ALBERT

Description: Background. The prognostic role of cell of origin profile (COO) assessed by immunohistochemistry (IHC) is controversial in Rituximab era. FIL conducted a phase III randomized trial aimed at investigating the benefit of intensification with high dose therapy plus autotransplant compared to R-dose-dense therapy as first line in young DLBCL at poor risk (aa-IPI 2-3). Clinical results were reported (Vitolo, ASH 2012). The aim of BIO-DLCL04 was to correlate the biological markers with PFS. Patients and Methods. From 2005 to 2010, 412 untreated DLBCL at aa-IPI 2-3 were enrolled. Central histology revision was mandatory and 13 patients were excluded due to different histologies. Biological markers were analyzed on DLBCL NAS; COO analysis was performed by IHC and cases were classified in germinal center (GC) and non-GC according to Hans' algorithm; COO determined by gene expression profile using the NanoString® nCounter® Analysis System based on 20-gene assay (Lymph2Cx) using formalin fixed paraffin embedded tissue is ongoing; BCL2, BCL6 and MYC anomalies were tested by IHC; final analysis by fluorescent in situ hybridization (FISH) is ongoing. Cases were deemed positive if at least 30% of lymphoma cells were stained with each antibody (with the exception of at least 40% for MYC). Results. At the time of this analysis, 223 DLBCL NAS were analyzed: 131 non-GC and 92 GC; BCL2, BCL6 and MYC anomalies were tested in 196, 74 and 107 cases respectively. Clinical characteristics for non-GC vs GC were: median age 51 years for both, male 49% vs 45%, aa-IPI 3 15% vs 25%, bone marrow involvement (BM) 16% vs 24%. R-HDC was performed in 45% of non-GC patients and in 49% of GC. Complete response was recorded in 105 (80%) non-GC patients and in 62 (67%) GC. At a median follow-up of 49 months, the 3-year PFS for non-GC vs GC was 75% (95% CI: 67-82) vs 57% (95% CI: 46-67) with crude hazard ratio, HR 0.55 (0.35-0.87), p.01 and adjusted (for age, gender, aa-IPI, BM) aHR 0.56 (0.35-0.88), p.013. No significant differences by treatment were reported. Overexpression of MYC by IHC had a relevant prognostic impact, with aHR 1.84 (0.99-3.44), p.054. By IHC, 3-years PFS for double negative vs single BCL2 or MYC overexpression vs double positive, was 85% vs 68% vs 51% respectively, with an aHR for double expressors compared to double negative of 3.91 (1.13-13.53), p.031. At the time of the present report, FISH analysis was conducted in 88 cases: 43 were triple negative, 37 single hit and 8 double/triple hit. By FISH, 3-years PFS for triple negative vs single hit vs double/triple hit was 74% vs 84% vs 25% respectively, with an aHR for double/triple hit compared to triple negative of 5.73 (2.05 to 16.02), p.001. Conclusions. In conclusion, with the limit of the analysis performed by IHC based on Hans' algorithm, BIO-DLCL04 showed an unexpected better outcome for non-GC compared to GC, irrespective of treatment arm. The ongoing analysis conducted by Nanostring will be more informative. The overexpression of MYC was an unfavourable risk factor, mainly if associated with BCL2 overexpression, irrespective of type of treatment. Moreover, double/triple hit patients represent a subgroup with extremely poor prognosis. High dose therapy plus autotransplant was not able to reverse the inferior outcome of neither double expressors nor double hit patients and new strategies are deemed for these poor prognosis patients. Disclosures No relevant conflicts of interest to declare.

Description: Background. Follicular lymphoma (FL) is usually classified as grade (G) I, II or III. Grade III is further subdivided into GIIIa and GIIIb, the latter almost exclusively consisting of CBs. However, this distinction is questioned. Recently, gene expression studies showed that clinical aggressiveness can be associated with specific molecular signatures partially independent from histological grade and that immune reactive cells can play a major role in the outcome determinism. However, to date, gene expression studies did not provide molecular rationale for histological grading and it is still debated if including FL GIIIb within FL or DLBCL. We studied the gene expression profile (GEP) of 43 FLs, 50 B-cell non-Hodgkin lymphomas (B-NHLs) of different histotypes, and 20 samples of normal B-lymphocytes in order to assess: the relationship of FL with normal B-cells and other B-NHLs; whether FL is a unique disease; and whether FL GIIIb is closer to FL or diffuse large B-cell lymphoma of the germinal center B-cell type (GCB-DLBCL). Methods. Forty-three FL cases were analyzed. Of these, 37 corresponded to cryopreserved tissue blocks and 6 to enriched neoplastic cells All the samples were obtained at the time of diagnosis, before treatment administration. In addition, samples of normal B-cell sub-populations including CB (N=5), centrocytes (CC, N=5), naïve, N (N=5), and memory cells, M (N=5) were studied. Finally, a panel of B-NHLs was analyzed including Burkitt’s lymphoma (BL, N=4), DLBCL, (N=16), mantle cell lymphoma (MCL, N=10), hairy cell leukemia (HCL, N=10), and B-cell chronic lymphocytic leukemia (B-CLL, N=10). Finally, in silico data concerning 37 cases of germinal center B-cell type (GCB) DLBCL were retrieved at http://www.ncbi.nlm.nih.gov/projects/geo/. For proper comparison with our samples, gene expression values were normalized “per gene” and “per chip”. For microarray analysis, fragmented cRNA was hybridized to HG-U133 2.0 plus microarray. Results. First, we found that the molecular profile of FL is intimately related to that of normal germinal centre B-cells, irrespectively of the histological grade. However, interestingly, several cell programs are regulated as in memory cells. Secondly, we observed that FL has a relatively homogeneous GEP that is distinct from that of other B-NHLs and does not include discrete molecular subgroups. However, by further clustering samples according to signatures differentially expressed among FLs or in FL vs. DLBCL, we showed that GI-IIIa tumors tend to cluster together, while GIIIb FL constitutes a distinct subgroup. Finally, we found that the molecular signature of GIIIb FL is indeed closer to that of the other FLs than to the one of GCB-DLBCL. These data support the hypothesis that GIIIb FL belongs to FL rather than DLBCL, and sustain the possible revision of FL histological grading, with the simple distinction into FL (GI-IIIa) and FL/large cell (GIIIb).

Description: In myeloid malignancies, the neoplastic clone outgrows normal hematopoietic cells toward BM failure. This event is also sustained by detrimental stromal changes, such as BM fibrosis and osteosclerosis, whose occurrence is harbinger of a dismal prognosis. We show that the matricellular protein SPARC contributes to the BM stromal response to myeloproliferation. The degree of SPARC expression in BM stromal elements, including CD146+ mesenchymal stromal cells, correlates with the degree of stromal changes, and the severity of BM failure characterizing the prototypical myeloproliferative neoplasm primary myelofibrosis. Using Sparc−/− mice and BM chimeras, we demonstrate that SPARC contributes to the development of significant stromal fibrosis in a model of thrombopoietin-induced myelofibrosis. We found that SPARC deficiency in the radioresistant BM stroma compartment impairs myelofibrosis but, at the same time, associates with an enhanced reactive myeloproliferative response to thrombopoietin. The link betwen SPARC stromal deficiency and enhanced myeloid cell expansion under a myeloproliferative spur is also supported by the myeloproliferative phenotype resulting from the transplantation of defective Apcmin mutant hematopoietic cells into Sparc−/− but not WT recipient BM stroma. Our results highlight a complex influence of SPARC over the stromal and hematopoietic BM response in myeloproliferative conditions.

Description: Abstract 3601 Richter syndrome (RS) represents the development of diffuse large B-cell lymphoma (DLBCL) in the context of chronic lymphocytic leukemia (CLL). Despite the availability of clinical predictors, the identification of RS patients projected to experience long survival represents an unresolved issue that may benefit from the development of novel RS prognosticators. The study was based on 86 RS classified as DLBCL after pathological review. Clonal relationship between CLL/RS pairs was assessed by immunoglobulin gene analysis. RS was clonally related to the CLL phase in 49/59 (83.1%) cases, and clonally unrelated in 10/59 (16.9%). In 27 cases, clonal relationship remained undetermined because of unavailability of paired CLL samples. Out of the molecular features investigated, the prevalence of TP53 disruption by mutations and/or deletion was significantly higher in clonally related RS (23/36, 63.9%) compared to clonally unrelated cases (1/8, 12.5%) (p=.015). Among immunogenetic features, usage of stereotyped VH CDR3 occurred in 25/49 (51.0%) clonally related RS, while was very rare among clonally unrelated cases (1/10, 10.0%) (p=.032). The difference in stereotyped VH CDR3 frequency occurred irrespective of IGHV gene mutation status. All clonally unrelated RS tested negative for EBV infection and none received alemtuzumab before DLBCL development. Clonally related and clonally unrelated RS were indistinguishable in terms of clinical features at presentation. However, RS survival was significantly shorter in clonally related (median: 14.2 months) versus clonally unrelated cases (median: 62.5 months) (p=.017) (Fig. 1A). This survival difference was observed even though clonally related RS received SCT in 8/48 (16.7%) cases, while clonally unrelated RS received in all cases conventional chemotherapy without SCT consolidation. Other variables that were associated with poor RS survival were TP53 disruption (p60 years (p=.005), ECOG PS 〉1 (p5 cm (p=.001), platelets 1.5 ULN (p=.002), and having received SCT (p=.027). None of the pathologic features of DLBCL diagnostic biopsies correlated with RS prognosis. After adjusting for clinical variables associated with RS survival, diagnosis of clonally unrelated RS translated into a 80% reduction in risk of death when compared to clonally related RS (HR: 0.22; p=.036). By recursive-partitioning analysis, clonal relationship, TP53 disruption and ECOG PS were the major determinants of RS survival, and classified RS patients according to risk of death (Fig. 1B). All clonally unrelated RS displayed a low risk of death (median survival 62.5 months; 95% CI: 33.7–91.3), irrespective of TP53 disruption and ECOG PS (Fig. 1B; Group 1). Among clonally related RS and RS with undetermined clonal relationship, patients presenting with ECOG PS ≤1 and no TP53 disruption had a low risk of death (median survival: 55.4 months) (Fig. 1B; Group 2). Patients presenting with ECOG PS ≤1 in the presence of TP53 disruption had an intermediate risk of death (median survival: 27.0 months) (Fig. 1B; Group 3). Patients presenting with ECOG PS 〉1 had a high risk of death, irrespective of TP53 disruption (median survival: 7.8 months) (Fig. 1B; Group 4). Based on c-statistics, the model including clonal relationship, ECOG PS and TP53 status had a 90% probability of correctly discriminating a priori RS survival (c-statistic=.90±.07). These observations suggest that clonally related and clonally unrelated RS are biologically and clinically distinct disorders, and that clonally unrelated RS should be considered as a secondary DLBCL arising de novo in the context of CLL, rather than a true RS. Therefore, we propose that the diagnosis of RS should be restricted to clonally related cases. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3879 Purpose. The use of early (interim) positron emission tomography (PET) restaging during front-line therapy in Hodgkin's lymphoma (HL) has considerably increased in clinical practice as an early recognition of treatment failure allows patients to be addressed to more intensive treatment regimens. Patients and Methods. Between June 1997 and June 2009, 304 newly-diagnosed Hodgkin's lymphoma patients (147 early-stage and 157 advanced-stage) were treated with the ABVD regimen at two Italian institutions. Patients underwent to a PET staging and restaging at baseline, after 2 cycles of therapy and at the end of the treatment. Results. 53 patients showed a positive interim PET and only 13/53 (24.5%) achieved a complete response (CR), whereas 251 patients showed a negative PET and 231/251 (92%) remained in CR. Comparison between interim PET-positive and interim PET-negative patients indicated a significant association between PET findings and 9-year progression-free survival (p=0.0000) and 9-year overall survival (p=0.0000), with a median follow-up of 31 months. Among the early-stage patients, 19 had a positive interim PET and only 4 (21%) achieved a CR; among the 128 negative interim PET patients, 122 (97.6%) obtained a CR. In the advanced-stage subset, 34 patients showed a persistently positive PET (with only 9/34, 26.4% in CR), whereas 123 showed a negative interim PET, with 109 (88.6%) remaining in CR. Conclusions. Our results confirm the role of early PET as a significant step forward for the management of both early and advanced-stage HL patients, offering the potential for an immediate switch to high-dose treatments, if required. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2650 Background and Rationale: The T-Cell Project (TCP) aims at verifying if a prospective collection of data in patients with Peripheral T-cell lymphomas (PTCLs) provides more accurate information to better define their prognosis. So far 885 patients have been accrued and diagnosis confirmed in 84% of the 350 reviewed cases. A dedicated online pathology form collecting a detailed biomarkers profile is filled out by research staff at peripheral sites with data from the local pathologist report. However, it is very difficult for people who are not accustomed to routinely reading pathology reports to interpret the findings correctly. To guarantee the data capture of biomarkers reproduces the pathology reports a control of quality of information entered in the pathology forms of the TCP was carried out. A similar evaluation was performed by the COMPLETE Registry, presenting the results in a parallel abstract. Methods: Biomarker data quality assessment concerns the review by an expert pathologist of peripheral capture of a suggested quite wide panel of biomarkers (54) used to diagnose patients registered in the TCP. The first 104 patients enrolled having complete registration data, availability of the original pathology report at the Trial Office and its data entered at the website constitute the sample of this analysis. A single mismatch between the site-entered data and the reviewer's findings, recorded on a separate forms, counts as an error. Results: On the whole, 5740 entries were reviewed, with a mean number of 9 immunophenotypic markers (range 3–22) and a mean number of 0.3 (range 0–4) gene rearrangement tests. In 35 (34%) cases out of the 104 reviewed no conflicting entry between what the site entered and what the reviewer determined was found. Patient disagreement of different extent was determined for the remaining cases: 1–2 errors, 35 (34%); 3–5 errors, 20 (19%); 6–10 errors, 8 (8%); 11–19 errors, 6 (5%). Where the site noted a finding was positive the reviewer was in agreement 73% of the time, noted they were negative in 15%, indeterminate in 1% and not assessed in 11%. For cases where the site noted a finding was negative the reviewer agreed with 97% of cases, noted they were positive in 1%, indeterminate in 0% and not assessed in 2%. The reviewer was in agreement with the site in 96% of the cases when the marker was indicated peripherally as not assessed, and for the remaining tests found the marker was positive, negative or indeterminate in 1%, 2% and 0% of cases respectively. The markers most difficult to interpret (at least 5% of total errors) are listed in the Table, reporting also the types of errors noted. With respect to the T-cell markers, the misreporting is mainly due to the difficulty in recognizing and thus entering the findings on the pathology report. For the suggested B-cell markers, a high rate of errors concerns the coding of the findings of the pathology report noted in the non-neoplastic populations surrounding the neoplastic cells. Of relevance, the very frequent mistaken interpretation of the EBV in situ hybridization (ISH), recorded as the result of the EBV immunoistochemistry test. Gene rearrangement studies are often missing and if present almost totally misinterpreted by the site, and reported as an immunoistochemistry test. The review by the expert pathologist accomplished a 11% of errors due to an ambiguous noting of the findings on the pathology report. Conclusion: The results of the biomarker quality assessment for the TCP confirm the difficulty for a correct interpretation of the pathology report in a relatively high rate of cases. For international projects the need for a periodic review emerges to guide site training and improve the accuracy of biomarker data capture in order to ensure database quality. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) cells may be differentiated into dendritic cells (DC) which have increased immunogenicity, but retain some immunosuppressive features of leukemic cells. Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed AML patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. We, then, in vitro generated DCs from 7 AML samples (AML-DCs) in the presence of GM-CSF, IL-4 and TNF-α. The cells we obtained were morphologically and phenotypically semi-mature DCs expressing CD40, CD80, CD86, HLA-DR and CD1a molecules and they were more efficient to induce T-cell proliferation and type 1 cytokine production than primary AML blasts. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. These data identify IDO-mediated catabolism as a tolerogenic mechanism in AML-DCs and have clinical implications for the use of AML-DCs as cellular vaccine against leukemia.

Description: Introduction The role of bone marrow (BM) involvement as prognostic factor in untreated young patients with diffuse large B-cell lymphoma at poor prognosis is still a matter of debate. Recent data showed an adverse prognostic role of BM involvement in DLBCL including patients both at low and high IPI score (Sehn L et al, J Clin Oncol 2011). On this basis, FIL analyzed the impact of BM involvement in the prospective randomized phase III trial DLCL04 (Vitolo U et al, Blood, ASH annual Meeting 2012) that included only young patients at high-risk age-adjusted IPI (aa-IPI) score 2 or 3. Patients and Methods Inclusion criteria were: age 18-65; untreated DLBCL or follicular grade IIIb; aa-IPI score 2 or 3. Patients were stratified according to aa-IPI and randomized at diagnosis to receive: R-CHOP14 x 8 cycles; R-MegaCHOP14 (1200 mg/sqm Cyclophosphamide, 70 mg/sqm Doxorubicin, standard dose Vincristine and Prednisone) x 6; R-CHOP14 x 4 + R-HDC (Rituximab + High Dose Cytarabine + Mitoxantrone + Dexamethasone) followed by BEAM and ASCT; R-MegaCHOP14 x 4 + R-HDC + BEAM and ASCT. BM biopsy and aspirate were mandatory at diagnosis; at the end of treatment, BM assessment was mandatory only in case of positivity at baseline. BM involvement was defined as concordant if marrow was involved by large B-cell and discordant if involved by small B-cells. Flow cytometry, immunohistochemistry, and/or molecular studies were utilized to confirm a clonal B-cell population. Results From June 2005 to September 2010, 399 patients were randomized to receive: 199 R-HDC+ASCT and 200 R-dose-dense chemotherapy without ASCT. All patients were evaluable for analysis. BM involvement was reported in 84 patients (21%): 39 (20%) in the R-HDC+ASCT group and 45 (22%) in the R-dose-dense chemotherapy group. Pattern of involvement was: concordant in 63 patients, discordant in 14 and not specified in 7 patients. Patients with BM involvement (BM positive: 84) compared to those without BM involvement (BM negative: 315) were significantly older (median age 53 years vs 47 years, p1 43% vs 45%, bulky 25% vs 33%, extranodal sites 〉 1 26% vs 33%, LDH higher than normal value 93% vs 89%). With a median follow-up of 49 months, 3-year PFS for the whole series of 399 patients enrolled in the trial was: 67% (95% CI: 62-72). Three-year PFS was significantly worse in BM positive vs. BM negative: 46% (95% CI:35-56%) vs. 73% (95% CI:67-77%) p