Agnieszka Tomaszewska

Role of Donor Activating KIR–HLA Ligand–Mediated NK Cell Education Status in Control of Malignancy in Hematopoietic Cell Transplant Recipients

Some cancers treated with allogeneic hematopoietic stem cell transplantation (HSCT) are sensitive to natural killer cell (NK) reactivity. NK function depends on activating and inhibitory receptors and is modified by NK education/licensing effect and mediated by coexpression of inhibitory killer-cell immunoglobulin-like receptor (KIR) and its corresponding HLA I ligand. We assessed activating KIR (aKIR)-based HLA I-dependent education capacity in donor NKs in 285 patients with hematological malignancies after HSCT from unrelated donors. We found significantly adverse progression-free survival (PFS) and time to progression (TTP) in patients who received transplant from donors with NKs educated by C1:KIR2DS2/3, C2:KIR2DS1, or Bw4:KIR3DS1 pairs (for PFS: hazard ratio [HR], 1.70; P = .0020, Pcorr = .0039; HR, 1.54; P = .020, Pcorr = .039; HR, 1.51; P = .020, Pcorr = .040; and for TTP: HR, 1.82; P = .049, Pcorr = .096; HR, 1.72; P = .096, Pcorr = .18; and HR, 1.65; P = .11, Pcorr = .20, respectively). Reduced PFS and TTP were significantly dependent on the number of aKIR-based education systems in donors (HR, 1.36; P = .00031, Pcorr = .00062; and HR, 1.43; P = .019, Pcorr = .038). Furthermore, the PFS and TTP were strongly adverse in patients with missing HLA ligand cognate with educating aKIR-HLA pair in donor (HR, 3.25; P = .00022, Pcorr = .00045; and HR, 3.82; P = .027, Pcorr = .054). Together, these data suggest important qualitative and quantitative role of donor NK education via aKIR-cognate HLA ligand pairs in the outcome of HSCT. Avoiding the selection of transplant donors with high numbers of aKIR-HLA-based education systems, especially for recipients with missing cognate ligand, is advisable.

Donor NK cell licensing in control of malignancy in hematopoietic stem cell transplant recipients

Among cancers treated with allogeneic hematopoietic stem‐cell transplantation (HSCT), some are sensitive to natural killer (NK) cell reactivity, described as the “missing self” recognition effect. However, this model disregarded the NK cell licensing effect, which highly increases the NK cell reactivity against tumor and is dependent on the coexpression of inhibitory killer cell immunoglobulin‐like receptor (iKIR) and its corresponding HLA Class I ligand. We assessed clinical data, HLA and donor iKIR genotyping in 283 patients with myelo‐ and lymphoproliferative malignancies who underwent HSCT from unrelated donors. We found dramatically reduced overall survival (OS), progression free survival (PFS), and time to progression (TTP) among patients with malignant diseases with the lack of HLA ligand cognate with this iKIR involved in NK cell licensing in corresponding donor (events 83.3% vs. 39.8%, P = 0.0010; 91.6% vs. 47.7%, P = 0.00010; and 30.0% vs. 17.3%, P = 0.013, for OS, PFS, and TTP, respectively). The extremely adverse PFS have withstand the correction when patient group was restricted to HLA mismatched donor‐recipient pairs. The incidence of aGvHD was comparable in two groups of patients. In malignant patients after HSCT the missing HLA ligand for iKIR involved in NK cell licensing in corresponding donor (“missing licensing proof”) induced extremely adverse survival of the patients due to the progression of malignancy and not to the aGvHD. Avoiding the selection of HSCT donors with the “missing licensing proof” in the malignant patient is strongly advisable.Am. J. Hematol. 89:E176–E183, 2014.

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.

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. β-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.

Infections due to alphaherpesviruses in early post-transplant period after allogeneic haematopoietic stem cell transplantation: Results of a 5-year survey

BACKGROUND Infections caused by human α-herpesviruses usually have a benign course with recurrencies. However, they may become dangerous in immunocompromised hosts. In this case, molecular methods constitute a reliable diagnostic tool enabling rapid assessment of the efficacy of antiviral treatment strategies. OBJECTIVES We estimated the frequency of alphaherpesviral DNAemia and the viral load during early post-transplantation period after alloHSCT; we also analyzed association of the DNAemia and chosen parameters of the patients. STUDY DESIGN A cohort of 190 alloHSCT recipients from two hospitals in Warsaw, Poland, was examined weekly during 100-day early post-transplantation period using quantitative real time PCR assays. A total of 2475 sera samples were evaluated for the presence of α-herpesviral DNA in patients, of whom 117 (62%) received unrelated grafts, while the remaining 73 (38%) received grafts from sibling donors. All patients received standard antiviral prophylaxis with acyclovir. In the examined group, anti-HSV-1, anti-HSV-2 and anti-VZV IgGs were examined prior to transplantation, RESULTS: Within the study period, DNA of α-herpesviruses was detected in 44 patients (23.2%). Most patients tested positive for HSV-1 DNA (43 patients, 22.6%), single patient for HSV-2, and no patient positive for VZV. Clinical symptoms such as pneumonia, skin changes, elevated levels of aminotransferases were observed in five patients, four of these patients presented symptoms of GvHD at the same time. (2,6%). Statistics shows that GvHD (P<0.001) and matched unrelated donor as a source of HSCT (P=0.048) are associated with the development of HSV-1 DNAemia. CONCLUSIONS Although our data demonstrate frequent reactivation of HSV-1 in the early post-transplant period, the rate of symptomatic infections was low. We did not find association between HSV-1 viremia and mortality, but significant association with GvHD and donor source was observed.

Analysis of the shedding of three β-herpesviruses DNA in Polish patients subjected to allogeneic hematopoietic stem cell transplantation: Six-year follow up.

BACKGROUND Infections with human β-herpesviruses are common worldwide and are still frequent in patients after hematopoietic stem cell transplantation. Some data suggest that HHV-6 and HHV-7 could take part in CMV reactivation from latency and/or progression of CMV disease in immunosupressed patients. OBJECTIVES The aims of this study were: (1) to summarise retrospectively the results of β-herpesviruses DNA detection in a large group of adult allogeneic haematopoietic stem cell transplant recipients; and (2) to find a potential correlation between viruses belonging to this subfamily. STUDY DESIGN AlloHSCT recipients (N=142) were examined in the early post-transplant period (median=89 days). The presence of CMV, HHV-6 and HHV-7 was confirmed through detection and quantification of viral DNA, isolated from 1679 sera samples. RESULTS CMV DNA alone was detected in 23.9% of patients, while single HHV-6 and HHV-7 were detected in 14.8% and 9.9% of individuals, respectively. The reactivation of more than one virus was identified in 31% of analysed patients. In cases of concurrent infection, HHV-7 was detected at the same time as HHV-6, and both of them were usually reactivated before CMV. The kinetics of virus reactivation and measured viral load may suggest a potential role of HHV-6 and HHV-7 as co-factors in CMV reactivation. CONCLUSIONS The observed kinetics of virus reactivation may strongly suggest a potential role of HHV-6 and/or HHV-7 as co-factors of CMV reactivation. The co-infection with these β-herpesviruses could predispose patients after hematopoietic stem cell transplantation to a longer and more severe CMV infection.

HLA-inferred extended haplotype disparity level is more relevant than the level of HLA mismatch alone for the patients survival and GvHD in T cell-replate hematopoietic stem cell transplantation from unrelated donor

Serious risks in unrelated hematopoietic stem cell transplantation (HSCT) including graft versus host disease (GvHD) and mortality are associated with HLA disparity between donor and recipient. The increased risks might be dependent on disparity in not-routinely-tested multiple polymorphisms in genetically dense MHC region, being organized in combinations of two extended MHC haplotypes (Ehp). We assessed the clinical role of donor-recipient Ehp disparity levels in N = 889 patients by the population-based detection of HLA allele phase mismatch. We found increased GvHD incidences and mortality rates with increasing Ehp mismatch level even with the same HLA mismatch level. In multivariate analysis HLA mismatch levels were excluded from models and Ehp disparity level remained independent prognostic factor for high grade acute GvHD (p = 0.000037, HR = 10.68, 95%CI 5.50-32.5) and extended chronic GvHD (p < 0.000001, HR = 15.51, CI95% 5.36-44.8). In group with single HLA mismatch, patients with double Ehp disparity had worse 5-year overall survival (45% vs. 56%, p = 0.00065, HR = 4.05, CI95% 1.69-9.71) and non-relapse mortality (40% vs. 31%, p = 0.00037, HR = 5.63, CI95% 2.04-15.5) than patients with single Ehp disparity. We conclude that Ehp-linked factors contribute to the high morbidity and mortality in recipients given HLA-mismatched unrelated transplant and Ehp matching should be considered in clinical HSCT.

Role of donor HLA class I mismatch, KIR-ligand mismatch and HLA:KIR pairings in hematological malignancy relapse after unrelated hematopoietic stem cell transplantation

HLA are functional in cancer immunosurveillance in adaptive and innate immunity pathways. In unrelated hematopoietic stem cell transplantation (HSCT) in 688 patients with hematological malignancies we compared antitumor efficacy of transplant in three models including the level of: (a) donor‐recipient HLA class I mismatch, (b) KIR‐ligand mismatch, (c) post‐transplant cognate HLA:KIR pairing. The effects were directly compared in multivariate models with backward elimination including all three effects in initial model. In final multivariate model HLA mismatch and KIR‐ligand mismatch levels were eliminated and HLA:KIR‐dependent NK cell licensing effect remained independent prognostic factor for DFS, relapse/progression incidence, and overall survival (OS). These results suggested that NK cell licensing via cognate HLA:KIR pairs is primarily functional in cancer immunosurveillance in HSCT.

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.

Monitorowanie zakażenia HHV-7 u pacjentów po transplantacji krwiotwórczych komórek macierzystych – ocena przydatności klinicznej

A. Tomaszewska *, T. Dzieciątkowski , S. Rynans , M. Przybylski , K. Halaburda , G. Mlynarczyk , W.W. Jedrzejczak , B. Marianska 1,4 1 Instytut Hematologii i Transfuzjologii, Klinika Transplantacji Komorek Krwiotworczych, Warszawa, Polska Katedra i Zaklad Mikrobiologii Lekarskiej WUM, Warszawa, Polska Miedzywydzialowe Studium Biotechnologii SGGW, Warszawa, Polska Katedra i Klinika Hematologii, Onkologii i Chorob Wewnetrznych WUM, Warszawa, Polska *Autor prezentujący i do korespondencji. Adres email: agnieszka_tomaszewska@onet.eu