Endoscopic pancreatic sphincterotomy in patients with IPMN-related recurrent pancreatitis: A single center experience.

BACKGROUND: Acute recurrent pancreatitis (ARP) is a rare manifestation of Intraductal Papillary Mucinous Neoplasms (IPMN) of the pancreas; ARP is a relative indication for pancreatic surgery in the setting of IPMN. Endoscopic pancreatic sphincterotomy (EPS) has been described as a minimal invasive treatment to reduce the episodes of ARP secondary to mucus migration in IPMN. METHODS: patients with IPMN-related ARP treated with ESP from January 2004 to December 2020 were retrospectively selected. Clinical and technical data were recorded. A clinical follow-up (minimum 12 months) was performed to assess the number of episodes of AP occurring after EPS. RESULTS: 25 patients were included. The mean follow-up after ESP was 93.4 months (SD± 56.6). The mean number of AP before and after EPS were respectively 3.29 (SD ± 1.04) and 0.51 (SD ± 0.71). A complete response (no further episodes of AP) and a partial response (>50% reduction of AP episodes) were obtained in 64% and 24% of the cases, respectively, with an overall response rate of 88%. One post-EPS bleeding and one minor-papilla stenosis were reported and were endoscopically managed. Two patients underwent pancreatic resection for the occurrence of high-risk stigmata for cancer progression. CONCLUSIONS: EPS is a safe and effective treatment to reduce the number of episodes of AP in selected patients with IPMNs-related ARP. Prospective trials are needed to confirm these data.

The impact of the multidisciplinary tumor board (MDTB) on the management of pancreatic diseases in a tertiary referral center.

BACKGROUND: The implementation of multidisciplinary tumor board (MDTB) meetings significantly ameliorated the management of oncological diseases. However, few evidences are currently present on their impact on pancreatic cancer (PC) management. The aim of this study was to evaluate the impact of the MDTB on PC diagnosis, resectability and tumor response to oncological treatment compared with indications before discussion. PATIENTS AND METHODS: All patients with a suspected or proven diagnosis of PC presented at the MDTB from 2017 to 2019 were included in the study. Changes of diagnosis, resectability and tumor response to oncological/radiation treatment between pre- and post-MDTB discussion were analyzed. RESULTS: A total of 438 cases were included in the study: 249 (56.8%) were presented as new diagnoses, 148 (33.8%) for resectability assessment and 41 (9.4%) for tumor response evaluation to oncological treatment. MDTB discussion led to a change in diagnosis in 54/249 cases (21.7%), with a consequent treatment strategy variation in 36 cases (14.5%). Change in resectability was documented in 44/148 cases (29.7%), with the highest discrepancy for borderline lesions. The treatment strategy was thus modified in 27 patients (18.2%). The MDTB brought a modification in the tumor response assessment in 6/41 cases (14.6%), with a consequent protocol modification in four (9.8%) cases. CONCLUSIONS: MDTB discussion significantly impacts on PC management, especially in high-volume centers, with consistent variations in terms of diagnosis, resectability and tumor response assessment compared with indications before discussion.

Fungal infections of the central nervous system and paranasal sinuses in onco-haematologic patients. Epidemiological study reporting the diagnostic-therapeutic approach and outcome in 89 cases.

Invasive fungal infections (IFI) of the Central Nervous System (IFI-CNS) and Paranasal Sinuses (IFI-PS) are rare, life-threatening infections in haematologic patients, and their management remains a challenge despite the availability of new diagnostic techniques and novel antifungal agents. In addition, analyses of large cohorts of patients focusing on these rare IFI are still lacking. Between January 2010 and December 2016, 89 consecutive cases of Proven (53) or Probable (36) IFI-CNS (71/89) and IFI-PS (18/89) were collected in 34 haematological centres. The median age was 40 years (range 5-79); acute leukaemia was the most common underlying disease (69%) and 29% of cases received a previous allogeneic stem cell transplant. Aspergillus spp. were the most common pathogens (69%), followed by mucormycetes (22%), Cryptococcus spp. (4%) and Fusarium spp. (2%). The lung was the primary focus of fungal infection (48% of cases). The nervous system biopsy was performed in 10% of IFI-CNS, and a sinus biopsy was performed in 56% of IFI-PS (P = 0.03). The Galactomannan test on cerebrospinal fluid has been performed in 42% of IFI-CNS (30/71), and it was positive in 67%. Eighty-four pts received a first-line antifungal therapy with Amphotericine B in 58% of cases, Voriconazole in 31% and both in 11%. Moreover, 58% of patients received 2 or more lines of therapy and 38% were treated with a combination of 2 or more antifungal drugs. The median duration of antifungal therapy was 60 days (range 5-835). A surgical intervention was performed in 26% of cases but only 10% of IFI-CNS underwent neurosurgical intervention. The overall response rate to antifungal therapy (complete or partial response) was 57%, and 1-year overall survival was 32% without significant differences between IFI-CNS and IFI-PS. The overall mortality was 69% but the IFI attributable mortality was 33%. Mortality of IFI-CNS/PS remains high but, compared to previous historical data, it seems to be reduced probably due to the availability of newer antifungal drugs. The results arising from this large contemporary cohort of cases may allow a more effective diagnostic and therapeutic management of these very rare IFI complications in haematologic patients.

Bloodstream infections in haematological cancer patients colonized by multidrug-resistant bacteria.

Infections by multidrug-resistant (MDR) bacteria are a worrisome phenomenon in hematological patients. Data on the incidence of MDR colonization and related bloodstream infections (BSIs) in haematological patients are scarce. A multicentric prospective observational study was planned in 18 haematological institutions during a 6-month period. All patients showing MDR rectal colonization as well as occurrence of BSI at admission were recorded. One-hundred forty-four patients with MDR colonization were observed (6.5% of 2226 admissions). Extended spectrum beta-lactamase (ESBL)-producing (ESBL-P) enterobacteria were observed in 64/144 patients, carbapenem-resistant (CR) Gram-negative bacteria in 85/144 and vancomycin-resistant enterococci (VREs) in 9/144. Overall, 37 MDR-colonized patients (25.7%) developed at least one BSI; 23 of them (62.2%, 16% of the whole series) developed BSI by the same pathogen (MDRrel BSI), with a rate of 15.6% (10/64) for ESBL-P enterobacteria, 14.1% (12/85) for CR Gram-negative bacteria and 11.1% (1/9) for VRE. In 20/23 cases, MDRrel BSI occurred during neutropenia. After a median follow-up of 80 days, 18 patients died (12.5%). The 3-month overall survival was significantly lower for patients colonized with CR Gram-negative bacteria (83.6%) and VRE (77.8%) in comparison with those colonized with ESBL-P enterobacteria (96.8%). CR-rel BSI and the presence of a urinary catheter were independent predictors of mortality. MDR rectal colonization occurs in 6.5% of haematological inpatients and predicts a 16% probability of MDRrel BSI, particularly during neutropenia, as well as a higher probability of unfavourable outcomes in CR-rel BSIs. Tailored empiric antibiotic treatment should be decided on the basis of colonization.

Preference for subcutaneous or intravenous administration of rituximab among patients with untreated CD20+ diffuse large B-cell lymphoma or follicular lymphoma: results from a prospective, randomized, open-label, crossover study (PrefMab).

Background: The aim of this study was to evaluate patient preference and satisfaction for the subcutaneous (s.c.) versus intravenous (i.v.) formulation of rituximab given with chemotherapy in previously untreated patients with CD20+ diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL). Patients and methods: Patients received eight cycles of rituximab according to 2 schedules: Arm A received 1 cycle rituximab i.v. (375 mg/m2) and 3 cycles rituximab s.c. (1400 mg) then 4 cycles rituximab i.v.; Arm B received 4 cycles rituximab i.v. (375 mg/m2) then 4 cycles rituximab s.c. (1400 mg). Alongside rituximab, both arms received 6-8 cycles of chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP), cyclophosphamide, vincristine, prednisone (CVP), or bendamustine as per standard local practice). Preference for s.c. or i.v. administration was evaluated using the Patient Preference Questionnaire (PPQ) at cycles 6 and 8. Patient satisfaction and convenience were assessed using the Cancer Therapy Satisfaction Questionnaire (CTSQ), and Rituximab Administration Satisfaction Questionnaire (RASQ) at cycles 4 and 8. Results: At the primary data cut-off (19 January 2015), the intent-to-treat population comprised 743 patients. The majority had DLBCL (63%) and baseline characteristics were balanced between arms. At cycle 8, 81% of patients completing the PPQ preferred rituximab s.c. Preference was not impacted by treatment sequence or disease type. Patient satisfaction as measured by RASQ was higher for s.c. versus i.v. CTSQ scores were similar between arms. Adverse events were generally balanced between administration routes and no new safety signals were detected. Conclusion: Most previously untreated patients with CD20+ DLBCL or FL preferred s.c. to i.v. rituximab administration. Patient satisfaction with rituximab treatment was generally greater with s.c. administration. Registered clinical trial number: NCT01724021 (ClinicalTrials.gov).

Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

PML is required for telomere stability in non-neoplastic human cells.

Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RARα in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis.

Osteolytic lesions, cytogenetic features and bone marrow levels of cytokines and chemokines in multiple myeloma patients: Role of chemokine (C-C motif) ligand 20.

The relationship between bone marrow (BM) cytokine and chemokine levels, cytogenetic profiles and skeletal involvement in multiple myeloma (MM) patients is not yet defined. This study investigated a cohort of 455 patients including monoclonal gammopathy of uncertain significance (MGUS), smoldering MM and symptomatic MM patients. Skeletal surveys, positron emission tomography (PET)/computerized tomography (CT) and magnetic resonance imaging (MRI) were used to identify myeloma bone disease. Significantly higher median BM levels of both C-C motif Ligand (CCL)3 and CCL20 were found in MM patients with radiographic evidence of osteolytic lesions as compared with those without, and in all MM patients with positive PET/CT scans. BM levels of CCL3, CCL20, Activin-A and Dickkopf-1 (DKK-1) were significantly higher in patients with high bone disease as compared with patients with low bone disease. Moreover, CCL20 BM levels were significant predictors of osteolysis on X-rays by multivariate logistic analysis. On the other hand, DKK-1 levels were related to the presence of MRI lesions independently of the osteolysis at the X-rays. Our data define the relationship between bone disease and the BM cytokine and chemokine patterns highlighting the tight relationship between CCL20 BM levels and osteolysis in MM.

Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients.

Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.

Haploidentical hematopoietic stem cell transplantation: state of art.

For patients with hematologic malignancies at high risk of relapse who do not have matched donors, a suitable alternative stem cell source is the HLA-haploidentical 2- or 3-loci mismatched family donor who is readily available for nearly all patients. Transplantation across the major HLA barrier is associated with strong T-cell alloreactions, which were originally manifested as a high incidence of severe GVHD and graft rejection. The present overview of the 7th symposium on haplidentical transplantation that took place at the Weizmann Institute on February 2014, shows how these obstacles to successful transplantation can now be overcome. The review also discusses the advantages and drawbacks of current options for full haplotype-mismatched transplantation and highlights innovative approaches for rebuilding immunity, reducing leukemia relapse and improving survival after transplantation. In addition, new modalities for immune tolerance induction following nonmyeloablative conditioning are discussed, showing new options for treatment of elderly patients who cannot tolerate myeloablative conditioning protocols, as well as novel strategies for immune tolerance and chimerism induction as a platform for cell therapy and organ transplantation.

T-cell depletion: from positive selection to negative depletion in adult patients.

Clinical trials have shown that a strategy for haploidentical transplantation based on the infusion of high numbers of T-cell-depleted hematopoietic progenitor cells and no post-transplant immunosuppression controls graft rejection and GvHD in patients with acute leukemia. Overall, event-free survival compares favorably with reports of transplants using sources of stem cells other than the matched sibling. Current studies are focussing on rebuilding post-transplant immunity to improve clinical outcomes separating GvHD from favourable donor immune responses.

Mutations and long-term outcome of 217 young patients with essential thrombocythemia or early primary myelofibrosis.

We investigated the influence of molecular status on disease characteristics and clinical outcome in young patients (⩽ 40 years) with World Health Organization (WHO)-defined essential thrombocythemia (ET) or early/prefibrotic primary myelofibrosis (early-PMF). Overall, 217 patients with ET (number 197) and early-PMF (number 20) were included in the analysis. Median follow-up time was 10.2 years. The cumulative incidence of thrombosis, hemorrhages and disease evolution into myelofibrosis/acute leukemia were 16.6%, 8.6% and 3% at 15 years, respectively. No differences were detectable between ET and early-PMF patients, although the latter cohort showed a trend for worse combined-event free survival (EFS). Mutation frequency were 61% for JAK2V617F, 25% for CALR and 1% for MPLW515K, and were comparable across WHO diagnosis; however, JAK2V617F allele burden was higher in the early-PMF group. Compared with JAK2V617F-positive patients, CALR-mutated patients displayed higher platelet count and lower hemoglobin level. CALR mutations significantly correlated with lower thrombotic risk (9.1% versus 21.7%, P = 0.04), longer survival (100% versus 96%, P = 0.05) and better combined-EFS (86% versus 71%, P = 0.02). However, non-type 1/type 2 CALR mutations ('minor' mutations) and abnormal karyotype were found to correlate with increased risk of disease evolution. At last contact, six patients had died; in five cases, the causes of death were related to the hematological disease and occurred at a median age of 64 years (range: 53-68 years). Twenty-eight patients (13%) were unmutated for JAK2, CALR and MPL: no event was registered in these 'triple-negative' patients.

In haematopoietic SCT for acute leukemia TBI impacts on relapse but not survival: results of a multicentre observational study.

The aim of this study was to determine whether parameters related to TBI impacted upon OS and relapse in patients with acute leukemia in CR who underwent haematopoietic SCT (HSCT) in 11 Italian Radiation Oncology Centres. Data were analysed from 507 patients (313 males; 194 females; median age 15 years; 318 with ALL; 188 with AML; 1 case not recorded). Besides 128 autologous transplants, donors included 192 matched siblings, 74 mismatched family members and 113 unrelated individuals. Autologous and allogeneic transplants were analysed separately. Median follow-up was 40.1 months. TBI schedules and HSCT type were closely related. Uni- and multi-variate analyses showed no parameter was significant for OS or relapse in autologous transplantation. Multivariate analysis showed type of transplant and disease impacted significantly on OS in allogeneic transplantation. Disease, GVHD and TBI dose were risk factors for relapse. This analysis illustrates that Italian Transplant Centre use of TBI is in line with international practice. Most Centres adopted a hyperfractionated schedule that is used worldwide (12 Gy in six fractions over 3 days), which appears to have become standard. TBI doses impacted significantly upon relapse rates.

Hypoxia-inducible factor (HIF)-1α suppression in myeloma cells blocks tumoral growth in vivo inhibiting angiogenesis and bone destruction.

Hypoxia-inducible transcription factor-1 (HIF-1α) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1α inhibition by a lentivirus short hairpin RNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1α suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1α inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1α. The effect of HIF-1α inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1α inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1α inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1α suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1α as a potential therapeutic target in MM.

Hema e-Chart registry of invasive fungal infections in haematological patients: improved outcome in recent years in mould infections.

The electronic surveillance system Hema e-Chart allowed us to prospectively collect data and to perform an analysis of invasive fungal infections (IFI) diagnosed in febrile patients as well as the procedures allowing their diagnosis and outcome according to the treatment given. Every patient admitted to 26 Italian Haematology Units with a new diagnosis of haematological malignancy and who was a candidate for chemotherapy was consecutively registered between March 2007 and March 2009. In all, 147 haematological patients with mycoses were identified. Yeasts were found in 23 infections; moulds were diagnosed in 17 proven, 35 probable and 72 possible mycoses. Galactomannan (GM) antigen was the most important test to diagnose probable mould infection; it was positive (cut-off >0.5) in 27 (77%) probable and in nine (53%) proven mould infections. Among patients with probable/proven mould infection who received no prophylaxis or non-mould-active prophylaxis with fluconazole, more patients (n = 26, 78.8%) had GM antigen positivity compared with patients (n = 10, 52.6%) given prophylaxis with mould-active drugs (p <0.05). First-line antifungal therapy was effective in 11/23 (48%) yeast infections and in 37/52 (71.2%) proven/probable mould infections. Twenty patients (14%) died within 12 weeks. The fungal attributable mortality was 30.4% and 17.3% in yeast and proven/probable mould infections, respectively. Among risk factors only age was independently associated (p 0.013) with mortality; sex, underlying haematological malignancy, previous prophylaxis and presence of neutropenia at diagnosis were not significant. A diagnosis of mould infection seemed to have a trend for a better outcome than the diagnosis of yeast infection (p 0.064).

Myeloma cells inhibit non-canonical wnt co-receptor ror2 expression in human bone marrow osteoprogenitor cells: effect of wnt5a/ror2 pathway activation on the osteogenic differentiation impairment induced by myeloma cells.

Multiple myeloma (MM) is characterized by the impaired osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Canonical Wnt signaling is critical for the regulation of bone formation, however, recent evidence suggests that the non-canonical Wnt agonist Wnt5a stimulates human osteoblastogenesis through its co-receptor Ror2. The effects of MM cells on non-canonical Wnt signaling and the effect of the activation of this pathway on MM-induced osteoblast exhaustion are not known and were investigated in this study. We found that the osteogenic differentiation of bone marrow hMSCs toward osteoprogenitor cells (PreOB) significantly increased Ror2 expression, and that MM cells inhibit Ror2 expression by PreOB in co-culture by inhibiting the non-canonical Wnt5a signaling. The activation of the non-canonical Wnt pathway in hMSCs by means of Wnt5a treatment and the overexpression of Wnt5 or Ror2 by lentiviral vectors increased the osteogenic differentiation of hMSCs and blunted the inhibitory effect of MM in co-culture. Consistently, Wnt5a inhibition by specific small interfering RNA reduced the hMSC expression of osteogenic markers. Our findings demonstrate that the Wnt5a/Ror2 pathway is involved in the pathophysiology of MM-induced bone disease and that the activation of the non-canonical Wnt5a/Ror2 pathway in hMSCs increases osteogenic differentiation and may counterbalance the inhibitory effect of MM cells.

Folate in gastrointestinal health and disease.

OBJECTIVE: Folate has heterogeneous functions and is involved in several activities in both animal and human body. It is an important constituent of our organism, and its bioavailability is mainly dependent from the correct function of our gastrointestinal tract. Our aim is to describe what happens to folate homeostasis in gastrointestinal health and disease, analyzing the alterations of folate metabolism in some specific conditions of intestinal and liver impairment. DISCUSSION: Folate absorption and metabolization involve the small intestine and the liver; in conditions of gastrointestinal tract disease (i.e. celiac disease, liver disease) folate function may be compromised with important consequences on the whole organism. Moreover, folate deficiency may produce gastrointestinal alterations too. For this reason, the gastrointestinal tract could be the responsible but also the victim of folate deficiency. CONCLUSIONS: The presence of folate deficiency should always be assessed in patients with a gastrointestinal disease. Further studies are needed to assess the role of folates in gastrointestinal tract diseases and in other gynecologic, neurologic, psychiatric, cardiovascular, ophthalmic and neoplastic diseases. Folates supplementation could be considered, in the future, as an effective complimentary therapy in several pathologic conditions.

Optimizing a photoallodepletion protocol for adoptive immunotherapy after haploidentical SCT.

In adults, one-haplotype-mismatched haematopoietic SCT (haploidentical HSCT) is associated with slow immune recovery due to decaying thymic function and extensive T-cell depletion of the graft. Although essential for preventing GVHD, T-cell depletion underlies the major reasons for transplant failure: leukemia relapse and infections, with infection-related mortality accounting for about 40% of non-leukemic deaths. Adoptive T-cell therapy would be helpful for these patients but to administer it without causing GVHD, alloreactive T cells need to be eliminated from donor T lymphocytes before infusion. In a preclinical study, to address this problem, we determined the efficacy of photodynamic purging of alloreactive T cells, by investigating combinations of parameters in order to achieve maximum allodepletion, preservation of T-regulatory cells and of pathogen and leukemia-specific T-cell responses in donor-vs-recipient MLR. We also needed to identify an optimal method to quantify the Ag-specific T-cell repertoires. Optimal procedures were identified. In particular, we compared limiting-dilution analyses (LDA) of proliferating T cells with H(3)-thymidine incorporation by bulk T cells and with flow cytometry CD25 expression, which is accepted as a T-cell activation marker. This study demonstrated that LDA is a reliable, predictable and sensitive method for measuring alloreactive, pathogen- and leukemia-specific T-cell frequencies.