Real-Time PCR Analysis of Chimerism in T Cell Subsets as an Early Predictor of Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation.

BACKGROUND: Graft-versus-host-disease (GvHD) is the major cause of morbidity and mortality after stem cell transplantation. The development of early prediction methods is therefore of importance. Our aim was to analyze the usefulness of early donor chimerism monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in T cells and in CD4+ and CD8+ (lineage chimerism) for GvHD prediction. MATERIAL AND METHODS: Chimerism was analyzed in 76 consecutive adult patients using RQ-PCR TaqMan technology on DNA extracted from Pan T, CD4+, and CD8+ cell subsets on Day 5, 10, 15 and 30 after allo-HSCT. RESULTS: The threshold of chimerism predictive for GvHD was the same for all tested cell subsets. In acute myeloid leukemia (AML) patients treated with myeloablative conditioning (MAC), the threshold predictive for acute graft versus host disease was 95% and 99% for Day 10 and Day 15, respectively. In patients treated with reduced intensity conditioning (RIC), the threshold predictive for chronic graft versus host disease was 98% on Day 10. The differences were statistically significant. CONCLUSIONS: Chimerism analysis in T cell subsets by RQ-PCR on Day 10 and Day 15 after transplantation is useful for prediction of aGvHD (AML patients after MAC) and cGvHD (patients after RIC). However, there was no difference in the results between chimerism in the T cell subsets. Our RQ-PCR protocol was highly sensitive and proved effective for analysis of lineage chimerism.

Co-infections with cytomegalovirus and human herpesvirus type 7 in adult Polish allogeneic haematopoietic stem cell transplant recipients.

Human herpesvirus 7 (HHV-7) is widespread around the world and may also be a possible cofactor for cytomegalovirus (CMV) infection in haematopoietic stem cell transplant (HSCT) recipients. In case of viral diseases where specific treatment is available, real-time PCR assays constitute reliable diagnostic tools enabling timely initiation of appropriate therapy and rapid assessment of the efficacy of antiviral treatment strategies. The presence of CMV and HHV-7 was confirmed by the detection of viral DNA isolated from 1,027 plasma samples. A group of 69 allogeneic HSCT (alloHSCT) recipients was examined in early post-transplant period using quantitative real-time PCR methods. Within the study period, 62 % of patients had at least once CMV DNA-emia, while HHV-7 DNA was found in 43 % of subjects. Co-infection between these ß-herpesviruses was detected in the plasma samples collected from 18 patients (26 %). Patients with concomitant HHV-7 DNA-emia had significantly higher number of CMV DNA copies compared with those without HHV-7 infection (1986 vs. 432 copies/ml, p < 0.001) but there was no difference in duration of CMV DNA-emia between these groups. On the other hand, while the load of HHV-7 DNA was comparable between patients with CMV DNA-emia and without CMV DNA-emia, the duration of HHV-7 DNA-emia was significantly longer in the first group (38.5 vs. 14 days, p < 0.001). HHV-7 DNA-emia is very frequently detected in Polish alloHSCT recipients. In those, who have subsequent CMV reactivation, the coexistence of the viruses may negatively affect the kinetics of infection with either of them. Therefore the investigation of concomitant HHV-7 DNA-emia could affect the prognosis of post-transplant patients suffering from CMV reactivation.

The significance of oral labial biopsy in hepatic graft-versus-host disease diagnosis in patients following allogeneic hematopoietic stem cell transplantation--a preliminary report.

BACKGROUND: Graft-versus-host disease (GvHD) is the most important complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) responsible for increased mortality. Recent studies have discussed the use of oral GvHD screening tests in the diagnosis of systemic GvHD. This is the first study of the significance of oral labial biopsy in diagnosis of hepatic graft-versus-host disease (H-GvHD) following allo-HSCT. MATERIAL/METHODS: Twenty-one patients after alloHSCT were selected: 12 patients with H-GvHD established clinically and in laboratory tests, and 9 patients without features of GvHD (control group). Histopathological and immunohistochemical evaluations of tissue samples included the following: 15 samples of oral mucosa (OM), 19 of lip salivary glands (LSG), and 5 of the liver, were performed in both groups. RESULTS: All patients had clinically normal oral mucosa and 4 patients with H-GvHD manifested with xerostomia symptoms. Histological examination of LSG and/or OM confirmed the GvHD diagnosis in 9 of 12 patients with H-GvHD. The microscopic changes included: mild inflammatory lymphocytic infiltrates and apoptotic bodies in OM, and inflammatory infiltrates of mild degree with minimal CD8+ T cells predominance in the invaded ducts epithelium in LSG. In the control group, 4 of 9 patients had mild chronic inflammation, which did not fulfill the criteria of GvHD. The histopathological image of liver biopsies correlated with the clinical GvHD diagnosis. CONCLUSIONS: The microscopic evaluation of LSG and/or OM biopsy confirms the clinical diagnosis of H-GvHD, regardless of the absence of clinical oral symptoms of GVHD. The histopathological features of oral GvHD may be subtle; the diagnosis requires a clinico-pathological and laboratory approach to exclude the other diseases with similar histopathological features.

Palifermin does not influence the incidence and severity of GvHD nor long-term survival of patients with hematological diseases undergoing HSCT.

BACKGROUND: Palifermin is known as the effective growth factor to reduce the incidence, duration and severity of oral mucositis (OM) following hematopoietic stem cell transplantation (HSCT). However, additional data on the long-term safety of palifermin and its potential influence on graft versus host disease (GvHD) are needed. MATERIAL/METHODS: In this multi-center, non-randomized, matched-control study we assessed early overall survival (OS), incidence and severity of acute/chronic GvHD (a/cGvHD) and incidence of secondary malignancies in 36 patients with hematological diseases treated with allogeneic HSCT and palifermin. RESULTS: The incidence of aGvHD was 28.2% and 38.4% (p=0.34) and cGvHD 41% and 53.8% (p=0.70) in the palifermin and control groups, respectively. The incidence of aGvHD grade 0-IV was 69.2%, 5.1%, 17.9%, 2.5%, 2.5% in the palifermin group and 61.5%, 12.8%, 12.8%, 10.2%, 5.2% in the control group, respectively (p>0.4 for each). The incidence of limited and extensive cGvHD was 17.9% and 23% in the palifermin versus 25.6% and 28.2% in the control group (p=0.32 and p=0.50, respectively). The estimated 3-year OS did not differ significantly between studied groups. We did not observe any secondary malignancies in these patients. CONCLUSIONS: Administration of palifermin doesn't seem to influence the incidence and severity of aGvHD/cGvHD, secondary malignancies occurrence and early OS in patients undergoing allogeneic HSCT.

Simultaneous human herpesvirus 6-associated encephalitis and Guillain-Barré syndrome in a patient after matched unrelated donor haematopoietic stem cell transplantation.

Viral infections are still a serious diagnostic and therapeutic problem in patients undergoing alternative donor transplants. ß-Herpesviruses (especially human herpesvirus type 5, 6 and 7) are recognized pathogens in this group of patients and may cause central nervous system disease. Guillain-Barré syndrome (GBS) is a very rare complication among stem cell transplant recipients and usually has been attributed to infection. We report a case of resolving simultaneous GBS and HHV-6-associated encephalitis in a haematopoietic stem cell transplant recipient with preceding reactivation of cytomegalovirus (CMV) infection. According to our knowledge this well-documented case is probably the first report from Poland.

Clinical evaluation of busulfan, cladribine and alemtuzumab as reduced intensity conditioning for stem cell transplantation.

BACKGROUND: Reduced intensity conditioning before allogeneic stem cell transplantation is an option for patients not eligible for myeloablative standard procedure. In recent years alemtuzumab, an anti CD52 antibody has gained much interest in this setting for its particular immunosuppresive properties.
MATERIAL/METHODS: We evaluated clinical results of allogeneic stem cell transplantation in nine patients (2 females and 7 males, median age 51, range 26-65) after reduced intensity conditioning consisting of busulfan, cladribine and alemtuzumab. The donors were HLA identical siblings (7) or unrelated (2). Peripheral blood stem cells mobilized with G-CSF were used. Cyclosporine alone was used for GvHD prevention after transplantation. RESULTS: 4 patients (44%) are alive at 24-42 months after transplantation and 3 patients (33%) remain in complete remission at 26-42 months, including one after donor lymphocyte infusion for decreasing hematopoietic chimerism. No acute graft versus host disease was observed and only 3 patients (33%) developed chronic graft versus host disease. High rate (56%) of CMV reactivation and 2 cases of tuberculosis were noted. All deceased patents died due to disease relapse and progression.
CONCLUSIONS: Reduced intensity conditioning with alemtuzumab and allogeneic stem cell transplantation offers potential cure to patients not eligible for conventional procedure. Better methodology of chimerism evaluation is mandatory to improve results of transplantations by implementation donor lymphocyte infusions.

Limited predictive value of real-time quantitative PCR cytomegalovirus monitoring in the blood. Fatal CMV pneumonia in an autologous stem cell transplant recipient previously treated with alemtuzumab.

BACKGROUND: Patients treated with alemtuzumab are at very high risk for cytomegalovirus (CMV) reactivation. Also, in those who develop reactivation short time before stem cell transplantation the risk of fatal complications is extremely high. CASE REPORT: We describe a 21-year-old patient with anaplastic large T-cell lymphoma who developed CMV reactivation after alemtuzumab treatment and received high-dose chemotherapy with autologous stein cell transplantation for progressive disease and severe bone marrow aplasia. Blood samples of the patient were tested regularly for CMV reactivation with real-time quantitative PCR. Even though it is considered the most sensitive available method it did not allow us to predict in advance development of fatal CMV pneumonia in this patient. CONCLUSIONS: The case report illustrates limitations of prognostic value of quantitative real-time PCR CMV assessment in blood samples.

Predictive value of RT-PCR PML-RARA transcript monitoring for extramedullary relapse of acute promyelocytic leukemia in the pleura, heart and pericardium after allogeneic SCT.

BACKGROUND: We report a patient with acute promyelocytic leukemia (APL) relapse in extremely rare sites--the pleura, heart and pericardium without evidence of bone marrow infiltration and with molecular evidence of disease after allogeneic stem cell transplantation (alloSCT). CASE DESCRIPTION: Presented patient underwent alloSCT in second complete hematological and cytogenetic remission with presence of promyelocytic leukemia-retinoic acid receptor A (PML-RARA) detected in reverse transcription-polymerase chain reaction (RT-PCR) with sensitivity of 10(-2). After transplant, this patient remained in complete hematological and cytogenetic remission but nested RT-PCR assays with detection thresholds of 10(-3)/10(-4) were positive for PML-RARA rearranged gene even chimerism tests showed 100% of donor profile. He was in a very good clinical condition and presented symptoms of transient limited chronic graft vs. host disease. Twenty one months after transplant, the leukemic relapse in the pleura, heart and pericardium was diagnosed. At that time, PML-RARA transcript detected in RT-PCR assay (10(-2)) was positive for the first time after transplant. During salvage chemotherapy he died because of cardiogenic shock. CONCLUSIONS: We conclude that detection of PML-RARA after alloSCT should be indication insightful diagnosis of medullary or extramedullary (EM) relapse. The imaging techniques of all possible sites of APL EM relapse have to be included.

[Quantitative real-time PCR--usefulness in detection and monitoring of CMV infection after hematopeietic stem cells transplant]. / Ulytecznosc ilosciowego badania technika real-time PCR do wykrywania i monitorowania zakaienia cytomegalowirusem po przeszczepieniu allogenicznych komórek krwiotwórczych.

UNLABELLED: It is recommended that all patients after allogeneic hematopoietic stem cell transplantation (alloHSCT) should be monitored for CMV infection markers. The aim of the study was to check the usefulness of quantitative DNA CMV monitoring after alloHSCT. MATERIAL AND METHOD: DNA CMV was tested by real-time PCR in sera and blood samples twice a week until 30th day after alloHSCT thereafter, once a week until 100th day and then, once every 2-3 weeks. 832 samples from 16 patients were tested. All patients were anti-CMV positive or/and received stem cells from seropositive donors. Introduction of antiviral treatment was based on initial viral load and its rate of increase. RESULTS: DNA CMV was detected in 13/16 patients; in 3 before 30h day after allo HSCT (group I) and in 10 (group II) after 30th day. In all patients from group I clinical symptoms were observed and DNA CMV was detected in sera and blood samples. Peak viral load was 2490-34 620 geq/ml. Although antiviral treatment was applied, reinfection was observed and infection lasted from 28 to 91 days. In 6 group II patients, clinical symptoms were observed and DNA CMV in sera and blood was detected for 16-56 days, DNA CMV peak load was 100-8950 geq/ml. In the remaining 4 patients, no clinical symptoms were observed--DNA CMV was detected in blood only for 7 to 14 days. In one patient with peak viral load 10,540 geq/ml, antiviral treatment was applied. In 3 with viral load of 400-2000 geq/ml, treatment was not introduced. The quantitative DNA CMV results were taken into account before the change of antiviral drugs for more effective drugs and the decrease of drug dose due to side effects. CONCLUSIONS: Application of quantititative DNA CMV testing allowed to optimise antiviral drug administration in immunosupressed patients after alloHSCT

Guillain-Barre syndrome--pathological connection with GvHD after allogeneic bone marrow transplantation.

The Guillain-Barre syndrome (GBS) could be a manifestation of neurotoxicity caused by multiple factors due to allogeneic bone marrow transplantations (alloBMT). In this paper we present a case of a 40-year old woman with chronic myeloid leukemia after alloBMT from her HLA identical brother. She developed the second grade of acute hepatic graft versus host disease (GvHD) shortly after alloBMT followed by chronic form. We observed Guillain-Barre syndrome probably triggering by GvHD in this patient.

[Autologous hematopoietic stem cell transplantation for the treatment of aggressive non-Hodgkin's lymphoma. The experience of Polish lymphoma research group]. / Autologiczna transplantacja komórek hematopoetycznych w leczeniu agresywnych chloniaków nieziarniczych Doswiadczenie Polskiej Grupy Badawczej Chloniaków (PLRG).

We analysed the outcome of 200 patients, aged 38 (13-72) years, with aggressive non-Hodgkin's lymphoma (NHL) treated with high-dose chemotherapy followed by autologous haematopoietic stem cell transplantation (autoHSCT) in ten oncohaematological centres belonging to Polish Lymphoma Research Group (PLRG). The source of stem cells for transplantation was peripheral blood (autoPBSCT, n = 153), bone marrow (autoBMT, n = 40) or both blood and bone marrow (n = 7). The probability of overall survival (OS) and progression-free survival at 10 years was 51% (+/- 7%). The transplant-related mortality rate equalled 7%. In multivariate analysis, the only factor influencing independently the probability of OS was disease status at transplantation (p < 0.00001). The outcome of patients transplanted in first or subsequent complete remission or first partial remission (PR) was significantly better compared with subjects given autoHSCT in PR 2 or those with primary or secondary refractoriness. Regarding histological subtypes, the highest OS rate (87%) was observed for anaplastic large T cell lymphoma. The outcome after autoBMT was better compared with autoPBSCT (OS probability: 67% vs. 43%), although the difference did not reach statistical significance. We conclude that high-dose therapy followed by autoHSCT is an effective option for high-risk aggressive NHL. Remission status is a major factor determining long-term outcome. This should be taken into account when referring patients for autoHSCT.