Introduction: Axi-cel and tisa-cel are reimbursed for the treatment of LBCL relapsed/refractory (R/R) after at least two treatments; brexu -cel for R/R MCL failing a BTK inhibitor. Methods: The CART-SIE is a multicenter study collecting data on all consecutive lymphoma patients treated with CAR-T. The aim of this analysis was to evaluate outcome [overall response rate (ORR), overall survival (OS), progression free survival (PFS)], and safety [cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS)] of the different CAR-T products. Results: From 2019 to 2022, 499 patients were enrolled and leukapheresed; 426 infused patients with adequate follow-up were analyzed. Clinical characteristics were as follows: median age 57 years (IQR: 46–65), stage III/IV 300 (70%); median number of prior lines 2 (IQR: 2–3), 295 (69%) refractory to the last treatment. According to local pathology reports, 44 (10%) were MCL and 382 (90%) LBCL, including 236 (55%) DLBCL, 89 (21%) HGBCL, 57 (13%) PMBCL. Median follow-up time for infused patients was 10.99 months (IQR: 4.18–18.03). Brexu-cel was infused in 43/44 (98%) MCL; the ORR at 30-days was 33/44 (75%), with 24 (56%) CRs; the 12-months OS and PFS were 76% (95% CI: 56–100) and 59% (95% CI: 35–97). In the 382 LBCL, axi-cel was infused in 192 (50%), and tisa-cel in 190 (50%); the ORR at 30-days was 255/382 (67%), with 174 (46%) CRs; the 12-months OS and PFS were 71% (95% CI: 66–76) and 45% (95% CI: 39–50). By histotype, the 12-months OS was 89% (95% CI: 81–98) in PMBCL, 70% (95% CI: 64–78) in DLBCL, 58% (95% CI: 47–71) in HGBCL; the 12-months PFS was 65% (95% CI: 53–80) in PMBCL, 42% (95% CI: 35–50) in DLBCL, 38% (95% CI: 29–50) in HGBCL. In the whole population, all grade CRS was observed in 355/426 (83%) patients, with 47 (11%) severe (grade 3–4); ICANS in 103 (24%) patients, with 39 (9%) severe (grade 3–4). Tocilizumab was administered in 272 (64%) and steroids in 108 (25%); 46 patients (11%) were admitted in the intensive care unit. Treatment related deaths were 9 (2%). CAR-T expansion kinetics by flow cytometry was evaluated in a cohort of 150 patients with a positive correlation with disease response and survival (p-value 0.0076 and 0.036 respectively). A sub-analysis comparing axi-cel and tisa-cel was performed in the DLBCL and HGBCL cases (although unbalanced for histotypes and disease status); no differences between axi-cel and tisa-cel were reported in 12-months OS 71% (95% CI: 62–80) and 64% (95% CI: 56–73) (log-rank p = 0.6792); an advantage of axi-cel compared to tisa-cel was observed in 12-months PFS 47% (95% CI: 38–57) and 37% (95% CI: 30–45), respectively (log-rank p = 0.0354). Conclusions: The outcome and safety of patients treated with CAR-T were similar to those reported by other real life studies with a low non-relapse mortality. CR rate in MCL seems lower than in pivotal trial. Encore Abstract - previously submitted to EHA 2023 Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies Conflicts of interests pertinent to the abstract. A. Chiappella Consultant or advisory role: Celgene-BMS, Gilead-Sciences, Ideogen, Janssen, Roche, SecuraBIO, Takeda Other remuneration: Lecture fees/educational activities: Astrazeneca, Celgene-BMS, Gilead-Sciences, Incyte, Janssen-Cilag, Novartis, Roche, Takeda

Introduction: In Italy, axicabtagene ciloleucel (axi-cel) is commercially available for the treatment of LBCL, including diffuse large B-cell lymphoma (DLBCL-NOS), high grade B-cell lymphoma (HGBCL), transformed follicular lymphoma (tFL), and PMBCL patients, relapsed/refractory (R/R) after at least two prior treatments. Methods: The CART-SIE is an ongoing prospective and retrospective study collecting data on the outcome of all consecutive lymphoma patients treated with CAR-T cells. The aim of this analysis was to compare the outcome of PMBCL and LBCL treated with axi-cel in the Italian real life. Results: From 2019 to 2022, 444 patients were infused. Axi-cel was administered in 192 patients: 57 PMBCL and 135 LBCL, including DLBCL-NOS (85), HGBCL (28) and tFL (22), respectively. The clinical characteristics for PMBCL versus LBCL showed: median age 35 versus 56 (p = 0.0001), bulky 33/57 (58%) versus 47/135 (35%) (p = 0.0035), limited stage 35/57 (61%) versus 41/135 (31%) (p = 0.0001), respectively. Median follow-up time for infused patients was 10.99 months (IQR 4.18, 18.03). The Overall Response Rate (ORR, complete CR + partial PR) at 30-days after the infusion was 44/57 (77%) with 30 (53%) CR in PMBCL, and 97/135 (72%) with 67 (50%) CR in LBCL, (p = 0.4206). The 12-months Overall Survival (OS) was 89% (95% CI: 81–98) in PMBCL versus 70% (95% CI: 62–80) in LBCL (log-rank p = 0.0016); by different histology subtypes, the 12-months OS was 89% (95% CI: 81–98) in PMBCL versus 74% (95% CI: 63–86) in DLBCL-NOS, 58% (95% CI: 41–81) in HGBCL, 76% (95% CI: 58–100) in tFL (log-rank p = 0.0013). The 12-months PFS was 65% (95% CI: 53–80) in PMBCL versus 47% (95% CI: 39–57) in LBCL (log-rank p = 0.0160); by different histology subtypes, the 12-months PFS was 65% (95% CI: 53–80) in PMBCL versus 40% (95% CI: 30–54) in DLBCL-NOS, 51% (95% CI: 35–75) in HGBCL, 65% (95% CI: 47–90) in tFL (log-rank p = 0.0420). All grades CRS was observed in 49/57 (86%) PMBCL and 118/135 (87%) LBCL patients, with 9 (16%) and 12 (9%) severe (grade 3–4) CRS, respectively; all grades ICANS were reported in 25 (44%) PMBCL patients and in 46 (34%) LBCL, with 12 (21%) and 14 (10%) severe (grade 3–4) ICANS, respectively. Tocilizumab was administered in 41 (72%) PMBCL and in 90 (67%) LBCL; steroids in 20 (35%) PMBCL and in 36 (27%) LBCL patients. Twelve (21%) PMBCL and 15 (11%) LBCL patients were admitted in the intensive care unit. Treatment related mortality was reported in 3 (5%) PMBCL and 3 (2%) LBCL patients. In a multivariable model with all clinically important and unbalanced variables, PMBCL maintained its significantly superior outcome as compared to LBCL, for OS and for PFS. Conclusions: CART-SIE is the first real-life study comparing the prognosis of patients affected by R/R PMBCL and LBCL treated with axi-cel. PMBCL patients had a superior OS and PFS compared to LBCL, with a similar incidence of CRS and ICANS. Encore Abstract—previously submitted to EHA 2023 Keywords: aggressive B-cell non-Hodgkin lymphoma, cellular therapies Conflicts of interests pertinent to the abstract A. Chiappella Consultant or advisory role: Celgene-BMS, Gilead-Sciences, Ideogen, Janssen, Roche, SecuraBIO, Takeda Other remuneration: Lecture fees/Educational activities: Astrazeneca, Celgene-BMS, Gilead-Sciences, Incyte, Janssen-Cilag, Novartis, Roche, Takeda

Background: Overexpression of BCL2 and MYC, and translocation of MYC, BCL2, and BCL6, are associated with poorer outcomes in patients with diffuse large B-cell lymphoma (DLBCL; Horn et al. Blood 2013). We previously reported progression-free survival (PFS) from POLARIX in subgroups of patients with DLBCL receiving Pola-R-CHP or R-CHOP, including patients with double expressor lymphoma (DEL; favoring Pola-R-CHP: hazard ratio [HR] 0.64, 95% confidence interval [CI] 0.42–0.97), and double- or triple-hit lymphoma (DHL/THL; favoring R-CHOP: HR 3.81, 95% CI 0.82–17.64) (Tilly et al. NEJM 2022). Aims: This is a prespecified exploratory analysis, to further analyze immunohistochemistry (IHC) expression status of BCL2 and MYC, and rearrangements (R) of BCL2, BCL6 and MYC as independent prognostic markers. The prognostic impact of DEL, and BCL2 and MYC overexpression within treatment arms was also explored. Methods: BCL2 and MYC protein expression were assessed by IHC and identified as IHC+ (≥50% [BCL2]/≥40% [MYC]) or IHC−; MYC-R, BCL2-R, and BCL6-R were detected by fluorescence in situ hybridization (Tilly et al. NEJM 2022). All patients provided informed consent. Pola-R-CHP is an investigational combination. Exploratory multivariate Cox regression models were adjusted for treatment, stratification factors (International Prognostic Index, bulky disease, geographic region), age >60 years, cell of origin, and biomarker evaluated, as appropriate. Results: The prevalence, univariate HR, and 2-year PFS estimates of BCL2+/MYC+ and BCL2-R/MYC-R/BCL6-R subgroups are presented (Table) except for BCL6-R, because all four PFS events in this subgroup were with Pola-R-CHP. Multivariate analyses results for Pola-R-CHP vs R-CHOP in patients with BCL2+ (HR 0.60, 95% CI 0.43–0.86) and those with MYC+ (HR 0.63, 95% CI 0.45–0.89) were similar to results of univariate analyses. Multivariate analyses of other subgroups were not performed due to the low patient number with BCL2-R/MYC-R/BCL6-R. While patients with DEL treated with Pola-R-CHP had improved PFS vs patients treated with R-CHOP (HR 0.64, 95% CI 0.42–0.97), the prognostic impact of DEL vs non-DEL was identified in the R-CHOP arm (univariate HR 1.53, 95% CI 1.06–2.21; multivariate HR 1.29, 95% CI 0.88–1.91) vs the Pola-R-CHP arm (univariate HR 1.10, 95% CI 0.72–1.69; multivariate HR 1.42, 95% CI 0.89–2.28). Similarly, patients with BCL2+ had inferior PFS vs patients with BCL2– in the R-CHOP arm (univariate HR 1.96, 95% CI 1.31–2.93; multivariate HR 1.74, 95% CI 1.14–2.66); however, no prognostic difference was detected in the Pola-R-CHP arm (univariate HR 1.30, 95% CI 0.85–2.01; multivariate HR 1.56, 95% CI 0.99–2.47). No prognostic impact of MYC+ vs MYC– was detected in either arm (R-CHOP: univariate HR 1.17, 95% CI 0.77–1.77; multivariate HR 1.00, 95% CI 0.65–1.55; Pola-R-CHP: univariate HR 0.83, 95% CI 0.54–1.27; multivariate HR 0.91, 95% CI 0.58–1.42). Image:Summary/Conclusion: Multivariate analyses support the benefit of Pola-R-CHP in patients with BCL2+ and MYC+ DLBCL. The poor prognostic impact associated with DEL, which was mainly driven by BCL2+, appears reduced in Pola-R-CHP- vs R-CHOP-treated patients.

Introduction: Axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) are anti-CD19 chimeric antigen receptor T cells (CAR-T) that proved very effective in relapsed/refractory (R/R) diffuse large B-cell (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL). to evaluate safety and efficacy of axi-cel and tisa-cel. Methods: The Italian Society of Hematology (SIE) is conducting a prospective observational trial aimed to: 1. register all DLBCL and PMBCL patients (pts) candidate to CAR-T in the Italian authorized centers; 2. evaluate the intention to treat overall response rate (ORR, complete [CR] and partial response [PR]), duration of response (DOR), progression free survival (PFS) and overall survival (OS); 3. evaluate safety in terms of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and long-term cytopenia; 5. evaluate different CAR-T products. The study was approved by ethics committees. Results: Since March 2019 to January 2021, 126 pts were leukapheresed and 113 infused. Clinical characteristics of the 113 infused pts were: median age 53 years (19-70), stage III/IV 77 (68%), 81 (72%) primary refractory; median number of prior lines was 3 (2-7), including 33 (29%) prior autologous stem cell transplantation. For histologies, 59 (52%) were DLBCL, 18 (16%) high-grade B-cell (HGBCL), 23 (20%) PMBCL, 13 (12%) transformed Follicular (tFL). Bridging therapy was delivered to 97 pts (86%) and all pts received lymphodepletion. Fifty-nine (52%) infused axi-cel and 54 (48%) tisa-cel. Median follow-up time for infused pts was 6.9 months (IQR: 3.13-11.78). At 30-days after the infusion, all the pts were evaluable for response: 45 (40%) CR, 35 (31%) PR, with ORR of 71%. For the evaluable pts, DOR was 73% (95%CI:62-85) at 6-months and 58% (95%CI:44-77) at 12-months. In the whole series, 6 and 12-months PFS were 54% (95% CI:45-65) and 46% (95%CI:35-59); 6 and 12-months OS were 80% (95%CI:71-88) and 75% (95%CI:65-86), respectively. With the limitation of small number, 6-months PFS were 48% for DLBCL, 62% for HGBCL, 66% for PMBCL and 59% for tFL; 6-months OS by histotype were 78% for DLBCL, 80% for HGBCL, 80% for PMBCL and 92% for tFL. No differences between axi-cel and tisa-cel were reported. Severe (grade 3-4) CRS was observed in only 6 (5%) pts, and severe ICANS in 11 (10%). Sixty-one (54%) pts received tocilizumab and 38 (34%) steroids. Cytopenia beyond 30 days was reported in 30 (27%) pts; 27 of them (24%) experienced viral or bacterial infections. No toxic deaths were recorded. Conclusions: In the real-life, axi-cel and tisa-cel showed an ORR similar to those of the registrative trials, even if almost all pts underwent bridging therapy. The response rate is similar across histotypes and between products. CAR-T toxicity is manageable, relapse beyond 6 months is a rare event. Cytopenias as well as infections are an emerging problem in real-life setting. EA – previously submitted to EHA 2021. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies, Immunotherapy No conflicts of interests pertinent to the abstract.

Background:Despite the overall good prognosis of follicular lymphoma (FL), approximately 20 to 30% of the cases experience early progression after first line therapy and suffer adverse survival (Casulo et al., 2015; Shi et al., 2017). Several efforts have been made to identify pre‐treatment factors useful to assess prognosis, however currently available parameters do not allow to accurately stratify patients before treatment. Lymphocyte to monocyte ratio (LMR) has been previously identified as an useful prognostic markers in diffuse large B‐cell lymphoma treated with Rituximab‐based chemotherapy (Rambaldi et al., 2013).Aims:In this study we aimed to evaluate the potential role of LMR in identifying patients with FL experiencing early progression after first line therapy.Methods:831 patients with a diagnosis of grade 1–3a FL were identified in the Bergamo Lymphoid Cancer Registry (NCT03131531) between 1983 to 2017. Cases managed with watchful observation after diagnosis (N = 145) or receiving local therapy (N = 121) or without adequate clinical information or follow‐up data (N = 117) were excluded from the analysis. The remaining patients (N = 448) with stage II‐IV disease and receiving first line chemotherapy at diagnosis were included in the study. The majority of the patients were treated with Rituximab‐based therapy (72%). Chemotherapy regimens included CHOP (N = 298), CVP (N = 76), Bendamustine (N = 22), Chlorambucil (N = 34) or others (N = 18). Rituximab maintenance was administered to 61 patients.Patients with early progression were defined as those with a PFS event before 30 months from first‐line treatment start (PFS30). Receiver operating characteristic (ROC) analysis was performed to assess the optimal cut‐off of LMR to predict PFS30. Kaplan‐Meyer curves, log‐rank tests and Cox models were used for survival analyses.Results:In the study population, 26% of the patients experienced early progression and were characterized by a shorter median OS compared to the other group (6.3 vs 17 years, P < 0.0001, Figure 1A). The optimal cut‐off of LMR to predict PFS30 was identified, with patients with LMR ≤2 or >5.2 (N = 176) being at higher risk for a PFS event compared to those with LMR >2 and ≤5.2 (N = 272) (Figure 1B). In multivariate analysis for PFS, LMR retained its significance independently from FLIPI (P = 0.0001). The simple addition of LMR to standard FLIPI permitted to refine patient prognosis in terms of PFS (Figure 1C) and OS (Figure 1D), with three groups identified i.e. low risk (FLIPI low and favorable LMR), intermediate risk (FLIPI low and unfavorable LMR; FLIPI int or high and favorable LMR) and high risk (FLIPI int or high and unfavorable LMR). Specifically, 57 (36%) patients with intermediate FLIPI were upgraded in the high risk category and 80 (54%) high risk FLIPI were downgraded into the intermediate one. Patients in the new high risk category were characterized by a 42% probability to have a PFS event before 30 months, compared to the 22% and 10% of the intermediate and low risk group, respectively. LMR was significantly associated with OS in univariate analysis (P = 0.047), but not in multivariate analysis with FLIPI (P = 0.11). The combined LMR and FLIPI index predicted survival with a 30‐months OS of 96%, 94% and 85% in the low, intermediate and high risk category, respectively (P = 0.0032).Summary/Conclusion:LMR is a significant prognostic factor for PFS in FL, independent from FLIPI and closely related to the use of Rituximab. The combination of LMR with currently available prognostic markers can be useful in identifying high patient before the start of first line treatment.image

This phase II, single-arm, multicenter study examined the efficacy and safety of coltuximab ravtansine (an anti-CD19 antibody drug conjugate) in 61 patients with histologically documented (de novo or transformed) relapsed or refractory diffuse large B-cell lymphoma who had previously received rituximab-containing immuno-chemotherapy. Patients had received a median of 2.0 (range 0-9) prior treatment regimens for diffuse large B-cell lymphoma and almost half (45.9%) had bulky disease (≥1 lesion >5 cm) at trial entry. Patients received coltuximab ravtansine (55 mg/m2) in 4 weekly and 4 biweekly administrations until disease progression or unacceptable toxicity. Forty-one patients were eligible for inclusion in the per protocol population. Overall response rate (International Working Group criteria) in the per protocol population, the primary end point, was 18/41 [43.9%; 90% confidence interval (CI:) 30.6-57.9%]. Median duration of response, progression-free survival, and overall survival (all treated patients) were 4.7 (range 0.0-8.8) months, 4.4 (90%CI: 3.02-5.78) months, and 9.2 (90%CI: 6.57-12.09) months, respectively. Common non-hematologic adverse events included asthenia/fatigue (30%), nausea (23%), and diarrhea (20%). Grade 3-4 adverse events were reported in 23 patients (38%), the most frequent being hepatotoxicity (3%) and abdominal pain (3%). Eye disorders occurred in 15 patients (25%); all were grade 1-2 and none required a dose modification. Coltuximab ravtansine monotherapy was well tolerated and resulted in moderate clinical responses in pre-treated patients with relapsed/refractory diffuse large B-cell lymphoma. (Registered at: clinicaltrials.gov identifier: 01472887).

Introduction: The Phase III GALLIUM study (NCT01332968) showed that obinutuzumab (GA101; G) significantly prolonged PFS in previously untreated follicular lymphoma (FL) pts relative to rituximab (R) when combined with chemotherapy (chemo; CHOP, CVP or bendamustine [B]). Grade 3–5 AEs and SAEs were more common with G-chemo. Updated results for each immunochemotherapy regimen are reported here. Methods: Pts were aged ≥18 years with documented, previously untreated FL (grades 1–3a), advanced disease (stage III/IV or stage II with tumour diameter ≥ 7 cm), ECOG PS 0–2, and requiring treatment according to GELF criteria. Chemo regimen was allocated by centre. Pts were randomised 1:1 (stratified by chemo, FLIPI-1 group and geographical region) to R 375 mg/m2 on day (D) 1 of each cycle (C) or G 1000 mg on D1, 8 and 15 of C1 and D1 of C2–8, for 6 or 8 cycles depending on chemo. Pts with CR or PR at end of induction (per Cheson 2007) continued to receive R or G every 2 months for 2 years or until progression. The cut-off date for this analysis was 10 September 2016. All pts gave informed consent. Conclusions: In treatment-naive FL pts, PFS was superior with G-chemo relative to R-chemo with consistent effects across chemo regimens. Some differences were seen in safety profiles between chemo regimens, but comparisons may be confounded by the lack of randomisation. Keywords: follicular lymphoma (FL); obinutuzumab; rituximab.

The clinical outcome of T-cell non-Hodgkin lymphoma (NHL) is poor and innovative treatments are needed. Tenascin-C is a large extracellular glycoprotein not expressed under physiological conditions, but overexpressed in cancer. Aim of the study was to evaluate tenascin-C expression within pathologic tissue of T-cell NHL and determine its clinical significance. We used an immunohistochemistry approach using the anti-tenascin-C monoclonal antibody Tenatumomab in 75 systemic T-cell NHL (including 72 mature and 3 precursor T-cell NHL), and 25 primary cutaneous T-cell NHL. Data were analyzed in terms of staining intensity, proportion of involved areas and histologic pattern, and results were correlated with clinical characteristics and outcome. Ninety-three percent of the cases were tenascin-C positive and 59% of systemic diseases were characterized by a predominant involvement (>50%). Stromal expression was detected in all the cases while vascular and vascular plus cytoplasmic expression was present in 49% and 23%. The constant overexpression of the tenascin-C gene was observed in two independent publicly available T-cell NHL gene expression datasets. In conclusions, tenascin-C represents an attractive target that sets the rationale to investigate the therapeutic activity of radiolabeled Tenatumomab in T-cell NHL.

In Italy, the incidence of invasive meningococcal disease (IMD) has remained stable since 2007 (around 0.3 cases/100,000 inhabitants). However, as reported for other European countries, an increase of serogroup Y Neisseria meningitidis has been observed. In this study we report IMD cases from 2007 to 2013 in Italy and investigate the clinical and epidemiological features of cases affected by serogroup Y. Molecular characteristics of serogroup Y strains are also described. During the study period, the proportion of IMD cases due to serogroup Y increased, ranging from 2% in 2007 to 17% in 2013 (odds ratio (OR): 8.8), whereby the five to 14 years age group was mostly affected (p < 0.001). Overall 81 serogroup Y IMD cases were identified, with a median age of 18 years, ranging from three months to 84 years. Of the 81 respective patient samples, 56 were further subject to molecular typing. The sequence type (ST)-23 complex (clonal complex (cc) 23) was predominant among serogroup Y meningococci (54/56 samples), and included nine different STs. Presumably, ST-23 was the founding genotype, with all the other STs presenting as single-locus variants. All cc23 isolates analysed harboured mutations in the lpxL1 gene; however, no associations among lpxL1 mutations, ST and age group were identified. Overall, these findings generate scientific evidence for the use of the quadrivalent meningococcal conjugate vaccine in the five to 14 years age group.

We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC50 of 0.2 μ M. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-γ by MSCs by 80–95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.

Objective. Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells.Materials and Methods. Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span, granulocyte macrophage colony-stimulating factor, interleukin-4, and calcium ionophore. All B-precursor ALL (N22) and AML (N13), but not T-cell ALL (M), differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2).Results. Upon in vitro culture, donor-derived CTL acquired a memory T phenotype, showing concomitant high CD45RA, CD45RO, CD62L expression. CD8(+) cells, but not CD4(+) cells, were granzyme, perforine, and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%, 30:1 E:T ratio), but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL, patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%, 1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern.Conclusions. Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Graft-vs-leukemia reactivity after donor lymphocyte infusion (DLI) can be mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on recipient hematopoietic cells. To study the diversity of cells involved in this immune response, hematopoietic cell reactive T cells were directly clonally isolated from peripheral blood of patients entering complete remission after DLI. T cells were briefly stimulated with bone marrow cells from patients pretransplant, and IFNgamma-secreting T cells were directly clonally isolated, and expanded. Cytotoxic T-lymphocyte (CTL) clones from individual patients used multiple distinct HLA-restricting molecules and varied in reactivity against patient-derived normal and/or malignant hematopoietic cells. For each patient, CTL clones specific for known immunodominant mHags as well as distinct unknown mHags were found. Within individual patients, CTL clones using the same HLA-restricting element could show differential recognition patterns, indicating further diversity in mHag reactivity. CTL clones from individual patients exhibiting identical specificities could show oligoclonal origin. In conclusion, the direct cloning technique shows that the response to hematopoietic cells after DLI is directed against multiple distinct mHags, including but not limited to known immunodominant mHags, implying that immunotherapy with T cells against multiple mHag specificities may be more effective in eradicating malignant cells.

ITF2357 (Italfarmaco, Italia) is a novel hydroxamic acid-based HDAC inhibitor (HDACi) that has shown reduced toxicity in phase I studies. We have investigated the cytotoxic and anti-proliferative activities of ITF2357 in multiple myeloma (MM) and acute myelogenous leukaemia cells (AML). ITF2357 had a strong cytotoxic activity in 8/9 MM and 6/6 AML cell lines, with a mean IC50 of about 0.2 μM, a concentration largely attained following oral administration of safe doses of ITF2357 to healthy individuals. In contrast SAHA, the prototypic hydroxamic HDACi, showed an IC50 of about 1 μM or above in all cases. The cytotoxic activity of ITF2357 was due to induction of apoptosis, as documented by detection of annexin V and cleaved caspase 3. The ITF2357 induced hyperacetylation of histones in cell lines resistant or sensitive to the cytoytoxic activity of the drug is under investigation in order to further define its mechanism of action. ITF2357 had also more potent cytotoxic activity compared to SAHA against freshly isolated CD138+ purified MM cells and AML samples, with an IC50 of about 0.1 μM in 3/3 MM and 13/15 AML cases. Sensitive AML cases included five cases of FAB M1, five M2 and three M4. Three of the sensitive cases carried a t(8;21) translocation, 2 an inv(16), 6 had a normal and 2 a complex karyotype. Furthermore four of the sensitive AML also carried a flt3 internal tandem duplication and 3 a type A mutation in the nucleophosmin 1 gene (NPM1). The 2 more resistant AML cases (one M1 and one M5, both with normal karyotype) showed nonetheless a response to the drug with an IC50 of about 0.5 μM. We have also developed a culture system to grow freshly isolated AML cells for at least 3 weeks in vitro on human mesenchymal cells (MSC). Interestingly, ITF2357 was cytotoxic for primary AML cells stimulated to grow in optimal conditions on MSCs, at the same dose as in standard short term cultures. In contrast ITF2357 was not cytotoxic for MSCs even at the 1 μM concentration. The strong cytotoxic activity of ITF2357 on MM and AML has provided the framework for ongoing phase I studies of ITF2357 in these malignancies.

SummaryGemtuzumab ozogamicin (GO) is a humanized anti‐CD33 antibody conjugated with the cytotoxic drug calicheamicin and approved for the treatment of relapsed acute myeloid leukaemia. As approximately 18% of acute lymphoblastic leukaemias (ALL) are also CD33 positive, we have investigated the cytotoxic activity of GO on CD33+ ALL cells in vitro and in vivo. 10 ng/ml GO induced 30–95% inhibition of thymidine uptake and 30–70% cell death in four freshly isolated and one in vivo passaged CD33+ ALL‐cell cultures. Furthermore, an in vivo model of a CD33+ ALL carrying the Philadelphia chromosome [t(9;22)] was established. 5 × 106 ALL‐2 cells inoculated in the tail vein of severe combined immunodeficient mice engrafted into haematopoietic organs, reaching a mean of 70%, 61% and 69% human CD45+ cells in bone marrow, spleen and liver, respectively, at 35 d. To test the therapeutic activity of GO, 50 or 100 μg immunotoxin was inoculated i.p. on days 7, 11 and 15 following tumour‐cell inoculation. GO treatment dramatically inhibited expansion of ALL‐2 cells in all tested organs and increased survival of tumour‐injected animals by 28–41 d, relative to controls. These data demonstrated that GO is active both in vitro and in vivo against CD33+ ALL cells.

Donor T cells recognizing hematopoiesis-restricted minor histocompatibility antigens (mHags) HA-1 and HA-2 on malignant cells play a role in the antileukemia effect of donor lymphocyte infusion (DLI) in patients with relapsed leukemia after allogeneic stem cell transplantation. We quantified the contribution of HA-1 and HA-2 specific T cells to the total number of leukemia-reactive T cells in three HA-2 and/or HA-1 positive patients responding to DLI from their mHag negative donors. Clinical responses occurring 5-7 weeks after DLI were accompanied by an increase in percentages HLA-DR expressing T cells within the CD8+ T cell population. To clonally analyze the leukemia-reactive immune response, T cells responding to the malignancy by secreting IFNgamma were isolated from peripheral blood, directly cloned, and expanded. Tetramer analysis and specific lysis of peptide-pulsed target cells showed that 3-35% of cytotoxic T lymphocyte (CTL) clones isolated were specific for HA-1 or HA-2. TCR VB analysis showed oligoclonal origin of the HA-1 and HA-2 specific CTL clones. The HA-1 and HA-2 specific CTL clones inhibited leukemic progenitor cell growth in vitro. The relatively high frequency of HA-1 and HA-2 specific T cells within the total number of tumor-reactive T cells illustrates relative immunodominance of mHags HA-1 and HA-2.

BACKGROUND AND OBJECTIVES One of the few measurable clinical results obtained by the use of somatic cells in onco hematology is the clear-cut effect donor leukocyte infusions (DLI) in patients with chronic myeloid leukemia (CML) who relapse after an allogeneic bone marrow transplantation (BMT). From then on much research has focused on the use of cells to treat different aspects of oncologic diseases from leukemia relapse to development in BMT recipients. METHODS AND INFORMATION SOURCES In this review we critically and schematically summarize the cell-based therapies which have led to a clinical application and recapitulate the results. RESULTS AND STATE OF THE ART Although the overall numbers of successfully treated patients is small, therapy has been shown to be safe and effective in a variety of clinical contexts in oncohematology. PERSPECTIVES Preliminary data will have to be validated in well designed clinical trials with cells generated by reproducible methods and in accreditated structures working according to Good Manufacturing Practices (GMP).