REVIEW ARTICLE
Microsatellite Instability Confounds Engraftment Analysis of Hematopoietic Stem-cell Transplantation Li-Hui Tseng, MD, PhD,*w Jih-Luh Tang, MD, PhD,w Lisa Haley, BS,* Katie Beierl, BS,* Christopher D. Gocke, MD,*z James R. Eshleman, MD, PhD,*z and Ming-Tseh Lin, MD, PhD*
Abstract: Polymorphic short tandem-repeat, or microsatellite, loci have been widely used to analyze chimerism status after allogeneic hematopoietic stem-cell transplantation. In molecular diagnostic laboratories, it is recommended to calculate mixed chimerism for at least 2 informative loci and to avoid microsatellite loci on chromosomes with copy number changes. In this report, we show that microsatellite instability observed in 2 patients with acute leukemia may confound chimerism analysis. Interpretation errors may occur even if 2 to 3 loci are analyzed because of length variation in multiple microsatellite loci. Although microsatellite loci with length variation should not be selected for chimerism analysis, the presence of microsatellite instability, like copy number alteration because of aberrant chromosomes, provides evidence of recurrent or residual cancer cells after hematopoietic stem-cell transplantation. Key Words: short tandem-repeat, microsatellite instability, allogeneic hematopoietic stem-cell transplantation, chimerism, leukemia relapse (Appl Immunohistochem Mol Morphol 2014;22:416–420)
S
hort tandem-repeat (STR), or microsatellite, loci are widely used for analysis of engraftment and chimerism status in patients after allogeneic hematopoietic stem-cell transplantation (HSCT).1,2 Mixed chimerism indicates the presence of recipient and donor cells in the posttransplant recipient peripheral blood or bone marrow.3,4 As chromosomal gains or losses can affect STR analysis and lead to errors in the calculation, we routinely check results of the cytogenetic analysis and/or fluorescent in situ hybridization (FISH) assay of the leukemia cells to avoid loci with gains or losses of chromosomes. In molecular diagnostic laboratories, it is advisable to use multiple microsatellite loci for mixed chimerism calcu-
From the Departments of *Pathology; zOncology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD; and wDepartment of Medical Genetics and Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan. The authors declare no conflict of interest. Reprints: Ming-Tseh Lin, MD, PhD, Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Park SB202, 600 North Wolfe St., Baltimore, MD 21287 (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins
416 | www.appliedimmunohist.com
lations, particularly for patients with no known cytogenetic tests of their leukemia cells.5,6 Microsatellite instability (MSI) is not common in acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).7,8 MSI is defined as a variation in length as a result of insertion or deletion of units of a microsatellite repeat in a neoplasm compared with the patients’ germline length.9 In colorectal cancer, mononucleotide microsatellites have shown that higher sensitivity for the detection of MSI, though tetranucleotide microsatellite loci, which are widely used for identity or chimerism analysis, can also show MSI.9–11 We reported 2 patients with acute leukemia. MSI, observed at relapse after HSCT, may lead to interpretative errors during chimerism analysis.
MATERIALS AND METHODS DNA was isolated from peripheral blood or bone marrow specimens using a Qiagen EZ1-automated nucleic-acid extractor and reagents (Qiagen, Valencia, CA). T lymphocytes were purified from peripheral blood using a RoboSep machine (Stemcell Technologies, Vancouver, BC, Canada) with anti-CD3 antibody, according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using the AmpFlSTR Identifiler kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions as described previously.12 Fifteen microsatellite loci (D8S1179 at chromosome 8, D21S11 at 21q11.2, D7S820 at 7q, and CSF1PO at 5q33.3-34 labeled with 6-FAM; D3S1358 at chromosome 3p, THO1 at 11p15.5, D13S317 at 13q22-31, D16S539 at 16q24-qter, and D2S1338 at 2q35-37.1 labeled with VIC; D19S433 at 19q12-13.1, vWA at 12p12-pter, TPOX at 2p25.3, and D18S51 at 18q21.3 labeled with NED; D5S818 at 5q21-31, and FGA at 4q28 labeled with PET) and the amelogenin locus at X (p22.1-22.3) and Y (p11.2) chromosomes were analyzed. A total of 1 mL of PCR products and 9 mL of deionized formamide/GeneScan-500 [LIZ] (Applied Biosystems) were mixed according to the manufacturer’s protocol, heated at 951C for 2 minutes, and placed on ice for at least 1 minute before electrokinetic injection on the ABI 3130xL or ABI3500Dx capillary electrophoresis instrument (Applied Biosystems), as described previously.13 Patient and donor samples were analyzed before transplant. Posttransplant chimerism is routinely calculated using an average of 2 to 3 informative
Appl Immunohistochem Mol Morphol
Volume 22, Number 6, July 2014
Appl Immunohistochem Mol Morphol
Volume 22, Number 6, July 2014
loci.12 The standard chimerism calculation is: (peak height of recipient-specific allele)/(sum of peak heights of recipient-specific and donor-specific alleles) 100%. Multiplex PCR amplification of 5 mononucleotide microsatellite loci (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide microsatellite loci (Penta C and Penta D) was performed using MSI Analysis System (Promega Corp., Medison, WI), according to the manufacturer’s instructions.9 Amplification products are analyzed by using ABI 3130xL capillary electrophoresis instrument (Applied Biosystems). The neoplasm is designated as MSI-high if novel allele lengths are identified in the neoplastic cells when compared with normal/germline at 2 or more microsatellite loci.9
RESULTS Case 1 A 58-year-old male patient with T-cell ALL received a nonmyeloablative allogeneic HSCT from his HLAhaploidentical male donor. The pretransplant analysis of the peripheral blood from the patient and the donor showed 6 informative microsatellite loci for the analysis of donor-dominant chimerism and 8 loci for the analysis of patient-dominant chimerism. Microsatellite analysis of the 3 selected loci for donor-dominant chimerism (D16S539, TPOX, and D5S818) showed no evidence of patient DNA in the peripheral blood and the CD3+ T cells isolated from peripheral blood at 1, 2, and 6 months posttransplant. However, analysis of the peripheral blood at 9 months posttransplant showed inconsistent results across the 3 selected loci: 3%, 76%, and 0% patient DNA for loci D16S539, TPOX, and D5S818, respectively (Fig. 1). Review of the clinical information revealed 75% of blasts detected by flowcytometry in the peripheral blood. In the context of relapse with 75% blasts in the sample by flow cytometry, we analyzed all the 8 informative loci for patient-dominant chimerism. Four loci (D21S11, D7S820, THO1, and D19S433) showed 20% to 25% donor DNA, consistent with 75% blasts in the peripheral blood. The other 4 loci showed 90% (D3S1358), 46% (D18S51), 100% (D5S818), and 100% (FGA) donor DNA. A comparison of current specimen, pretransplant patient, and donor samples revealed length variation in at least 6 of the 15 microsatellite loci used in the AmpFlSTR Identifiler kit (Fig. 2). Single nucleotide polymorphism array showed no loss of heterozygosity of any of the chromosome arms bearing the microsatellite loci included in the AmpFlSTR Identifiler kit (data not shown). Length variations of the patient’s leukemia cells were observed in all 5 mononucleotide microsatellite loci, consistent with MSI-high (Fig. 3).
Case 2 A 64-year-old female patient with AML with a normal karyotype received a myeloablative unrelated HSCT at relapsed disease status. The pretransplant analysis of the peripheral blood from the patient in remission and the donor showed 9 informative loci for the r
2014 Lippincott Williams & Wilkins
MSI Confounds Engraftment Analysis of HSCT
evaluation of donor-dominant chimerism and 8 loci for the evaluation of patient-dominant chimerism. At 1 month posttransplant, a bone marrow examination showed 20% blasts. Chimerism analysis of the informative D8S1179 locus showed 2 dominant 133-base and 159-base alleles from the donor, a minor 147-base allele from the patient, and a 155-base allele of unknown origin (Fig. 4). The peak height of the 155-base allele [1221 relative fluorescence units (RFUs)] was similar to that of the 147-base allele (943 RFUs) and was 32.2% of that of the 159-base allele (3776 RFUs). As the peak height of a tetranucleotide microsatellite stutter is usually
Microsatellite Instability Confounds Engraftment Analysis of Hematopoietic Stem-cell Transplantation Li-Hui Tseng, MD, PhD,*w Jih-Luh Tang, MD, PhD,w Lisa Haley, BS,* Katie Beierl, BS,* Christopher D. Gocke, MD,*z James R. Eshleman, MD, PhD,*z and Ming-Tseh Lin, MD, PhD*
Abstract: Polymorphic short tandem-repeat, or microsatellite, loci have been widely used to analyze chimerism status after allogeneic hematopoietic stem-cell transplantation. In molecular diagnostic laboratories, it is recommended to calculate mixed chimerism for at least 2 informative loci and to avoid microsatellite loci on chromosomes with copy number changes. In this report, we show that microsatellite instability observed in 2 patients with acute leukemia may confound chimerism analysis. Interpretation errors may occur even if 2 to 3 loci are analyzed because of length variation in multiple microsatellite loci. Although microsatellite loci with length variation should not be selected for chimerism analysis, the presence of microsatellite instability, like copy number alteration because of aberrant chromosomes, provides evidence of recurrent or residual cancer cells after hematopoietic stem-cell transplantation. Key Words: short tandem-repeat, microsatellite instability, allogeneic hematopoietic stem-cell transplantation, chimerism, leukemia relapse (Appl Immunohistochem Mol Morphol 2014;22:416–420)
S
hort tandem-repeat (STR), or microsatellite, loci are widely used for analysis of engraftment and chimerism status in patients after allogeneic hematopoietic stem-cell transplantation (HSCT).1,2 Mixed chimerism indicates the presence of recipient and donor cells in the posttransplant recipient peripheral blood or bone marrow.3,4 As chromosomal gains or losses can affect STR analysis and lead to errors in the calculation, we routinely check results of the cytogenetic analysis and/or fluorescent in situ hybridization (FISH) assay of the leukemia cells to avoid loci with gains or losses of chromosomes. In molecular diagnostic laboratories, it is advisable to use multiple microsatellite loci for mixed chimerism calcu-
From the Departments of *Pathology; zOncology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD; and wDepartment of Medical Genetics and Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan. The authors declare no conflict of interest. Reprints: Ming-Tseh Lin, MD, PhD, Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Park SB202, 600 North Wolfe St., Baltimore, MD 21287 (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins
416 | www.appliedimmunohist.com
lations, particularly for patients with no known cytogenetic tests of their leukemia cells.5,6 Microsatellite instability (MSI) is not common in acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).7,8 MSI is defined as a variation in length as a result of insertion or deletion of units of a microsatellite repeat in a neoplasm compared with the patients’ germline length.9 In colorectal cancer, mononucleotide microsatellites have shown that higher sensitivity for the detection of MSI, though tetranucleotide microsatellite loci, which are widely used for identity or chimerism analysis, can also show MSI.9–11 We reported 2 patients with acute leukemia. MSI, observed at relapse after HSCT, may lead to interpretative errors during chimerism analysis.
MATERIALS AND METHODS DNA was isolated from peripheral blood or bone marrow specimens using a Qiagen EZ1-automated nucleic-acid extractor and reagents (Qiagen, Valencia, CA). T lymphocytes were purified from peripheral blood using a RoboSep machine (Stemcell Technologies, Vancouver, BC, Canada) with anti-CD3 antibody, according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using the AmpFlSTR Identifiler kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions as described previously.12 Fifteen microsatellite loci (D8S1179 at chromosome 8, D21S11 at 21q11.2, D7S820 at 7q, and CSF1PO at 5q33.3-34 labeled with 6-FAM; D3S1358 at chromosome 3p, THO1 at 11p15.5, D13S317 at 13q22-31, D16S539 at 16q24-qter, and D2S1338 at 2q35-37.1 labeled with VIC; D19S433 at 19q12-13.1, vWA at 12p12-pter, TPOX at 2p25.3, and D18S51 at 18q21.3 labeled with NED; D5S818 at 5q21-31, and FGA at 4q28 labeled with PET) and the amelogenin locus at X (p22.1-22.3) and Y (p11.2) chromosomes were analyzed. A total of 1 mL of PCR products and 9 mL of deionized formamide/GeneScan-500 [LIZ] (Applied Biosystems) were mixed according to the manufacturer’s protocol, heated at 951C for 2 minutes, and placed on ice for at least 1 minute before electrokinetic injection on the ABI 3130xL or ABI3500Dx capillary electrophoresis instrument (Applied Biosystems), as described previously.13 Patient and donor samples were analyzed before transplant. Posttransplant chimerism is routinely calculated using an average of 2 to 3 informative
Appl Immunohistochem Mol Morphol
Volume 22, Number 6, July 2014
Appl Immunohistochem Mol Morphol
Volume 22, Number 6, July 2014
loci.12 The standard chimerism calculation is: (peak height of recipient-specific allele)/(sum of peak heights of recipient-specific and donor-specific alleles) 100%. Multiplex PCR amplification of 5 mononucleotide microsatellite loci (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide microsatellite loci (Penta C and Penta D) was performed using MSI Analysis System (Promega Corp., Medison, WI), according to the manufacturer’s instructions.9 Amplification products are analyzed by using ABI 3130xL capillary electrophoresis instrument (Applied Biosystems). The neoplasm is designated as MSI-high if novel allele lengths are identified in the neoplastic cells when compared with normal/germline at 2 or more microsatellite loci.9
RESULTS Case 1 A 58-year-old male patient with T-cell ALL received a nonmyeloablative allogeneic HSCT from his HLAhaploidentical male donor. The pretransplant analysis of the peripheral blood from the patient and the donor showed 6 informative microsatellite loci for the analysis of donor-dominant chimerism and 8 loci for the analysis of patient-dominant chimerism. Microsatellite analysis of the 3 selected loci for donor-dominant chimerism (D16S539, TPOX, and D5S818) showed no evidence of patient DNA in the peripheral blood and the CD3+ T cells isolated from peripheral blood at 1, 2, and 6 months posttransplant. However, analysis of the peripheral blood at 9 months posttransplant showed inconsistent results across the 3 selected loci: 3%, 76%, and 0% patient DNA for loci D16S539, TPOX, and D5S818, respectively (Fig. 1). Review of the clinical information revealed 75% of blasts detected by flowcytometry in the peripheral blood. In the context of relapse with 75% blasts in the sample by flow cytometry, we analyzed all the 8 informative loci for patient-dominant chimerism. Four loci (D21S11, D7S820, THO1, and D19S433) showed 20% to 25% donor DNA, consistent with 75% blasts in the peripheral blood. The other 4 loci showed 90% (D3S1358), 46% (D18S51), 100% (D5S818), and 100% (FGA) donor DNA. A comparison of current specimen, pretransplant patient, and donor samples revealed length variation in at least 6 of the 15 microsatellite loci used in the AmpFlSTR Identifiler kit (Fig. 2). Single nucleotide polymorphism array showed no loss of heterozygosity of any of the chromosome arms bearing the microsatellite loci included in the AmpFlSTR Identifiler kit (data not shown). Length variations of the patient’s leukemia cells were observed in all 5 mononucleotide microsatellite loci, consistent with MSI-high (Fig. 3).
Case 2 A 64-year-old female patient with AML with a normal karyotype received a myeloablative unrelated HSCT at relapsed disease status. The pretransplant analysis of the peripheral blood from the patient in remission and the donor showed 9 informative loci for the r
2014 Lippincott Williams & Wilkins
MSI Confounds Engraftment Analysis of HSCT
evaluation of donor-dominant chimerism and 8 loci for the evaluation of patient-dominant chimerism. At 1 month posttransplant, a bone marrow examination showed 20% blasts. Chimerism analysis of the informative D8S1179 locus showed 2 dominant 133-base and 159-base alleles from the donor, a minor 147-base allele from the patient, and a 155-base allele of unknown origin (Fig. 4). The peak height of the 155-base allele [1221 relative fluorescence units (RFUs)] was similar to that of the 147-base allele (943 RFUs) and was 32.2% of that of the 159-base allele (3776 RFUs). As the peak height of a tetranucleotide microsatellite stutter is usually
