Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction.

We have developed a real-time quantitative polymerase chain reaction (PCR) assay using TaqMan technology (Applied Biosystems, Foster City, CA) for monitoring donor cell engraftment in allogenic hematopoietic stem cell transplant recipients. For this purpose, we selected 19 specific sequence polymorphisms belonging to 11 human biallelic loci located on 9 different chromosomes. Using a set of specially designed primers and fluorogenic probes, we evaluated the 19 markers' informativity on a panel of 126 DNA samples from 63 recipient/donor pairs. In more than 90% of these pairs, discrimination between recipient and donor genetic profile was possible. By using serial dilutions of mixed DNAs, we evaluated the linearity and sensitivity of the method. A linear correlation with r higher than 0.98 and a sensitivity of 0.1% proved reproducible. Fluorescent-based PCR of short tandem repeats (STR-PCR) and real-time PCR chimerism assay were compared with a panel of artificial cell mixtures. The main advantage of the real-time PCR method over STR-PCR chimerism assays is the absence of PCR competition and plateau biases, and results evidenced greater sensitivity and linearity with the real-time PCR method. Furthermore, different samples can be tested in the same PCR run with a final result in fewer than 48 hours. Finally, we prospectively analyzed patients who received allografts and present 4 different clinical situations that illustrate the informativity level of our method. In conclusion, this new assay provides an accurate quantitative assessment of mixed chimerism that can be useful in guiding early implementation of additional treatments in hematopoietic stem cell transplantation.

Stage I-IIE primary non-Hodgkin's lymphoma of the testis: results of a prospective trial by the GOELAMS Study Group.

Sixteen patients with aggressive primary testicular involvement were analyzed separately from a prospective multicenter series of 494 patients with stage I/II aggressive nonlymphoblastic lymphoma. The treatment strategy included 3 cycles of anthracycline-based chemotherapy followed by regional radiation therapy on inguinal, iliac, and para-aortic lymph nodes and central nervous system (CNS) prophylaxis by intrathecal chemotherapy and brain irradiation. Chemotherapy was stratified by age group. Patients aged 18-60 years received the Groupe Ouest Est d'Etude des Leucemies Aigues et Maladies du Sang (GOELAMS) 02 protocol: 3 monthly cycles of VCAP (vindesine 3 mg/m2 day 1, doxorubicin 80 mg/m2 day 2, cyclophosphamide 1500 mg/m2 day 2, and prednisone 80 mg/m2 days 1-5). Patients aged 61-75 years received the GOELAMS 03 protocol: 3 monthly cycles of VECP-Bleo (vindesine 3 mg/m2 day 1, epirubicin 60 mg/m2 day 1, cyclophosphamide 750 mg/m2 day 1, prednisone 50 mg/m2 days 1-7, and bleomycin 10 mg/m2 days 1 and 5). Sixteen patients had testicular involvement (3.3%). Median age was 62 years (range, 29-73 years). The histological subtypes were diffuse large-cell lymphoma in all cases. Ann Arbor stage was IEA in 11 patients, IEB in 3 patients, and IIEA in 2 patients. All patients achieved a complete response. One patient died from septic shock during the last course of chemotherapy. After a median follow-up period of 73.5 months, the probability of disease-free survival (DFS) and overall survival (OS) were 70% and 65%, respectively for all patients. Disease-free survival and OS were 66% and 83% in patients = 60 years of age, and 74% and 56% in patients > 60 years of age. Relapse occurred in extranodal sites in 4 cases and in abdominal lymph nodes in the last case. Relapse in the CNS occurred in only 1 patient and in the contralateral testis in 1 patient. We found no correlation between OS, DFS and extent of testicular involvement, Ann Arbor stage, International Prognostic Index score, or lactate dehydrogenase level. This is the first report of a prospective study in which treatment of testicular non-Hodgkin's lymphoma was precisely defined at diagnosis. Compared to other series, a combination of orchiectomy with 3 cycles of CHOP (cyclophosphamide/doxorubicin/vincristine/prednisone)-derived chemotherapy, regional radiation therapy, and CNS prophylaxis seems to improve prognosis. The improvement in prognosis seemed to be due in part to irradiation, including the pelvic and lomboaortic lymphatic areas, and in part to CNS prophylaxis.