Posttherapy Surveillance of B-Cell Precursor A c u t e Lymphoblastic Leukemia Value of Polymerase Chain Reaction and Limitations of Flow Cytometry

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1 Hematopatholocjy / PCR SURVEILLANCE OF TREATED B-CELL PRECURSOR ALL Posttherapy Surveillance of B-Cell Precursor A c u t e Lymphoblastic Leukemia Value of Polymerase Chain Reaction and Limitations of Flow Cytometry Key Words: B-cell precursor acute lymphoblastic leukemia; Flow cytometry; Hematogones; Minimal residual disease; Polymerase chain reaction Abstract Flow cytometric immunophenotypic analysis is critical in diagnosis and classification of acute leukemia and has been used after therapy to monitorfor minimal residual disease. However, the presence of normal B-cell precursors, hematogones, particularly in the context of treated pediatric B-cell precursor acute lymphoblastic leukemia (BP-ALL), may confound such evaluation. In this study, the value of more specific genotypic markers (polymerase chain reaction evaluation of antigen receptor genes) was assessed to resolve this issue. Flow cytometric analysis of enriched mononuclear cells revealed l%to 0% precursor B cells (PBCs), based on expression ofl ormorepanbb cell antigens in addition to CD, CD, and terminal deoxynucleotidyl transferase in all patients studied. Inasmuch as this mimicked the immunophenotype of the original leukemic clone, PBCs, in isolation, were considered suspicious for minimal residual disease. However, of the posttherapy specimens (79%) revealed no monoclonally rearranged antigen receptor genes, and 7 of these patients had trackable genotypic markers at presentation. Accordingly, by PCR these 7 patients had complete molecular remission, supported by clinicalfollow up of 6 to 7 months. Among the remaining patients with PCRnegative disease, continue in remission, confirming the interpretation of false-positive flow cytometric analysis. In conclusion, flow cytometric monitoring of posttherapy bone marrow specimens from patients with BP-ALL may be misleading, if considered in isolation, in falsely suggesting the presence of minimal residual disease. Rather, PCR for antigen receptor gene rearrangements is a valuable and specific tool, helpful in differentiating hematogones from minimal residual disease in patients with treated BP-ALL whose bone marrow harbors increased PBCs. The most common form of acute lymphoblastic leukemia (ALL) is the B-cell precursor type (BP-ALL). In most cases, morphologic remission can be induced with chemotherapy, but relapse is inevitable in many patients, reflecting inability to document the absence of residual leukemia by morphologic features alone. Although current technology is redefining "remission," the presence of leukemic blast cells at a level typically undetectable with conventional morphologic evaluation is referred to as minimal residual disease. Flow cytometric immunophenotypic analysis (FCM) is essential in the diagnosis of ALL, and has been used to monitor minimal residual disease after induction therapy.' However, the presence of normal B-cell precursors, or hematogones, in bone marrow from patients with treated BP-ALL may confound evaluation of minimal residual disease. Hematogones are benign precursor B-lymphoid cells (PBCs) normally present in bone marrow, particularly in children. Morphologically, hematogones are small to medium-sized lymphoid cells with scant, agranular cytoplasm and nuclei showing homogeneous chromatin condensation, with absent to inconspicuous nucleoli. - Immunophenotypically, hematogones are characterized by an immature B-lineage profile, in that they express the pan-b cell antigens such as CD 9 and CD0, as well as the precursor antigens CD and CD, and the nuclear enzyme terminal deoxynucleotidyl transferase (TdT).5- The population of bone marrow hematogones is reported to be substantially increased in a variety of nonneoplastic and neoplastic hematologic disorders..l~':, Thus hematogones share morphologic and immunophenotypic similarities with leukemic blast cells, making the distinction between them extremely difficult. If considered in isolation, FCM can be misleading, and can be interpreted as indicating minimal Am J Clin Pathol 999; Bhaskar V.S. Kallakury, MD, Dan-Paul Hartmannn PhD, Jeffrey Cossman, MD, Joseph E. Gootenberg, MD, and Adam magg, MM

2 Kallakury et al / PCR SURVEILLANCE OF TREATED B-CELL PRECURSOR ALL residual disease. This is particularly important because h e m a t o g o n e s have been reported to be substantially increased in bone marrow from pediatric patients with ALL in continued morphologic remission, or subsequent to bone marrow transplantation. "6 In patients undergoing chemotherapy for ALL, hematogones have even been mistakenly reported as residual leukemic blast cells. 5 Materials and Methods Patients and Clinical Specimens After obtaining informed consent from either patients or legal gaurdians, bone marrow specimens from patients with BP-ALL diagnosed at Georgetown University Medical Center between January 995 and February 998 were studied after the following selection criteria were met: I A prior diagnosis of BP-ALL had been established. I All patients were being monitored after initiation of chemotherapy, and were in various phases of treatment (ie, postinduction, consolidation, maintenence, posttherapy). I Bone marrow aspirates or biopsy samples from all patients demonstrated morphologic remission with less than 5% blast cells. I Flow cytometry revealed at least % PBCs in posttherapy marrow specimens, raising the consideration of hematogones vs leukemic blast cells (minimal residual disease). A conesponding Wright-Giemsa-stained cytocentrifuge preparation from the enriched mononuclear cells of each sample analyzed at flow cytometry was examined to confirm morphologic remission. Relevant clinical and follow-up information was obtained from the patients' medical records. Flow Cytometric Analysis Density gradient-enriched bone marrow mononuclear cells were subjected to single-color and dual-color FCM with standard procedures. In brief, 0 u L of the cell suspension was incubated at C for 0 minutes with ul each of fluorescein isothiocyanate (FITC) and phycoerythrin-labeled monoclonal antibodies and heat-inactivated 760 Am J Clin Pathol 999;: Polymerase Chain Reaction D was extracted from the enriched bone marrow mononuclear cells with standard procedures and subjected to PCR analysis for both immunoglobulin heavy chain (IgH) gene and T-cell receptor y chain (TCR-y) gene rearrangements according to published p r o t o c o l s. 8 ' 9 For IgH rearrangement, a single pair of V H and JH consensus primers (5'-CTG-TCG-ACA-CGG-CCG-TGT-ATT-ACT-G-' and 5'-AAC-TGC-AGA-GGA-GAC-GGT-GAC-C-,, respectively) was used. Reactions were carried out in a total volume of 0 ul containing final concentrations of the following: approximately ug template D, 50 pmol of each primer, 00 um of deoxyribonucleoside triphosphate (dntp), mmol/l TRIS hydrochloride (ph 8.), 50 mmol/l potassium chloride,.5 mmol/l magnesium chloride, 0.00% gelatin, and.5 urn Taq D polymerase (Boehringer Mannheim, Indianapolis, IN). After 5 minutes of initial denaturation at 9 C, 0 cycles of PCR were performed (9 C for 0 min, 60 C for 0 min, and 7 C for 0 min), concluding with final elongation (7 C for 7 h). TCR-y PCR analysis was performed with a single pair of TV and TJ consensus primers (5'-AGG-GTT-GTGT T G - G A A - T C A - G G - ' and 5'-CGT-CGA-CAA-CAAGTC-TTG-TTC-CAC-', respectively) according to a previously described protocol but with certain modifications. 9 In brief, approximately ug of template D was amplified in a 0-uL reaction mixture containing final concentrations of mmol/l TRIS HC, 50 mmol/l KC, mmol/l MgCL, 500 Limol/L dntp, umol/l each of primers, and um Taq polymerase (Boehringer Mannheim). D amplification was performed for 0 cycles (9 C for 0 min, 55 C for 5 min, and 7 C for 5 min). D from known positive The purpose of this study was to determine the specificity of immunoglobulin and T-cell receptor gene rearrangement status with polymerase chain reaction (PCR) compared with FCM in characterizing PBCs in posttherapy bone marrow specimens from patients with BP-ALL in morphologic remission, and thus to differentiate hematogones from minimal residual disease. rabbit s e r u m. Cells were w a s h e d, r e s u s p e n d e d, and analyzed with an automated flow cytometer (FACScan; Becton Dickinson, Mountain View, CA) equipped with LYSIS I software (Becton Dickinson). Initial gating was performed on small to m e d i u m - s i z e d cells with low complexity and subsequently analyzed with the appropriate fluorescent monoclonal antibodies. The antibodies used included CD, CD, CD, CD5, CD7, CD8, CD, CD9, C D 0, C D, C D, C D, H L A - D R, and K and X immunoglobulin light chains. Staining for the nuclear enzyme TdT was performed using an FITC-labeled antibody (Supertechs, Rockville, MD) after cell fixation and permeabilization. Positive and negative controls, as appropriate, were p r o c e s s e d in p a r a l l e l. H i s t o g r a m s were obtained using the FACScan r e s e a r c h data software (Becton Dickinson) and were analyzed to determine the percentage of PBCs (defined as those cells expressing a pan-b cell antigen such as CD 9 and CD0, as well as CD, CD, and TdT) in each of the specimens.

3 Hematopathology / ORIGIL ARTICLE Results Clinicopathologic Data at Presentation Twenty-one bone marrow specimens from patients with previously documented BP-ALL and who fulfilled the aforementioned selection criteria were included in the study Table II. Twelve patients were children (<8 years), and were adults. In patients, the initial diagnosis of BP-ALL was made at Georgetown University Medical Center; in the other, the diagnosis was made elsewhere, and the disease was in morphologic remission when the patients were first seen at our hospital. In all of these patients, the leukemic blast cells had a PBC immunophenotype; all expressed at least, and usually, pan-b cell antigens (CD 9, CD0, CD, CD), as well as the precursor antigens CD and TdT Furthermore, in 8 of these patients the blast cells were also CD positive. Antigen receptor gene rearrangement status at initial diagnosis was known in 9 of patients whose initial diagnosis was made at this institution, but was not known in the remaining patients. Posttherapy Specimen Evaluation Posttherapy FCM and PCR results were obtained between and 8 months (average 9. months) after initial diagnosis (see Table ). Light microscopic evaluation of the cytocentrifuge preparations of enriched mononuclear cells processed for FCM and the corresponding unmanipulated bone marrow aspirates or biopsy samples confirmed morphologic remission in all patients, with less than 5% blast cells. In patients, an atypical lymphocytosis (possible hematogones) was noted, whereas in the remaining patients no such population was morphologically discernible. Precursor B cells were identified in all patients at FCM, and ranged between % and 0% (mean.%). Their immunophenotypic profile was essentially similar to that expressed by the leukemic blast cells at presentation, with coexpression of or more precursor antigens (CD, CD, TdT) on these B cells. This raised the consideration of Hgs vs minimal residual disease. However, in of these patients PCR revealed no evidence of monoclonal IgH or TCR-y gene rearrangements. Repeat follow-up FCM and PCR of 7 additional bone Table II Summary of Flow Cytometric Analysis and Gene Rearrangement in Patients With Treated Precursor B-Cell Acute Lymphoblastic Leukemia Posttherapy Follow-Up Follow-Up Presentation Patient 5 G / Si 9 Age (y) PCR Time Since Presentation (mo) Percentage of Precursor B Cells PCR Time Since Last Evaluation (mo) IgH+,TCR-y+ igh+ igh+ IgH+.TCR-Y+ igh + lgh+ igh+,tcr-yigh+,tcr-y+ igh lgh+,tcr-y+ gl I ightcr-y+,lghighigh-.tcr-yigh-.tcr-yighigh-,tcr-yighighlgh+,tcr-y+ IgHigH Outcome Died posttransplantation Relapsed Relapsed + = Positive for monoclonal gene rearrangement; - = negative for monoclonal gene rearrangement; IgH = immunoglobuiin heavy chain gene; TCR-y = T-cell receptor y chain gene; = not available. Am J Clin Pathol 999;:759-7B6 76 control (B-cell non-hodgkin lymphoma and T-cell ALL) and negative control (reactive tonsil) specimens was concurrently processed with each PCR run. PCR was also performed using primers of the erb-b- gene to demonstrate the presence of amplifiable D.8 A -ul aliquot from each of the IgH and TCR-y PCR reactions was subjected to 6% polyacrylamide gel electrophoresis, stained with ethidium bromide, and visualized under ultraviolet light. The image was digitally captured with an image analyzer (Gel Doc 00; Bio-Rad, Hercules, CA). The PCR was scored positive if the amplification products resulted in or appropriately sized (80 to 0 bp for IgH, 60 to 90 bp for TCR-y) discrete bands. By contrast, the presence of only a smear with no discrete signals was interpreted as negative. The IgH and TCR primers used in this assay detect approximately 85% and 90%, respectively, of rearrangements at these loci. The analytical sensitivity of these PCR assays is in the range of 0.%, with the ability to detect cell with a monoclonally rearranged antigen receptor in a background of,000 cells with polyclonal rearrangements.8'9

4 SURVEILLANCE OF TREATTE D-CEEL PRECURSOR A L L B 50% Log y 50% Log In the remaining of cases, the presence of PBCs at FCM was indeed associated with a monoclonal antigen receptor gene rearrangement that was identical to that documented at presentation in of the patients (presentation data not known in the third patient), indicating molecular evidence of residual BP-ALL. An example of a posttherapy bone marrow specimen in which positive IgH and TCR-y PCR identical to that at presentation indicated minimal residual disease that subsequently evolved to morphologic relapse months later is shown in llmage. Clinical Follow-Up The average duration of follow-up subsequent to initial presentation was 7.8 months (range to 7 months). Of the patients in whom a diagnosis of hematogones was made on the basis of negative gene rearrangement analysis, continued in morphologic remission at last follow-up. In patient, disease relapsed a year later, at which time the bone e 50 /c Lot -rafe&f i r&pes fjris»9 * J W ^ * «T i jtt M jtftbvjjmtlic T ^ w r. ^KSssPB*^ ' FL«D I B FL«D9 tt _5$S* -* FLIVTdT9 > w I -Mi isl mmm 0 00 FSC-K 00 6( I io ib CD FITC ANTI-TdT-FITC Image I I. Flow cytometric analysis of the bone marrow aspirate in a - year-old patient with B-cell precursor acute lymphoblastic leukemia (BP-ALL) shows CD-positive, CD9-positive, TdT-positive precursor B cells (PBCs) in the initial specimen (panels A-C) associated with a monoclonal IgH rearrangement by polymerase chain reaction (PCR) (panel D: lane, size markers; lane, negative control; lane, positive control; lane, patient). A posttherapy sample of bone marrow in morphologic remission from the same patient shows PBCs with a profile identical to that noted at presentation (panels E-G), raising the consideration of residual or recurrent acute leukemia. However, PCR for IgH rearrangement was negative (panel H: lane, size markers; lane, negative control; lane, positive control; lane, patient), supporting the diagnosis of polyclonal PBCs (hematogones) rather than relapsed BP-ALL. 76 Am J Clin Pathol 999;9: American Sooiety oo flinical Pathologists marrow specimens from 5 of these patients yielded similar results (detectable PBCs but negative PCR; data not shown). In 7 of these patients, the leukemic clone at presentation harbored a PCR-detectable antigen receptor gene rearrangement, confirming the remission status of the posttherapy samples. Posttherapy PCR results were obtained on average 7 months (range to 8 months) after initial presentation. Although in the other patients the presentation gene status was not known, mean follow-up of.5 months (range to 9 months) confirmed clinical and morphologic remission in of the. Thus, in of the patients with negative results at PCR, the positive FCM results favored the presence of hematogones rather than minimal residual disease. An example in which PBCs in the posttherapy sample were immunophenotypically identical to the original BP-ALL blast cells, but were determined to represent hematogones on the basis of absence of gene rearrangement (original BP-ALL positive for IgH rearrangement), is shown in llmage II.

5 Hematopathology / ORIGIL ARTICLE e < M ^^^ ^ - * w ^ Image Leukemic blast cells in the presentation bone marrow aspirate (panel A) from a -year-old patient with B-cell precursor acute lymphoblastic leukemia are associated with monoclonal rearrangements of both IgH and TCR-y genes (panel B: lanes and 5, size markers; lanes and 6, negative IgH and TCR controls; lanes and 7, positive IgH and TCR controls; lanes and 8, patient sample positive for IgH and biallelic TCR-y rearrangements). A posttherapy follow-up marrow sample years later shows morphologic remission (panel C) but molecular evidence of relapse by polymerase chain reaction (PCR) analysis (panel D: lanes and 5, size markers; lanes and 6, negative IgH and TCR controls; lanes and 7, positive IgH and TCR controls; lanes and 8, positive IgH and biallelic TCR-y rearrangements identical to that at presentation). A repeat bone marrow aspirate months later revealed frank morphologic relapse (panel E) associated with PCR abnormalities identical to those noted previously (panel F: lanes and 5, size markers; lanes and 6, negative IgH and TCR controls; lanes and 7, positive IgH and TCR controls; lanes and 8, positive weak signal for IgH and strong signal for biallelic TCR-y rearrangements in patient sample). American Society of Clinical Pathhlogists Am J Clin Pathol 999;99:

6 Kaiiakury et al /PCR SURVEILLANCE OF T REATED B-CELL P RECURSOR ALL marrow specimen showed monoclonally rearranged IgH and TCR-y genes (data not shown). Of the patients in whom PCR positivity in the posttherapy samples suggested minimal residual disease, had morphologic relapse months later, another underwent bone marrow transplantation and died of graft-related complications, and the third continued in morphologic remission months after (posttherapy) evaluation. The presence of increased PBCs in bone marrow of patients with treated BP-ALL may pose a diagnostic dilemma with regard to differentiating hematogones from residual or recurrent leukemia. This is particularly relevant in patients with disease in morphologic remission and who are being monitored with FCM for evidence of minimal residual disease. Differentiating hematogones from BPALL blast cells can be challenging, given their morphologic similarities and often identical immunophenotypic profiles. Further confounding this situation is the reported substantial increase in hematogones in some children after chemotherapy for ALL. Although the current study was performed with single-color and dual-color FCM and the distinction could not be made with certainty, others have used novel strategies such as multidimensional FCM and reported success in distinguishing hematogones from leukemic blast cells in patients with treated ALL.0 Multidimensional FCM is more informative than single-color or dual-color FCM because it permits simultaneous evaluation of the relationship and staining intensity of multiple cell surface antigens, thus enabling differentiation of normally maturing precursor cells from aberrant neoplastic cells. In this regard, multidimensional FCM may be particularly useful if the evaluation of the diagnostic and followup specimens is performed at the same institution with a similar reagent pairing panel. Because this is not always feasible, more robust, objective, and reproducible (between institutions) assays may be preferred. Other investigators have used markers of neoplasia or clonality, such as karyotypic abnormalities, D ploidy, and PCR studies, to distinguish hematogones from leukemic blast cells at initial presentation, but these techniques have not been reliably used to make this distinction in posttherapy bone marrow specimens. Anastasi et al,6 in a study of the analysis of suspect cells after therapy for ALL, used the presence of cytogenetic abnormalities at presentation to evaluate for the presence of residual disease. Two patients in their study had numerical chromosomal abnormalities at presentation, which were demonstrated in the atypical cells in patient, confirming the 76 Am J Clln Pathol 99; : Discussion presence of leukemic blast cells. Although this approach may be relevant in patients with relatively increased blast cells, it lacks the more universal applicability and level of sensitivity of PCR to evaluate for minimal residual disease (blast cell counts within normal limits). The utility of PCR as a sensitive marker of minimal residual disease is well documented; it can detect in or 5 cells, but its role as a specific and reliable marker in successfully distinguishing between hematogones and small numbers of leukemic blast cells has not been firmly established. Our findings indicate that when PCR is negative for monoclonally rearranged IgH or TCR genes in posttherapy specimens, particularly in those with trackable markers, it is more likely that PBCs noted at FCM are hematogones rather than leukemic blast cells. The presence of demonstrable antigen receptor gene rearrangements in the corresponding initial bone marrow aspirates of most of these patients further supports this conclusion. That all of the patients in this study with a positive PCR at presentation and a negative PCR in follow-up samples that contained PBCs were in morphologic remission at last evaluation (up to 5 months after posttherapy analysis) confirms this interpretation. Among the patients with a negative PCR at follow-up, only had disease relapse (approximately months after posttherapy analysis). Given the positive PCR result for both IgH and TCR gene rearrangements in that specimen demonstrating morphologic relapse (data not shown), it is unlikely that the previous PCR results in the posttherapy sample were false-negative. In another patient, testicular relapse developed, and results of IgH PCR from that site were positive, although the bone marrow continued to be PCR-negative for leukemic blast cells. This patient remains in sustained marrow remission 6 years after initial diagnosis. It should be noted, however, that a positive PCR result at presentation and a negative PCR result in the posttherapy sample in the setting of increased PBCs clearly does not exclude the presence of clonal cells, because mutations or ongoing rearrangments may affect primer annealing, resulting in a false-negative PCR. The significance of a positive PCR result in posttherapy marrow samples from patients with BP-ALL who harbor increased PBCs is not known. Although a positive PCR result may indicate the presence of a small population of clonal cells, whether it necessarily reflects an increased predilection for subsequent morphologic relapse is uncertain. Preliminary studies suggest that detection of minimal residual disease at the end of therapy correlates with future clinical relapse, although relapse may be delayed. In a subsequent retrospective study of 50 patients with childhood BP-ALL, PCR-detectable minimal residual disease predicted event-free survival independent of standard clinical and cytogenetic parameters. A

7 Hematopathology / ORIGIL ARTICLE From the Departmenss of Pathology and Pediatrics (Division of Hematologic Oncology,, and Lomhardi Cancer Center, Georgetown University Medical Center, Washington, DC. Presented in part at the 87th Annual Meeting of the United States and Canadian Academy of Pathology, Boston, MA, February 998. Address reprint requests to Dr Bagg: Department of Pathology, 67 Basic Science Building, Georgetown University Medical Center, 900 Reservoir Road NW, Washington, DC References. Imamura N, Kuramoto A. Detection of minimal residual disease in acute lymphoblastic leukemia by flow cytometry with monoclonal antibodies. Am] Hematol. 99;8:-.. Davis RE, Longacre TA, Cornbleet PJ. Hematogones in the bone marrow of adults: immunophenotypic features, clinical settings and differential diagnosis. Am J Clin Pathol. 99;:0-.. Caldwell CW, Poje E, Helikson MA. B-cell precursors in normal pediatric bone marrow. Am ] Clin Pathol. 99;95: Schmitt C, Eaves CJ, Lansdorp PM. Expression of CD on human B cell precursors. Clin Exp Immunol. 99;85: Ryan DH, Kossover S, Mitchell S, et al. Subpopulations of common acute lymphoblastic leukemia antigen-positive lymphoid cells in normal bone marrow identified by hematopoietic differentiation antigens. Blood. 986;68: Ryan DH, Chappie CW, Kossover SA, et al. Phenotypic similarities and differences between CALLA-positive acute lymphoblastic leukemia cells and normal marrow CALLApositive B cell precursors. Blood 987;70: Hokland P, Nadler LM, Griffin JD, et al. Purification of common acute lymphoblastic leukemia antigen positive cells from normal human bone marrow. Blood. 98;6: Greaves MF, Hariri G, Newman RA, et al. Selective expression of the common acute lymphoblastic leukemia (gp 0) antigen on immature lymphoid cells and their malignant counterparts. Blood. 98;6: Greaves M, Delia D, Janossy G, et al. Acute lymphoblastic leukemia associated antigen expression on non-leukemic "lymphoid cells." Leukemia Res. 980;:5-.. Hirt A, Morell A, Frei H, et al. Proliferation of lymphoid precursor cells in the bone marrow of patients with various disorders of the immune system. Exp Hematol. 988;6:8-.. Stekhoven JHS, Langenhuysen CAM, Bakkeren JAJM, et al. Morphology and incidence of the "post-therapeutic lymphoid cells" in the bone marrow of children with acute lymphoblastic leukemia. Am] Pathol. 986;:6-5.. van den Doel LJ, Pieters R, Huismans DR, et al. Immunological phenotype of lymphoid cells in regenerating bone marrow of children after treatment for acute lymphoblastic leukemia. Eur J Hematol. 988;: Ifrah N, Boucheix C, Marie JP, et al. Persistence of bone marrow lymphocytosis after induction treatment in common acute lymphoblastic leukemia: Marker analysis and significance. Cancer. 986;58: Anastasi ], Vardiman JW, Rudinsky R, et al. Direct correlation of cytogenetic findings with cell morphology using in-situ hybridization: an analysis of suspicious cells in bone marrow specimens of two patients completing therapy for acute lymphoblastic leukemia. Blood. 99;77: Nolan C, ed. Molecular Cloning: A Laboratory Manual. New York, NY: Cold Spring Harbor Laboratory Press, Sioutos N, Bagg A, Michaud GY, et al. Polymerase chain reaction versus Southern blot hybridization: detection of immunoglobulin heavy-chain gene rearrangements. Diagn Mol Pathol. 995;: Benhatter J, Delacretaz F, Martin P, et al. Improved polymerase chain reaction detection of clonal T-cell lymphoid neoplasms. Diagn Mol Pathol. 995;:8-.. Longacre TA, Foucar K, Crago S, et al. Hematogones: a multiparameter analysis of bone marrow precursor cells. Blood. 989;7: Wells DA, Sale GE, Shulman HM, et al. Multidimensional flow cytometry of marrow can differentiate leukemic from normal lymphoblasts and myeloblasts after chemotherapy and bone marrow transplantation. Am 7 Clin Pathol. 998;: Muehleck SD, McKenna RW, Gale PF, et al. Terminal deoxynucleotidyl transferase (TdT)-positive cells in bone marrow in the absence of hematologic malignancy. Am] Clin Pathol. 98;79: Yamada M, Wasserman R, Lange B, et al. Minimal residual disease in childhood B-lineage lymphoblastic leukemia: persistence of leukemic cells during the first 8 months of treatment. N Engl J Med. 990;:8-55. Am J Clin Pathol l999;: prospective study5 of minimal residual disease in childhood B-cell ALL reported that PCR-based levels of leukemic cells at the end of induction therapy served as a better prognostic indicator than did conventional risk factors, corroborating the previously published data. Conversely, molecular evidence of disease may persist for as long as years after completion of therapy in some patients with childhood ALL in sustained remission.6 In conclusion, increased PBCs detected at FCM, if considered in isolation, may be misleading in suggesting the presence of minimal residual disease in posttherapy bone marrow samples in patients with previously diagnosed BPALL. In contrast, PCR for IgH and TCR gene rearrangements can be a valuable and ssecific tool li nistinguishing hematogones from leukemic blast cells in the setting of minimal residual disease after chemotherapy. Although some patients with BP-ALL may not have trackable antigen receptor gene rearrangements, reverse transcriptase PCR of frequently occurring translocations, such as t(:), can be used to detect those few cases in which antigen receptor gene rearrangements are either undetectable with standard PCR or have undergone secondary changes.

8 Kallakury et al / PCR SURVEILLANCE OF TREATED B-CELL PRECURSOR ALL. Beishuizen A, Verhoeven MAJ, van Wering ER, et al. Analysis of Ig and T-cell receptor genes in 0 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis. Blood. 99;8:8-7.. Potter MN, Steward CG, Oakhill A. The significance of detection of minimal residual disease in childhood acute lymphoblastic leukemia. Br J Hematol. 99;8:-8. - Steenbergen EJ, Verhagen OJHM, van Leeuwen EF, et al. Prolonged persistence of PCR-detectable minimal residual disease after diagnosis or first relapse predicts poor outcome in childhood B-precursor acute lymphoblastic leukemia. Leukemia. 995;9: Jacquy C, Delepaut B, Van Daele S, et al. A prospective study of minimal residual disease in childhood BTineage acute lymphoblastic leukemia: MRD level at the end of induction is a strong predictive factor of relapse. Br J Hematol. 997;98: Roberts WM, Estrov Z, Ouspenskaia MV, et al. Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia. N EnglJ Med. 997;6: Am J Clin Pathol l999;: