ALBERT

Description: Saliva is an interesting, non-conventional, valuable diagnostic fluid. It can be collected using standardized sampling device; thus, its sampling is easy and non-invasive, it contains a variety of organic metabolites that reflect blood composition. The aim of this study was to validate a user-friendly method for the simultaneous determination of low molecular weight metabolites in saliva. We have optimized and validated a high throughput, direct, low-cost reversed phase liquid chromatographic method with diode array detection method without any pre- or post-column derivatization. We indexed salivary biomolecules in 35 whole non-stimulated saliva samples collected in 8 individuals in different days, including organic acids and amino acids and other carbonyl compounds. Among these, 16 whole saliva samples were collected by a single individual over three weeks before, during and after treatment with antibiotic in order to investigate the dynamics of metabolites. The concentrations of the metabolites were compared with the literature data. The multianalyte method here proposed requires a minimal sample handling and it is cost-effectiveness as it makes possible to analyze a high number of samples with basic instrumentation. The identification and quantitation of salivary metabolites may allow the definition of potential biomarkers for non-invasive “personal monitoring” during drug treatments, work out, or life habits over time.

Description: Abstract 3945 Poster Board III-881 Introduction Non Hodgkin Lymphoma could be clinically divided as low grade/indolent NHL (LG NHL) and high grade/aggressive NHL (HG NHL). These diseases are chemo and radio-sensitive and improvements have been achieved by immunotherapeutic approaches. However some patients will relapse and a follow up strategy has to be planned in order to detect and treat them. Several aspects should be considered in planning a follow up including safety, specificity, sensitivity, costs and impact on the patient's psychology: an optimal follow up should mediate between these ones. The more diffuse follow up have been planned years before the introduction of innovative methods and imaging techniques, suggesting the opportunity to revise these programs. Methods We collected data about 418 NHL patients -both low and high grade- treated at our institution from 1990 to 2005 who achieved a complete remission according to Cheson criteria and who entered a follow up program which schedule is planned for 5 years divided in two periods: first two years evaluation every 3 months and in the following three years every sixth month. At each visit physical examinations, blood testing (blood count, chemistry) are performed; for imaging techniques we alternate a whole body CT scans to ultrasounds and chest X-ray coupled. Analyzing time to relapse (TTR), we tried to optimize our follow up schedule trough a computation application known as multi-objective analysis. The first step of this method has been to choose and try to quantify the costs of a follow up which reflect its effectiveness. We considered as costs the expected time between relapse and its detection (Ca) and the expected number of performed examinations before failure or censoring occurs (Cb). The total follow up costs could be summarized in a vector Cref: (Ca,Cb). After doing that we described survival analysis, relapse rate and their onset time in order to be suitable for the informatic analysis. We used a log logistic parametric model to do that. Next we shaped our ideal follow up as a structure based one, which is currently the most used in medical practice: a first period where examinations are more tightly spaced, followed by a second period where they are performed further apart. We describe such follow-up schedule “S” as (d1, k, d2) : d1 is the time between the first k examinations; d2 is the time between the remaining examinations. Applying the log-logistic model we calculated the Cref and schedule for our follow up: Cref was (Ca,Cb)=(8.5,6.2); this means that, on average, every patient entering the follow-up will perform 6.2 examinations, and among those who incur in a relapse, the relapse will be detected 8.5 units of time (eg. weeks) after its onset. The follow up structure was (13,8,26). We then calculated all the possible combination of (d1, k, d2) values from 1 to d1 = 25, k= 24 and d2 = 60. Results 360000 follow up schedules had been detected after searching all the possible combinations for d1, k and d2. For each one Ca e Cb costs have been calculated and than compared by multi-objective analysis to those of the current schedule, We look for both follow up structured and “free” follow up, where “free” means that intervals between examination is continuously variable. When comparing follow-up schedules, we apply the rule of Pareto-dominance: the schedule S1 is superior to the S2 schedule if and only if the cost values for S1 are both lower than the cost values for S2. The method then takes into account only those schedules which improve the current one, with respect to both the Ca and the Cb costs. After multi-objective analysis six follow up were detected. These were as following: (16,10,22), (15,8,20), (15,6,19), (16,8,19), (16,5,18), (16,4,18) and are shown in Fig. 1. Conclusions no differences in follow up schedules emerged when we considered separately LG and HG lymphoma patients. Maximum improving of our follow was just 4% and all the new schedules had wide time frequency visit in the first period and a narrow one in the second period: this was a consequence of the higher relapse distribution in the first period and this follow up organization lead to a lower total number of visit without risk of lose any relapse. This is the first application of this analysis to hematological patients confirming the validity of a follow up schedule should be shaped in two different period. Disclosures: No relevant conflicts of interest to declare.

Description: Allogeneic stem-cell transplantation is a potential curative option in multiple myeloma (MM). Reduced-intensity conditioning regimens (allo-RIC) result in a lower transplant related mortality (TRM) compared to conventional conditioning, despite of a higher relapse rate. Several prospective studies compared single or tandem autologous stem cell transplantation (SCT) with planned tandem autologous-reduced intensity allogeneic SCT, with discordant results in overall and progression-free survival (OS and PFS). Many studies were conducted using a “mini-allo-SCT”, a regimen containing a low-dose total body irradiation (TBI) and Fludarabine (Flu). Moreover, introduction of new drugs (bortezomib, thalidomide or lenalidomide) in the first decade of 2000 changed the biological history of MM. We analyzed the results of ten-year experience with mini-allo-SCT in patients with MM in our institution. Patients, materials and methods: Between June 2000 and December 2010, 21 patients (9 M, 12 F, median age 54 – range 36-66) received a mini-allo-SCT, 17 from an HLA identical sibling donor, 4 from a MUD full-matched. The source of stem cell was the peripheral blood in all patients. All grafts were not manipulated. At the time of diagnosis, Durie-Salmon (DS) stage was I in 5 patients (23.8%), II in 3 patients and III in 13 patients (61.9%). Disease status at the time of transplant was partial response (PR) in 17 patients (81%), 13 of them in first PR, 4 in second or more PR; 4 patients received allo-SCT as salvage therapy in active ore refractory disease. Eleven patients (52.4%) underwent to transplant after one line of treatment, 5 patients after 2 lines, 5 patients after 3 or more lines. Five patients (23.8%) were treated with new drugs. Auto-SCT is included in previous lines. Nine patients received one auto-SCT before the mini-allo-SCT; ten patients (47.6%) underwent transplantation after two auto-SCT. Two patients were allo-grafted frontline. Conditioning regimen was Flu-TBI in all patients. Graft versus Host Disease (GvHD) prophylaxis consisted on cyclosporine and MMF in all. Results: Overall response rate was 76%, 5 PR and 11 complete remission (CR). One patient developed progression next allo-SCT. Four patients died in the first 100 days after allo-SCT, and they are censored for OS and PFS analyses. Of 17 pre-transplant PR, 11 achieved CR (64%), 4 maintained PR, 2 died before response evaluation. Of 4 patients who underwent allo-SCT in active disease, only 1 obtained a PR, whereas the other 3 patients developed progression or were not-valuable. Six patients (28.6%) developed acute GvHD, but no one died for complicated acute GvHD. Eleven patients (52.4%) had chronic GvHD. Follow up range was from 4 to 96 months. The median time was 19 months. The relapse/progression rate in course of follow up was 29%. Two patients progressed after PR (40%), 3 after CR (5.9%). At the time of the last follow-up 8 patients died (47%), 3 of them for progression of MM. Survival analyses: TRM at 1 and 3 years was respectively 24% and 31%. OS at five years was 51%, with a plateau trend after 3 years (Fig. 1). In univariate statistical analysis, early DS stage at diagnosis (I-II), double auto-transplant, development of chronic GvHD have a significant impact (p value .05 ) Fig. 3a:. OS in patients previously treated or not with new drugs ( p value 〉 .05 ); Fig. 3b: PFS in patients previously treated or not with new drugs ( p value 〉 .05 ) Disclosures No relevant conflicts of interest to declare.

Description: Delayed T cell reconstitution after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Optimal T cell recovery requires a functional thymus, and strategies to enhance T cell reconstitution have the potential to improve overall outcome in allo-HSCT recipients, however, at the present time such strategies are limited. Hence one of the most significant clinical challenges is the need for rapid regeneration of thymopoiesis following induced immunodepletion and transplantation. Zinc is the second most abundant trace metal in the body, binding to more than 300 proteins involved in DNA synthesis and repair, gene transcription, cell proliferation as well as differentiation and apoptosis. Zinc deficiency (ZD) is a clinical condition causing immunosuppression and thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). Furthermore, mild ZD is one of the causes of the reduction in thymic function in the elderly and the role of zinc in tissue regeneration after damage has been clearly demonstrated in liver, skin, and intestinal diseases. In a pilot clinical trial, we demonstrated that patients receiving oral zinc supplementation after autologous HSCT showed increased thymic-dependent T cell reconstitution in the absence of adverse clinical events (Iovino 2018, Leuk Res). Although a clear clinical benefit was observed, the mechanisms underlying this process are poorly understood. Thus, we used a murine model to evaluate the effect of zinc supplementation in thymic reconstitution after acute damage. Using a model of thymic damage caused by sub-lethal total body irradiation (SL-TBI, 550 cGy), we found that mice that received zinc supplementation demonstrated increased thymic cellularity when compared to untreated age-matched mice (Fig. 1a). Importantly, this finding was also confirmed in a clinically-applicable model of MHC-matched allogeneic HSCT (Fig. 1b). We have previously demonstrated endothelial cells (EC), which are extremely resistant to damaged, are able to trigger thymic endogenous reconstitution after damage by producing regenerative factors such as BMP4, which targets thymic epithelial cells (TECs), a key population crucial for T cell development (Wertheimer 2018, Science Immunol). Interestingly, in our model of zinc administration, we found an increase in the number of regeneration-initiating ECs (Fig. 1c), and increased proliferation of TECs (Fig. 1d), which can occur in response to BMP4. Consistent with the hypothesis that zinc supplementation is activating the BMP4 pathway, when stimulated in vitro for 24 hours with supraphysiological doses of zinc sulfate, ex vivo propagated ECs (exECs) were directly induced to produce BMP4 (Fig. 1e), suggesting a likely mechanism by which zinc supplementation promotes thymic reconstitution. In conclusion, we demonstrate a mechanism by which zinc supplementation can improve thymic function and offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4772 Introduction Non Hodgkin Lymphomas (NHL) are commonly divided in two large groups known as high grade (HG) and low grade (LG) lymphomas. Specific therapeutic strategies are potentially curative, but a tailored treatment should be planned from the diagnosis on the basis of the prognostic features. Some clinical features at the diagnosis or relapse may be grouped to build prognostic indexes. These indexes are “build” starting from features present at diagnosis and related to patients and disease onset, but lacks to give any information about disease history and chemosensitivity. For these purposes others methods have been evaluated and probably the two that gave major help to detect slow responders or resistant patients are minimal residual disease (MRD) and PET scans. Here we develop and apply a partitioning recursive algorithm, known as HCS, by which possibly detect new possible prognostic factors. Methods Our dataset comprised 651 NHL patients followed at our Institution from 1990 to 2005, divided in High grade (HG NHL, n=343; 52,7%) and Low grade Lymphoma (LG NHL, n= 308; 47,3%). Only patients who enter in a follow up program were considered: therefore patients with at least a partial response. We considered as variables for algorithm analysis: age, sex, histological subtype, IPI status and bone marrow involvement, treatment approaches (poli-chemotherapy, mono-chemoterapy, radiotherapy, surgery, purine based chemotherapy, monoclonal antibodies, transplant approach, oral chemotherapy), response to therapy, previous successful treatments, previous relapses, previous failed therapies. Data were analyzed by a recursive partitioning algorithms. A partitioning recursive algorithm. This tool is thought to splits data in different subgroups that behave in a different way. It works starting from data and utilized them to create all the possible combinations of splits available. Among them it chooses the best one by statistics and finally applies it to patients' datasets. For these reason it is defined as “partitioning”. Afterwards the algorithm starts again the analysis on the subset previously detected and that's because it is called “recursive”. Results The most important split emerged to be the quality of response: patiets were splitted between patients in partial remission (PR) and complete remission (CR). Among PR patients, one subset (subset 1) with worse prognosis were found: both comprised patients with HG NHL not treated with monoclonal antibodies and/or transplant. Therefore the remaining PRs patients, treated by transplant approach and immunotherapy, had a better outcome. In CR group subset 2 has been detected, comprising HD NHL patients not treated by oral chemo alone were detected. Those patients, who had a better outcome, had been treated aggressively with polichemotherapy and/or autologous transplant. Differences between detected groups are statistically significant with p. value 0,03 maximum. Splits are shown in Figure 1 and 2. Conclusion Application of computer science analysis to NHL patients has been successfull. Quality of response emerged as the most important prognostic factor but among both PRs and CRs patients those with better outcome had HG NHL diagnosis and were treated by autologous transplantation or/and immunotherapy. This analysis confirms data available about transplant as a good approach for NHL patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4493 Introduction Acute GVHD involving the gastrointestinal tract is now the major cause of non-relapse mortality following allogenic transplant. Diagnosis remains problematic for some patients with pathology in the mid-gut; there is significant sampling error with mucosal biopsy; we lack objective measures of physiologic improvement or worsening in the gut; and duration of immune suppressive therapy remains imprecise. To address these issues, we have serially evaluated intestinal pathology in patients with acute GVHD using a reproducible ultrasound technique as a proof of principle study. Specifically, we examined the hypothesis that contrast-enhanced ultrasound (CEUS) could detect an enhancement of microcirculation during active intestinal acute GVHD as a diagnostic tool and that CEUS could be used to serially assess physiologic changes after treatment. Methods Four patients (pts) with hematologic malignancy (1 each with ALL, AML, mantle cell lymphoma, and myeloma) received a matched unrelated donor allogenic transplant after a myeloablative (N=2), reduced intensity (N=1), or non-myeloablative (N=1) conditioning. GVHD prophylaxis consisted of cyclosporine with either short course methotrexate (N=3) or mycophenolate mofetil (N=1). All patients developed biopsy-proven intestinal GVHD that was steroid refractory in 2 patients. At GVHD onset, patients were scanned with transabdominal ultrasonography and subsequently by CEUS using a linear phased-array 7.5-MHz transducer. A second generation echo-contrast agent (SonoVue®, Bracco), which consists of microbubbles stabilized by phospholipids and filled with sulphur hexafluoride, was injected i.v. as a bolus (2.4 mL) followed by 5 mL saline flush. Microbubbles have a mean diameter of 2.5 μm and remain within the vascular space allowing real-time imaging of microcirculation. Results In all patients, routine ultrasonography revealed mucosal edema involving the terminal ileum in 3 patients (wall thickness 5.1, 5.8 and 7.9 mm) and the colon in 4 patients (ascending colon 5.8, 6, 8.4 and 18 mm; transverse colon 6 and 12.6 mm; descending colon 11 mm). CEUS at GVHD onset showed an arterial phase (AP) complete enhancement of the entire wall section from the mucosal to the serosal layer (terminal ileum) in 2 patients. There was absence of enhancement only in the outer border of the muscularis propria in 1 patient (pt); there was absence of enhancement both in the outer and in the inner border of the colon wall and enhancement only of the intermediate layer in 1 pt. All patients showed late parenchymal phase (PP) wash out. These enhancement patterns have previously been described in active Crohn's disease (Serra C. et al; 2007). CEUS follow-up findings on serial examinations: 1) allowed us to monitor residual disease in one pt after Infliximab, showing persistent AP enhancement of microcirculation suggesting residual GVHD activity which required further treatment with eventually complete remission; 2) CEUS showed normalization and/or decreased microcirculation enhancement in pts responding to treatment (N=2); 3) CEUS showed AP microcirculation enhancement when GVHD flared (N=2); 4) CEUS showed persistence of AP enhancement of microcirculation after Rituxan (4 doses 375mg/m2/weekly) despite a decrease in ileum wall thickness and in agreement with only a slight improvement of symptoms in one pt when GVHD flared, 5) CEUS showed no improvement of intestinal microcirculation wall enhancement in steroid refractory aGVHD patients who eventually died (N=2). In one of them there was persistence of AP phase enhancement despite improvement of symptoms suggesting still active disease. Conclusions Contrast enhanced US showed intestinal microcirculation wall enhancement and delayed washout at the onset of GVHD symptoms in areas of the intestine inaccessible to endoscopic evaluation. CEUS findings on serial examinations correlated with incomplete responses and flares of GVHD symptoms (microcirculation enhancement) and responses to therapy (decreased microcirculation activity). CEUS may be useful for both diagnosis and prognosis, as it provides both anatomic and physiologic information about intestinal GVHD. These findings prompted us to design a prospective study to evaluate clinical usefulness of CEUS in diagnosis and follow-up of intestinal acute GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: INTRODUCTION Multiple myeloma (MM) is considered an incurable disease. Despite the introduction of novel agents allowed deeper response, high-dose chemotherapy and autologous stem cell transplantation (ASCT) remain the standard of care for patients (pts) in good clinical conditions. The most used strategies to mobilize stem cells from bone marrow (BM) into peripheral blood are high-dose cyclophosphamide (HD-CTX) plus G-CSF and G-CSF plus plerixafor (G-CSF+P). The goal of this retrospective study is to investigate whether the two different mobilization strategies have an impact on the clearance of monoclonal PCs in the apheresis products and on pts' outcome. PATIENTS AND METHODS We analyzed 62 pts (median age 61, range 41-75, 37 males and 25 women) diagnosed with MM and treated with ASCT between Mar 2014 and Mar 2018 at our Hematology Division (Pisa, Italy). All pts received induction therapy with at least 4 cycles of bortezomib, thalidomide and dexamethasone (VTD). 9/62 pts obtained a less than partial response (PR) and received lenalidomide-based regimens. After induction, 8 (12,9%) pts achieved complete remission (CR), 26 (41,9%) were in PR, 28 (45,2%) obtained a very good partial response (VGPR). 43/62 fit pts received HD-CTX (2-3 g/sqm) on day 1 followed by G-CSF (30 MU/day) started on day 4 until day 7, increased to 60 MU/day from day 8 until the end of apheresis. In 19/62 pts, after 4 days of G-CSF (60 MU/day) administration and not sufficient mobilization, we added plerixafor (0,24 mg/kgbw) for up to 4 consecutive days. In 43/62 pts we collected apheresis samples (10μl) analyzed through flow citometry to enumerate clonal residual PCs. The panel used to asses clonality included: CD138 Per-Cp, CD38 APC, CD19 PE-Cy7, CD45 APC-Cy7, cytoplasmic immunoglobulin K chain and L chain. RESULTS At the end of the peripheral blood stem cell (PBSC) collection, pts treated with HD-CTX presented a higher CD34+ absolute count (p=0.0489) and achieved the threshold of 5x106 CD34+ cells/kgbw in a significantly (p=0.006) higher percentage. We found a nearly significant (p=0.0517) lower count of CD34+ PBSCs in pts who received lenalidomide-based regimens before the mobilization. Performing flow citometry on apheresis samples, we observed that the number of the harvested clonal PCs showed a significant correlation (p=0.0115) with the occurrence of post-ASCT relapse. ROC curve analysis investigating the predictive effect of the number of pathological PCs on disease relapse showed an area under the curve of 0,6978 (95% CI 0.5392-0.8564; p=0.0267). Neither BM residual PCs detectable on BM biopsies performed before apheresis (r=-0.1323; p=0.609) nor the type of mobilization scheme (p=0.707) had an impact on the proportion of clonal PCs in the graft. Additionally, we did not observe any statistically significant difference in progression free- (PFS) (p=0.8276) and overall survival (OS) (p=0.2475) between the HD-CTX and G-CSF+P groups. DISCUSSION PBSC mobilization has a succession rate 〉 85%. Despite the use of HD-CTX to increase PBSC yields and decrease tumor burden, there is not clear evidence of a superior mobilization strategy. Additionally, HD-CTX has a not negligible toxicity and approximately 10% of the pts require hospitalization. Conversely, G-CSF+P is a safe and effective approach also in poor mobilizers. In our study, we observed a significative difference in the apheresis yields (p=0.0489) and in the percentage of pts who achieved the threshold of 5x106 CD34+ cells/kgbw (p=0.006) in favor of HD-CTX. Additionally, the detection of harvested residual clonal PCs could be a promising strategy to recognise pts more likely to relapse after ASCT. Nonetheless, we failed to demonstrate a superior effect of HD-CTX in the clearance of harvested clonal PCs, in agreement with the absence of a different pts' outcome amongst the two mobilization strategies. In conclusion, the choice between the two regimens is challenging and requires careful consideration of multiple factors. Overall, young fit pts, especially in the high-risk setting, should be treated with all appropriate modalities including chemiomobilization followed by double-ASCT. Conversely, in pts candidate to a single-ASCT it is reasonable to use G-CSF+P, since HD-CTX does not improve PFS and OS and add toxicity. The absence of an in-vivo purging effect on apheresis products of chemiomobilization further strengthens a chemotherapy-free mobilization. Disclosures Galimberti: Roche: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau.