Identification of amplified clonal T cell populations in the blood of patients with chronic graft-versus-host disease: positive correlation with response to photopheresis.

Chronic graft-versus-host disease (cGVHD) is a multiorgan disorder with skin manifestations resembling scleroderma. Since photopheresis, a treatment that induces an anticlonotypic immune response, has proven to be effective in both cutaneous T cell lymphomas with circulating clonal T cells and in cGVHD, we have searched for circulating clonal T cell populations in patients with cGVHD, and determined whether T cell clonality in the blood is associated with therapeutic response. We screened blood samples from 27 patients after HLA-matched allogeneic bone marrow transplantation (allo-BMT), 10 without cGVHD and 17 with extensive cGVHD, for clonal T cell receptor gamma (TCR gamma) gene rearrangements using fluorescent-based polymerase chain reaction (PCR) and automated high-resolution capillary electrophoresis. Amplified populations of clonal T cells with unique TCR gamma gene rearrangements were found in six of 10 (60%) allo-BMT patients without cGVHD and 13 of 17 (76.5%) allo-BMT patients with cGVHD (P = 0.41), as compared to none of 10 (0%) healthy controls. Twelve patients with cGVHD were treated by photopheresis, and the presence of amplified populations of clonal T cells was found to be associated with a cutaneous response to photopheresis, as eight of eight (100%) clone-positive vs none of four (0%) clone-negative patients experienced a clinically significant cutaneous response to treatment (P = 0.001). Our findings suggest that patients with cGVHD that have detectable expanded clonal T cell populations in their peripheral blood, may be more likely to respond to treatment by photopheresis.

Heteroduplex formation in SMN gene dosage analysis.

Most spinal muscular atrophy patients lack both copies of SMN1 exon 7 and most carriers have only one copy of SMN1 exon 7. We investigated the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is an assay to determine copy number of SMN1 exon 7 that utilizes multiplex quantitative polymerase chain reaction (PCR) with DraI digestion to differentiate SMN1 from SMN2. Heteroduplex formation in PCR is a well-described phenomenon. In addition to demonstrating the presence of heteroduplexes by sequence analysis of purified SMN1 bands, we compared the SMN1 signals in various genotype groups (total n = 260) to those in a group lacking SMN2 (n = 13), and we estimated the relative amounts of SMN1/SMN2 heteroduplexes. The SMN1 signal increased as SMN2 copy number increased despite a constant SMN1 copy number, although not all pairwise comparisons showed a statistically significant difference in the SMN1 signal. In conclusion, SMN1/SMN2 heteroduplexes form in SMN gene dosage analysis, falsely increasing the SMN1 signal. External controls for SMN gene dosage analysis should be chosen carefully with regard to SMN2 copy number. The effect of heteroduplex formation should be considered when performing quantitative multiplex PCR.

Identification of clonal T cells in the blood of patients with systemic sclerosis: positive correlation with response to photopheresis.

OBJECTIVES: To search for circulating clonal T-cell populations in patients with systemic sclerosis (SSc), and to determine whether T-cell clonality in the blood predicts therapeutic response to photopheresis. DESIGN: Analysis of clonal T-cell receptor gamma gene rearrangements before photopheresis treatment and blinded clinical evaluation of cutaneous response to photopheresis in a case series. SETTING: University hospital setting. PATIENTS: Thirteen consecutive patients with SSc. INTERVENTIONS: Photopheresis in 11 patients. MAIN OUTCOME MEASURES: Clonality of T cells in the blood before photopheresis and clinical response to photopheresis. RESULTS: Screening of blood samples from 13 SSc patients for clonal T-cell receptor gamma gene rearrangements revealed a monoclonal T cell population in 6 (46%) of 13 SSc patients. Clinical response to photopheresis in 11 patients was evaluated in a blinded manner using skin severity scores. Clonality of T cells appeared to be associated with a higher chance of response to photopheresis therapy, as 4 (67%) of 6 patients in the clone-positive group vs 1 (20%) of 5 in the clone-negative group experienced a clinically significant response to treatment. CONCLUSIONS: A high proportion of patients with SSc have detectable expanded clonal T-cell populations in the peripheral blood, and such patients appear more likely to respond to photopheresis.

Detection of clonal T-cell receptor gamma gene rearrangements using fluorescent-based PCR and automated high-resolution capillary electrophoresis.

BACKGROUND: Analysis of T-cell receptor gamma (TCR gamma) gene rearrangements by PCR is a powerful tool for detecting clonal T-cell populations for the diagnosis of lymphoid neoplasms. We report a method for TCR gamma PCR analysis using capillary electrophoresis (CE). METHODS AND RESULTS: To define the threshold for identification of a predominant monoclonal population within a polyclonal background, we developed a novel objective parameter of the peak height ratio (Rn) of the peak of interest and the average of the two immediate flanking peaks. After evaluation of monoclonal, reactive, and normal T-cell populations, an Rn of 3.0 or greater was determined to be consistent with a monoclonal population, whereas an Rn between 1.9 and 3.0 was considered an intermediate range. This CE method was compared with the standard denaturing gradient gel electrophoresis (DGGE) method using previously evaluated clinical specimens. Eleven of 12 clinical specimens (92%) with a definitive diagnosis of T-cell lymphoma were monoclonal by CE, with 100% concordance with the DGGE method. Of nine specimens morphologically suspicious for T-cell lymphoma, five specimens were positive by CE analysis compared with four specimens by DGGE. In addition, 14 specimens for staging from patients with known T-cell lymphoma were studied using both the CE and DGGE methods, with a concordance of 86%. CONCLUSION: CE is a powerful and efficient method for analysis of clonality by TCR gamma PCR.

Larger trinucleotide repeat size in the androgen receptor gene of infertile men with extremely severe oligozoospermia.

Androgens are significant regulators of human spermatogenesis. Their action is mediated through the androgen receptor (AR), which binds to the androgen responsive element on DNA and regulates gene transcription. Men become infertile with spinobulbar muscular atrophy (Kennedy disease) caused by a trinucleotide repeat expansion, > or = 40 CAG repeats, in the AR gene located on the X chromosome. In this prospective study, we investigated whether the variable size, larger repeats, of this trinucleotide could alter AR function and result in impaired spermatogenesis. A total of 69 infertile men were studied. Clinical and laboratory analysis showed idiopathic, nonobstructive azoospermia in 16 men, extremely severe oligozoospermia in 27 men (< 1 million sperm/mL), and severe oligozoospermia in 26 men (1 to 5 million sperm/mL). Fertile control men (n = 45) were selected by documented paternity proven by linkage analysis. Leukocyte DNA was analyzed by polymerase chain reaction (PCR) amplification across the AR repeat region. Accurate size determination of the PCR product using an ABI 373 DNA sequencer allowed precise calculation of CAG repeat sizes. The AR gene was not analyzed for other types of mutations. The difference in CAG repeat size between infertile men and proven fertile controls was statistically significant, P = .03. Patients with extremely severe oligozoospermia had significantly longer CAG repeat tracts (mean, 25.4 +/- 4.0; P = .0005; range 20-39) than controls (mean, 22 +/- 2.8; range 12-30) or patients with severe oligozoospermia (mean, 22.2 +/- 2.3; range 18-26). None of the 26 infertile men with sperm counts < 1 million/mL had < or = 19 CAG repeats compared with 6 out of 45 controls (13%; P = .06). This study suggests that some men with severe impairment of spermatogenesis have longer trinucleotide repeats in the AR gene. Although direct evidence is missing, lower affinity between androgen and the AR protein or decreased AR protein availability with longer repeats could be responsible for a diminished androgen effect on spermatogenesis. Two of the patients in the extremely severe oligozoospermia group had 35 and 39 CAG repeats, respectively (normal range is 11 to 33). Although not yet considered a mutation, longer trinucleotide repeats are unstable and might either expand or contract between generations. If they expand, conception through the use of intracytoplasmic sperm injection (ICSI), could result in the son of an

Allogeneic cell therapy for patients who relapse after autologous stem cell transplantation.

Allogeneic donor leukocytes can be used after nonmyeloablative conditioning to exploit their graft-versus-tumor (GVT) activity in the setting of reduced conditioning-regimen toxicity. This approach may be particularly useful for patients who relapse after autologous stem cell transplantation (SCT). However, GVT activity, toxicity, and ability to establish mixed chimerism may differ in patients who were heavily pretreated prior to SCT compared with patients treated earlier in the course of their disease. We have performed a series of studies of nonmyeloablative allogeneic transplantation and present data on the subset of 14 patients treated for relapse after autologous SCT: 4 patients received no conditioning and unstimulated donor leukocyte infusions (DLI), 10 patients received conditioning with fludarabine and cyclophosphamide followed by unstimulated or granulocyte-colony-stimulating factor (G-CSF)-stimulated allogeneic peripheral blood stem cells (PBSCs), 4 patients received no graft-versus-host disease (GVHD) prophylaxis, and 10 patients received cyclosporine GVHD prophylaxis. All but 1 patient had sustained donor chimerism at least 30 days after allogeneic cell therapy (ACT), and 8 patients had more than 80% donor chimerism after ACT. Acute GVHD developed in 11 patients (grade III-IV, n = 6). Aplasia was more frequent in the patients receiving unstimulated PBSCs, despite the development of mixed chimerism. There were 6 complete responses and 4 partial responses; response was independent of conditioning and growth-factor stimulation of the donor graft. Five patients died of treatment-related causes and 4 patients died from progressive disease. Four patients remained alive 27 to 194 weeks (median, 66 weeks) after ACT. Prior autologous SCT may define a subset of patients at particularly high risk for GVHD and other toxicity after ACT. However, these data show that ACT with either DLI or G-CSF-stimulated blood cells results in direct GVT activity in some patients with Hodgkin's disease, myeloma, and non-Hodgkin's lymphoma, even after relapse from autologous SCT. Most patients developed donor chimerism with minimal conditioning. Alternative prophylactic regimens that control GVHD while maintaining GVT are needed to improve outcomes in these heavily pretreated patients.

Analysis of the factor V Leiden mutation using the READIT Assay.

BACKGROUND: A variety of methods exist for the detection of single-nucleotide polymorphisms (SNPs) present in amplified segments of genomic DNA. We show the application of a novel SNP scoring tool for analysis of the factor V Leiden mutation. METHODS AND RESULTS: We have developed a novel method for analyzing SNPs. The luciferase-based technique, known as the READIT Technology (Promega Corp, Madison, WI), was used to analyze 510 residual human samples sent for factor V Leiden testing from three independent testing laboratories. A blinded retrospective analysis of the factor V Leiden mutation was used to determine the accuracy and throughput capabilities of the technology. One hundred percent concordance was observed between the READIT Assay and genotype assignments made in the testing laboratories. In addition, greater than 6 SDs of separation were observed between the means of wild-type and heterozygote sample populations. Repetitive sample measurements with representative wild-type, heterozygote, and mutant samples showed that greater than 9 SDs separated the means of heterozygote and homozygote sample populations. Confidence intervals based on the means of wild-type, heterozygote, and mutant sample populations were determined. CONCLUSION: Perfect concordance using the READIT Assay showed its effectiveness as a SNP scoring tool. The design of the factor V READIT Assay was straightforward, requiring the design of two unmodified oligonucleotides that differ at the 3' penultimate position to form perfect hybrids with the wild-type or Leiden form of the factor V sequence. The use of previously published amplification primers and conditions minimized the time needed to optimize and validate the assay. The READIT Calculator supplied with the assay allowed automated genotype assignments and statistical analysis from the READIT Assay data. Confidence-interval analysis validated the ability to distinguish between wild-type, heterozygote, and mutant samples using the READIT Assay.

Hepatosplenic gamma-delta T-cell lymphoma as a late-onset posttransplant lymphoproliferative disorder in renal transplant recipients.

We report 2 cases of renal transplant recipients in whom hepatosplenic gamma-delta T-cell lymphoma (gamma-delta HSTCL) developed 5 and 10 years after transplantation. Both patients had marked hepatosplenomegaly, B symptoms (weight loss, fever, and night sweats), and abnormal peripheral blood findings, including anemia in both, thrombocytopenia and leukoerythroblastic changes in 1, and leukocytosis in the other. Markedly atypical lymphoid infiltrate of intermediate to large cells was observed in the spleen, liver, and bone marrow. The malignant cells showed typical immunophenotype of gamma-delta T cells (CD2+, CD3+, CD4-, CD8-, CD7+, gamma-delta T-cell receptor-positive, and alpha-beta T-cell receptor-negative) with clonal T-cell receptor gene rearrangement and were of the V-delta-1 subset. In addition, the cells contained a cytolytic granule-associated protein, TIA-1, and Fas ligand, indicating cytotoxic T-cell differentiation. The malignant T cells in both cases were of host tissue origin. Both cases were negative for Epstein-Barr virus genome using Southern blot analysis. The patients did not respond to reduction of immunosuppression. Despite initial response to chemotherapy, both patients died within 6 months of diagnosis. Our findings indicate that gamma-delta HSTCL can occur as a late complication in transplant recipients.

Bone marrow engraftment analysis after allogeneic bone marrow transplantation.

BME analysis of allo-BMT patients is used to confirm engraftment and detect MC after transplant. With frequent monitoring, the detection of MC by BME analysis may alert clinicians to a high risk of relapse and allow early intervention with a rapid taper of immunosuppression or DLI therapy. In pancytopenic patients BME analysis can help differentiate relapse from drug toxicity or infection. Although many methods have been used for BME analysis, PCR amplification of STR loci is the choice of many clinical laboratories because it is informative, quantitative, relatively rapid, and sensitive. The sensitivity of BME analysis is dependent upon many factors that need to be optimized by the laboratory performing the analysis. Although BME analysis is complex, the clinical significance for allo-BMT patients warrants the effort to develop, validate, and perform BME analysis in support of the allo-BMT service.

Detection of leukemia-associated MLL-GAS7 translocation early during chemotherapy with DNA topoisomerase II inhibitors.

Leukemias with MLL gene translocations are a complication of primary cancer treatment with DNA topoisomerase II inhibitors. How early translocations appear during primary cancer treatment has not been investigated. We tracked the leukemic clone with an MLL gene translocation during neuroblastoma therapy in a child who developed acute myeloid leukemia. The karyotype of the leukemic clone showed del(11)(q23). We used panhandle PCR-based methods to isolate the breakpoint junction involving MLL and an unknown partner gene. Marrow DNA from neuroblastoma diagnosis and DNA and RNA from serial preleukemic marrows were examined for the translocation. The karyotypic del(11)(q23) was a cryptic t(11;17). GAS7, a growth arrest-specific gene at chromosome band 17p13, was the partner gene of MLL. Two different MLL-GAS7 fusion transcripts were expressed. The translocation was already detectable by 1.5 months after the start of neuroblastoma treatment. The translocation was not detectable in the marrow at neuroblastoma diagnosis or in peripheral blood lymphocyte DNAs of six normal subjects. GAS7 is a new partner gene of MLL in treatment-related acute myeloid leukemia. MLL gene translocations can be present early during anticancer treatment at low cumulative doses of DNA topoisomerase II inhibitors. Although MLL has many partner genes and most have not been characterized, panhandle PCR strategies afford new means for detecting MLL gene translocations early during therapy when the partner gene is unknown.

Sequence-based identification of Mycobacterium species using the MicroSeq 500 16S rDNA bacterial identification system.

We evaluated the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (PE Applied Biosystems), a 500-bp sequence-based identification system, for its ability to identify clinical Mycobacterium isolates. The organism identity was determined by comparing the 16S rDNA sequence to the MicroSeq database, which consists primarily of type strain sequences. A total of 113 isolates (18 different species), previously recovered and identified by routine methods from two clinical laboratories, were analyzed by the MicroSeq method. Isolates with discordant results were analyzed by hsp65 gene sequence analysis and in some cases repeat phenotypic identification, AccuProbe rRNA hybridization (Gen-Probe, Inc., San Diego, Calif.), or high-performance liquid chromatography of mycolic acids. For 93 (82%) isolates, the MicroSeq identity was concordant with the previously reported identity. For 18 (16%) isolates, the original identification was discordant with the MicroSeq identification. Of the 18 discrepant isolates, 7 (six unique sequences) were originally misidentified by phenotypic analysis or the AccuProbe assay but were correctly identified by the MicroSeq assay. Of the 18 discrepant isolates, 11 (seven unique sequences) were unusual species that were difficult to identify by phenotypic methods and, in all but one case, by molecular methods. The remaining two isolates (2%) failed definitive phenotypic identification, but the MicroSeq assay was able to definitively identify one of these isolates. The MicroSeq identification system is an accurate and rapid method for the identification of Mycobacterium spp.

Hepatosplenic and subcutaneous panniculitis-like gamma/delta T cell lymphomas are derived from different Vdelta subsets of gamma/delta T lymphocytes.

Gamma/delta T cell lymphomas (gamma/delta TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most gamma/delta TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor delta (TCRS) gene repertoire of hepatosplenic gamma/delta TCL (gamma/delta HSTCL) and subcutaneous panniculitis-like gamma/delta TCL (gamma/delta SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 gamma/delta HSTCL and 4 gamma/delta SPTCL by polymerase chain reaction and immunostaining to determine their usage of the Vdelta subtypes (Vdelta1-6). It is noteworthy that 10 of 11 gamma/delta HSTCL expressed the Vdelta1 gene. The remaining case also expressed T cell receptor delta (TCRS) as determined by flow cytometry and TCRdelta rearrangement in Southern blot. However, the Vdelta gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified Vdelta gene. In striking contrast to the gamma/delta HSTCL, all 4 gamma/delta SPTCL expressed the Vdelta2 gene. Our data demonstrate that gamma/delta HSTCL are preferentially derived from the Vdelta1 subset of gamma/delta T lymphocytes, whereas gamma/delta SPTCL are preferentially derived from the Vdelta2 subset. The pattern of Vdelta gene expression in HSTCL and SPTCL corresponds to the respective, predominant gamma/delta T cell subsets normally found in the spleen and skin. This finding suggests that gamma/delta TCL are derived from normal gamma/delta T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific Vdelta gene segment usage may provide a molecular tool to distinguish better among various types of gamma/delta TCL lymphoma particularly in the clinically advanced, widely disseminated cases.