 of 21 staging and 16 of 17 follow-up specimens. Among seven patients in remission by 1 month, PSPs identified MRD in follow-up specimens. This study demonstrated the feasibility of PSPs to identify disease in staging and follow-up specimens, which could be used to develop strategies for MRD analysis in a larger setting.

High frequency of leukemic clones in newborn screening blood samples of children with B-precursor acute lymphoblastic leukemia

AbstractThe detection of leukemia cells on newborn genetic screening cards (“Guthrie cards”) of a small group of patients and several sets of identical twins developing acute lymphoblastic leukemia (ALL) with identical phenotypic and chromosomal markers has provided evidence that childhood ALL cases may arise in utero. We conducted a retrospective study of a randomly selected group of childhood B-precursor ALL patients to determine the frequency of the presence of “leukemic” clones prenatally in ALL cases by testing newborn screening cards. The 17 ALL patients analyzed had a median age of 46 months (range, 18 months to 13 years) and had median presenting white blood cell (WBC) counts of 10 950/µL (range, 2900-70 300/µL) at diagnosis. A clonal rearrangement of the immunoglobulin heavy chain (IgH) gene was identified in diagnostic lymphoblasts and sequenced and patient-specific primers were used to amplify DNA from blood samples on the patient's newborn screening cards. Twelve of the 17 (71%) analyzed newborn cards had detectable IgH rearrangements amplified by seminested polymerase chain reaction. DNA sequencing confirmed that the IgH rearrangements detected matched the IgH sequences identified from diagnostic leukemia cells, indicating the presence of a “leukemic” clone at birth. There were no differences in age or presenting WBC counts between the cases with or without positive newborn screening cards. All 6 patients with hyperdiploid ALL had detectable “leukemic” clones on their cards. The results of our study support the notion that a high proportion of childhood B-precursor ALL cases arise in utero, although postnatal events are also important factors in leukemogenesis.

Molecular emergence of acute myeloid leukemia during treatment for acute lymphoblastic leukemia

Therapy-related acute myeloid leukemias (t-AML) with translocations of the MLL gene are associated with the use of topoisomerase II inhibitors. We established the emergence of the malignant clone in a child who developed t-AML with a t(11;19) (q23;p13.3) during treatment for acute lymphoblastic leukemia (ALL). The MLL-ENL and the reciprocal ENL-MLL genomic fusions and their chimeric transcripts were characterized from samples collected at the time of t-AML diagnosis. We used PCR with patient-specific genomic primers to establish the emergence of the MLL-ENL fusion in serially obtained DNA samples. The MLL-ENL fusion was not detectable in bone marrow at the time of ALL diagnosis or after 2 months of chemotherapy (frequency <8.3 x 10(-7) cells(-1)). The genomic fusion was first detected in bone marrow after 6 months of treatment at a frequency of one in 4,000 mononuclear bone marrow cells; the frequency was one in 70 cells after 20 months of therapy. At the first detection of MLL-ENL, the only topoisomerase II inhibitors the patient had received were one dose of daunorubicin and two doses of etoposide. The MLL-ENL fusion was not detectable in blood at the time of ALL diagnosis or after 0.7, 2, 8, 10, and 12 months of therapy but was detectable in blood at 16 months (one in 2.3 x 10(4) cells). Recombinogenic Alu sequences bracketed the breakpoints in both fusions. These data indicate that the malignant clone was not present before therapy, arose early during chemotherapy, and was able to proliferate even during exposure to antileukemic therapy.

Molecular monitoring of minimal residual disease in patients in long-term complete remission after allogeneic stem cell transplantation for multiple myeloma

In the present study, we used a polymerase chain reaction-based (PCR-based) strategy to retrospectively analyze the presence of residual myeloma cells in serial posttransplant bone marrow samples obtained from 13 patients in remission after allogeneic hemopoietic stem cell transplantation (allo SCT). For this purpose, patient-specific primers were generated from complementarity determining regions 2 and 3 of the rearranged IgH gene. The level of sensitivity of the PCR-based assay ranged from 1 in 10(5) to 1 in 10(6) normal marrow cells. Following transplantation, 9 of 12 patients who attained stringently defined complete remission (CR) remained persistently PCR(-) for a median of 36 months, and 4 of the patients remained PCR(-) up to the latest analysis, which was performed at 48, 72, 72, and 120 months, respectively, after allo SCT. None of the patients in the PCR(-) subgroup experienced a disease relapse, and only 1 of 4 PCR(+) patients experienced a relapse. It is concluded that allo SCT has the potential ability to induce sustained serological and molecular CR in selected patients with multiple myeloma.

Molecular monitoring of minimal residual disease in patients in long-term complete remission after allogeneic stem cell transplantation for multiple myeloma

AbstractIn the present study, we used a polymerase chain reaction–based (PCR-based) strategy to retrospectively analyze the presence of residual myeloma cells in serial posttransplant bone marrow samples obtained from 13 patients in remission after allogeneic hemopoietic stem cell transplantation (allo SCT). For this purpose, patient-specific primers were generated from complementarity determining regions 2 and 3 of the rearranged IgH gene. The level of sensitivity of the PCR-based assay ranged from 1 in 105 to 1 in 106 normal marrow cells. Following transplantation, 9 of 12 patients who attained stringently defined complete remission (CR) remained persistently PCR− for a median of 36 months, and 4 of the patients remained PCR− up to the latest analysis, which was performed at 48, 72, 72, and 120 months, respectively, after allo SCT. None of the patients in the PCR− subgroup experienced a disease relapse, and only 1 of 4 PCR+ patients experienced a relapse. It is concluded that allo SCT has the potential ability to induce sustained serological and molecular CR in selected patients with multiple myeloma.

Minimal residual disease is common after allogeneic stem cell transplantation in patients with B cell chronic lymphocytic leukemia and may be controlled by graft-versus-host disease.

Following allogeneic stem cell transplantation (SCT), we studied the presence of donor and recipient derived cells within the CD19+ B cell fraction, in patients with B cell chronic lymphocytic leukemia (CLL). The chimeric status of the six patients studied was further investigated with minimal residual disease (MRD) detection, by sequencing and using patient-specific primers derived from junctional regions of clonally rearranged immunoglobulin heavy-chain (IgH) receptor genes. To date, five of six patients are alive with a median follow-up time of 24 months (range 15-60) post-SCT. All patients experienced acute and chronic graft-versus-host disease and responded clinically to SCT. All patients were MRD positive after SCT, which correlated to mixed chimerism within the CD19+ cell fraction in all samples except one (25/26). High levels of tumor necrosis factor-alpha (TNF-alpha) and soluble interleukin-2 receptor (sIL-2R) indicated advanced disease, and patients with increased levels pre- and post-SCT were also those with the most long-lasting PCR-detectable MRD post-SCT. Hence, a high tumor burden pre-SCT may reflect the long duration of detectable MRD in patients with B-CLL after SCT. A durable anti-leukemic effect was probably important in these patients.

Kaposi's sarcoma-associated herpesvirus gene sequences are detectable at low copy number in primary amyloidosis

Primary amyloidosis (AL), like multiple myeloma (MM), results from a clonal proliferation of plasma cells. Recent detection of Kaposi's sarcoma-associated herpesvirus (KSHV) gene sequences in MM patients, although controversial, suggested that KSHV may also be present in AL. In the present study, we assayed for KSHV gene sequences in patients with primary AL independently in 2 laboratories. Nested polymerase chain reaction (PCR) was performed on DNA isolated from 21 bone marrow (BM) core biopsy samples to amplify orf26 andorf72, 2 regions of the KSHV genome. Eighteen of 21 (86%) BMcore biopsy samples were KSHV PCR positive. BM aspirates from 16 of these 21 AL patients were cultured for 4-6 weeks to generate long term bone marrow stromal cells (LT-BMSCs), and 13 of 16 (81 %) LT-BMSCs were also KSHV PCR positive. Results in all but 1 sample were consistent in the 2 laboratories. Sequencing of the PCR products in the 2 laboratories confirmed 94-98% and 95-98% homology to the published orf 26 and orf 72 KSHV gene sequences respectively, with inter-patient base pair differences. Despite the presence of KSHV gene sequences, only 4/18 (22%) KSHV PCR positive patients demonstrated KSHV lytic antibodies by immunoblot assay. A sensitivity assay performed on the BCBL-1 cell line confirmed the presence of KSHV at a very low copy number in AL. PCR using patient specific light chain gene primers also amplified DNA isolated from 2 AL BM core biopsies and 3 AL LT-BMSCs which were KSHV PCR positive, suggesting the presence of clonotypic cells. Our results therefore demonstrate KSHV gene sequences albeit at a very low copy number in the majority ofBM core biopsies and LT-BMSCs from AL patients, and serological responses in only a minority of cases. Ongoing studies to identify viral transcripts and gene products will determine the biological relevance of KSHV in AL disease pathogenesis.