Background: Patients with low tumour burden follicular lymphoma (FL) are managed with an initial watchful waiting (WW) approach. The way to better predict the time-to-treatment (TTT) is still under investigation for its possible clinical impact. This study explored whether radiomic features extracted from baseline 18F-FDG PET/CT could predict TTT in FL patients on WW. Methods: Thirty-eight patients on initial WW (grade 1–3a) were retrospectively included from 2010 to 2019. Eighty-one PET/CT morphological and first-level intensity radiomic features were extracted from the total metabolic tumour burden (TMTV), the lesion having the highest SUVmax and a reference volume-of-interest placed on the healthy liver. Models using linear regression (LR) and support vector machine (SVM) were constructed to assess the feasibility of using radiomic features to predict TTT. A leave-one-out cross-validation approach was used to assess the performance. Results: For LR models, we found a root-mean-squared error of 29.4, 28.6, 26.4 and 26.8 and an R2 of 0.03, 0.08, 0.21 and 0.20, respectively, incrementing the features from one to four. Accordingly, the best model included three features: the liver minimum SUV value, the liver SUV skewness and the sum of squared SUV values in the TMTV. For SVM models, accuracies of 0.79, 0.63, 0.76 and 0.68 and areas under the curve of 0.80, 0.72, 0.77 and 0.63 were found, respectively, incrementing the features from one to four. The best performing model used one feature, namely the median value of the lesion containing the SUVmax value. Conclusions: The baseline PET/CT radiomic approach has the potential to predict TTT in FL patients on WW. Integrating radiomics with clinical parameters could further aid in patient stratification.

Splenomegaly is a quite common clinical feature of Philadelphia (Ph) negative chronic myeloproliferative neoplasms (MPNs) and its presence may, in some cases, drives treatment decision. Most importantly, palpable splenomegaly is a minor criterion for both pre-fibrotic/early primary myelofibrosis and primary myelofibrosis (PMF) diagnosis, even if clinical assessment by physical examination is poorly reliable and accurate. On the other hand, despite the International Working Group-Myeloproliferative Neoplasms Research and Treatment and European LeukemiaNet guidelines defined spleen response criteria by palpation, they also recognized the highly subjective nature of spleen size assessment by physical examination, and recommended objective confirmation of volume reduction via computed tomography or magnetic resonance imaging (MRI). In particular, spleen volume (SV) reduction of at least 35

Since the introduction of rituximab in the late 1990s, significant progress has been made in advancing targeted therapies for B cell lymphomas, improving patients’ chance of being cured and clinicians’ therapeutic armamentarium. A better understanding of disease biology and pathogenic pathways, coupled with refinements in immunophenotypic and molecular diagnostics, have been instrumental in these achievements. While traditional chemotherapy remains fundamental in most cases, concerns surrounding chemorefractoriness and cumulative toxicities, particularly the depletion of the hemopoietic reserve, underscore the imperative for personalized treatment approaches. Integrating targeted agents, notably monoclonal antibodies, alongside chemotherapy has yielded heightened response rates and prolonged survival. A notable paradigm shift is underway with innovative-targeted therapies replacing cytotoxic drugs, challenging conventional salvage strategies like stem cell transplantation. This review examines the landscape of emerging targets for lymphoma cells and explores innovative therapies for diffuse large B cell lymphoma (DLBCL). From Chimeric Antigen Receptor-T cells to more potent monoclonal antibodies, antibody–drug conjugates, bispecific antibodies, checkpoint inhibitors, and small molecules targeting intracellular pathways, each modality offers promising avenues for therapeutic advancement. This review aims to furnish insights into their potential implications for the future of DLBCL treatment strategies.

Background: CD79b is a B-cell-specific antigen that is crucial to the B-cell receptor and is considered a key target for treatment in aggressive B-cell lymphomas. Methods: While immunohistochemical studies have shown widespread expression of CD79b in mature B-cell-derived lymphomas, flow cytometry allows for precise measurement and differentiation between surface and intracellular localization. Results: In our comparative analysis, we discovered that CD79b expression percentages and mean fluorescence intensity (MFI) were lower in a group of 127 cases of aggressive B-cell lymphomas compared to a control group of benign reactive hyperplasia. We also observed significant variability in the surface expression of CD79b among lymphoma cases, with 18% showing predominantly intracellular positivity. There was a strong correlation between the surface expression of CD79b and clonal light chains. Notably, primary mediastinal B-cell lymphomas exhibited significantly lower surface CD79b expression compared to other lymphoma subtypes (median 0.8% IQR 0-48.5 vs. 80% IQR 24-97, p = 0.0005). Furthermore, patients over 60 years old and those with a higher Revised International Prognostic Index (R-IPI) had significantly higher CD79b expression, both of which are associated with a significant benefit from adding an anti-CD79b drug conjugate to first-line chemotherapy in diffuse large B-cell lymphomas. Conclusions: In conclusion, the quantitative flow cytometric analysis of CD79b surface expression in aggressive B-cell lymphomas provides clinically relevant information, highlighting its potential usefulness in guiding therapeutic decisions.

Image-guided core needle biopsies (IG-CNB) represent a minimally invasive approach for obtaining tissue in patients with lymphadenopathy and suspected lymphoma. Despite their utility, diagnostic challenges persist, with lower efficacy compared with excisional biopsies. Our study aimed to evaluate the potential utility of incorporation of flow cytometry (FC) alongside immunohistochemistry (IHC) when performing IG-CNB for suspected lymphoproliferative diseases. Analyzing 170 consecutive cases, guided by ultrasound (n = 94) or computer tomography (n = 76), we employed a diagnostic algorithm, already established in our laboratory practice, utilizing three antibody cocktail-equipped tubes tailored for defining lymphomas, particularly those of B-cell origin. FC expedited the diagnostic process, yielding presumptive results in 87.6% of cases within 48 h, with a positive predictive value of 98%. Addition of FC to routine IHC enhanced the diagnostic rate from 91.2% to 95.3%, reducing IG-CNB failure rate by 45%, from 8.8% to 4.7%. This enhancement was particularly notable for deep-seated sites and in the setting of suspected disease recurrences. Consequently, FC emerges as a valuable adjunctive tool, allowing for the improvement of diagnostic performance, with a particular focus on the ability to quantify the expression of surface markers for targeted therapies, and holding the potential to diminish the necessity for repeat excisional biopsies subsequent to IG-CNB procedures.

EUS-FNB has been introduced in clinical practice as a less invasive diagnostic approach with respect to surgery. We performed a single-center retrospective study on the diagnostic efficacy of EUS-guided FNB, including 171 patients with lymph nodes, splenic, and extranodal lesions that underwent EUS for FNB at our institution. Excluding 12 patients who did not undergo FNB and 25 patients with a previous diagnosis of a solid tumor, we included 134 patients with clinical/radiological suspect of a lymphoproliferative disease, including 20 patients with a previous history of lymphoma. Out of the 134 biopsies, material of diagnostic quality was obtained in 111 procedures (84.3%). Histological examination of the EUS-FNB samples produced an actionable diagnosis in 100 cases (74.6%). Among the patients without an actionable diagnosis, a second, different diagnostic procedure produced a further eight diagnoses of lymphoma. Therefore, the sensitivity of EUS-FNB for diagnosing lymphomas was calculated to be 86.4% (51/59). Assignment of lymphomas to WHO classification subtypes was possible in 47/51 (92%) of the cases. In conclusion, EUS-FNB is an effective procedure for the histological characterization of lesions that are suspected to be lymphoproliferative disease, allowing for an actionable diagnosis in 75% of cases.

Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 2673 Infused bags from responders at M3 and M6 showed a higher percentage of CAR+CD8+ cells than non-responders (56% vs 27% p=0.02 and 55.4% vs 27.3% p 0.034 respectively). As a consequence, we observed significant difference in the ratio CD4+/CD8+ CAR+ in infused bags between responders at M3 vs non-responders (ratio 0.74 vs 2.47, p=0.011) (Figure 1A) We used a ROC analysis to identify an ideal ratio CD4+/CD8+ CAR+ cut-off among infused CAR-T to discriminate responders at M3: we found that a ratio of 1.12 had 100% sensitivity and 70 % specificity in identifying responses at M3 (AUC 82%, p=0.001). (Figure 1B) In a logistic regression analysis, the CD4+/CD8+ CAR+ ratio was confirmed as a variable to predict response at M3 (Odds Ratio 23.3 p=0.012) and M6 (Odds Ratio 10 p=0.028). Summary/Conclusions We found that a higher percentage of CAR+CD8+ T cells and a lower ratio CD4+/CD8+ CAR+ in the infusion bags are significant predictors for response at 3 and 6 months in pts treated with CD19-CAR-T. We observed that a very low percentage of CAR+CD8+EM T cells may be associated with early treatment failure at 1 month. These data support the role of CD8-mediated cytotoxic mechanism of action by CAR-T cells, and may be useful for strategies of potential empowerment of manufactured products in order to raise CAR-T efficacy. HemaSphere | 2023;7(S3) EHA2023 Hybrid Congress Copyright Information: (Online) ISSN: 2572-9241 © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) which allows third parties to download the articles and share them with others as long as they credit the author and the Abstract Book, but they cannot change the content in any way or use them commercially. Abstract Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx.Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 2674

CD4+ and CD8+ chimeric antigen receptor T cells (CAR-T) play different roles in the in vivo anti-tumour response, but the role of the CD4+ /CD8+ ratio among infused CAR-T has not been clearly defined yet. We analysed leftovers from infused anti-CD19 CAR-T bags of 31 patients with aggressive B-cell lymphomas. The median ratio was 1.44, lower for brexu-cel compared to tisa-cel and axi-cel. The CAR+CD4+ /CD8+ ratio was influenced by lactate dehydrogenase levels at apheresis, not by age, previous treatments or the CD4+ /CD8+ ratio in peripheral blood. Patients with a response at 3 months after CAR-T (M3) had a lower CAR+CD4+ /CD8+ ratio in the infused products compared to non-responders (ratio 0.74 vs. 2.47, p = 0.011). A CAR+CD4+ /CD8+ ratio higher than the cut point of 1.12 was associated with an increased risk of treatment failure at M3 (OR 23.3, p = 0.012) and M6 (OR 10, p = 0.028). The median 6-month PFS was 76% for patients with a ratio lower than 1.12% vs. 31% for the others. The prognostic role of the CAR+CD4+ /CD8+ ratio was independent of the costimulatory domain (CD28 vs. 4-1BB) of the product (OR 16.41, p = 0.041). Our data indicate a crucial role for CD8+ CAR-T and the CAR+CD4+ /CD8+ ratio in predicting CAR-T efficacy.

Asymptomatic patients with follicular lymphoma (FL) and a low tumour burden can be followed without initial therapy, a strategy called watchful waiting (WW). Prediction of the time to treatment (TTT) is still a challenge. We investigated the prognostic value of baseline total metabolic tumour volume (TMTV) and whole-body total lesion glycolysis (WB-TLG) to predict TTT in patients with FL on WW. We conducted a retrospective study of 54 patients with FL (grade 1–3a) diagnosed between June 2013 and December 2019, staged with FDG PET/CT, and managed on WW. Median age was 62 years (range 34–85), stage was advanced (III–IV) in 57

Topic: 18. Indolent and mantle-cell non-Hodgkin lymphoma - Clinical Background: First-line immunochemotherapy of Follicular lymphoma (FL) combining anti-CD20 antibodies Rituximab or Obinutuzumab with Bendamustine (R/O-B) followed by maintenance therapy results in high rates of response and long progression-free survival. A major adverse effect of this regimen is long-lasting lymphopenia as a risk factor for infections. Aims: We performed a cross-sectional and longitudinal study on lymphocyte T cell subsets during anti-CD20 maintenance therapy using flow-cytometry. Associations between T cell subset counts and infectious events were analyzed. Methods: We enrolled 55 patients (pts) with high tumor burden FL between 2017 and 2021 who received first-line treatment with anti-CD20 plus Bendamustine (R/O-B) or CHOP regimen (R-CHOP), followed by maintenance. We collected peripheral blood samples at the following time points: end of induction (EoI), at +6, +12, +18 and +24 months after. We analyzed T cell subsets defined by the maturation status (Naïve, Central Memory, Effector Memory, and TEMRA) in addition to absolute CD4+ and CD8+ T cell counts. Results: We enrolled 55 pts: 49% females, 51% males, median age 60 years (range 50- 80), who received R/O-B (76%) and R-CHOP (24%). At the EoI median CD4+ counts were lower in the R/O-B group compared to R-CHOP (119/mmc vs 402/mmc; p =0.0001). Despite the trend of CD4+ counts to increase over time following R/O-B, they remained significantly lower at +6 (155/mmc vs 637/mmc p=0.0008) and +12 (264.6/mmc vs 497.3/mmc; p=0.027), when compared to R-CHOP. Most strikingly, the median CD4+ naive count was extremely low in R/O-B group at EoI (3.43/mmc vs 71.3/mmc; p=0.003) and up to +12 (26/mmc vs 173/mmc; p=0.04), while no differences were observed at +18 and +24. We didn’t find differences in T CD8+ counts and subsets between R/O-B and R-CHOP group at any time. Gender and age had a significant impact on T cell recovery in R/O-B treated pts. Males (M) had prolonged lower CD4+ counts respect to females (F) (146/mmc vs 316/mmc at +12 for M vs F, p=0.018; 230/mmc vs 425/mmc at +24 for M vs F, p=0.026). Moreover, the proportion of naïve CD4+ T cells remained lower in M than in F (from 9% vs 15% at +6, p=0.05, to 11% vs 35.4% at +24, p=0.045). As age increases lower naïve CD4+ count is observed at +6 (p=0.0019) and +12 months (p=0.016). Infectious episodes were registered during the 2 year-observation period in 47% of pts. 60% of infections were bacterial, 30% reactivation of VZV, and 10% fungal. The cumulative incidence rate of infection was significantly different between R/O-B and R-CHOP (59% vs 7 % p= 0.003). In univariate analysis age>60 years and more than 12 months to reach CD4+>200/mmc were identified as risk factors for infections in the R/O-B group (respectively OR 4.87, CI 1.23 -19, p =0.024; OR 7, CI 95% 1.59-30, p =0.014). These variables were confirmed in multivariate analysis (respectively OR 4.34, CI 95% 1.012-18.6, p=0.048; OR 1.13, CI 95% 1-1.28, p=0.05). Summary/Conclusion: FL pts undergoing first-line therapy with R/O-B experienced a prolonged and deep CD4+T and lymphopenia, in particular of the naïve CD4+ T cell subset compared with pts treated with R-CHOP. Elderly Male pts showed a particularly delayed recovery of CD4+ T cell counts. Prolonged CD4+ T cell lymphopenia over 12 months and age>60 ys are risk factors for infections, and could be indicators for the necessity to prolong anti-microbial prophylaxis. Moreover, benefits of the R/O-B regimen should be weighed against the risks of infections in particular among elderlies. Keywords: Follicular lymphoma

The head-and-neck area is one of the most common sites for nodal and extranodal localization of lymphomas. Clinical presentation of lymphomas in the head and neck region varies from slowly growing indolent lymphomas to highly aggressive lymphomas causing compression of the upper airways for which a timely diagnosis is warranted. The aim of this study was to evaluate the predictive value of flow cytometry (FC) in the diagnosis of head and neck lesions suspicious for lymphoma. We analyzed cell suspensions of 50 excisional biopsies using FC and compared the results with histological examination. Using a sequential three-tube antibody panel, we found a high level of diagnostic concordance between FC and histology in the 30 non-Hodgkin lymphomas (NHL). When no aberrant B or T cell population was identified in FC, indirect signs were helpful to predict diagnoses other than NHL, such as Hodgkin Lymphoma, metastatic lesions of epithelial tumors or reactive hyperplasia. In conclusion, FC can provide useful diagnostic information in a short turnaround time when clinical evaluation by hematologists and otolaryngologists raised the suspicion of lymphoma.

Progressive multifocal leukoencephalopathy (PML) is a rare fatal disease caused by polyomavirus JC occurring in the context of a deep and prolonged immunosuppression. Rituximab is one of the monoclonal antibodies labelled with a black box warning for the risk of PML. The estimated incidence rate of PML ranges from 1·39 to 1·87 per 10·000 rituximab-exposed patients (Focosi et al., 2019). Although PML is classified as a 'very rare' complication of rituximab treatment, the combination with other drugs may significantly modify this risk. In the last decade, the combination of bendamustine and rituximab (BR) has become the preferred regimen chosen by many hematologists to treat patients with high tumour burden follicular lymphomas (FL). Similarly, maintenance therapy administering rituximab every two or three months for two years has become part of standard first line treatment of FL. Here we report our monocentric experience of three cases of PML occurring in patients with FL treated with BR followed by rituximab maintenance. This retrospective analysis included 47 patients with high tumour burden FL (22 males, 25 females; median age 59 years, range 28–82 years), diagnosed at our institution from 2014 to 2018, and treated with rituximab maintenance following six cycles of BR induction chemotherapy. All patients were HIV-negative. All patients treated with rituximab maintenance had at least a partial response to BR induction treatment (40 complete response and seven partial response). At last follow-up, 41 patients are alive and 6 patients had died. Among the causes of death, three patients died of PML, one of lymphoma, one of gastrointestinal bleeding and one of unspecified cause. The probability of OS at a median follow-up of 3 years was 91% with significantly lower probability for patients aged >70 years when compared to younger patients (78% vs. 95%, respectively; P = 0·001). Characteristics of the three patients developing PML are summarized in Table 1. The onset of PML symptoms occurred during rituximab maintenance in two patients and at the end of maintenance in a third patient. Time between onset of symptoms and definitive diagnosis of PML was approximately 1 month. Definitive diagnosis of PML was based on the demonstration of the JC virus using PCR in the CSF, combined with the typical clinical and imaging findings (Table 1 and Fig 1) according to consensus criteria of the American Academy of Neurology Neuroinfectious Disease Section (Berger et al., 2013). At the time of PML diagnosis, lymphocyte counts were lower than normal. Immunophenotyping of peripheral blood, available in two patients, showed a marked reduction of the CD4+ cell count <200/μl (Table 1). B cells detected by CD19 were almost absent. Our observation of three cases of PML in a cohort of 47 patients with FL treated with BR followed by R maintenance appears unusually high and, considering the limit of a small monocentric cohort, it does not allow to calculate actual incidence rates. A low and persistent CD4+ cell count in peripheral blood has been described as the major risk factor for PML in HIV-positive patients (Engsig et al., 2009). JC virus is ubiquitous in humans (50–70% of the general population has a positive serology) and remains in a latent state in several organs, including the brain. CD4+ cells appear to play a central role in the control of JC virus infection in the brain and drive CD8+ T-cell activation into JC virus-specific effector cells through a T Helper 1-type response (Pavlovic et al., 2018). Lymphocytopenia and CD4+ cell deficit are well-known sequelae of BR treatment (García Muñoz et al., 2014; Martínez-Calle et al., 2019). In a cohort of 295 patients with CLL treated with BR, median times to lymphocyte count recovery (≥1000/μl) and CD4+ recovery (≥200/μl) were 26 and 24 months, respectively (Martínez-Calle et al., 2019). In the GALLIUM study, bendamustine combined with either the anti-CD20 antibody obinutuzumab or rituximab followed by anti-CD20 maintenance induced a marked reduction of CD3+ and CD4+ T cells, while patients treated with CHOP or CVP showed negligible changes in T cell counts. Recovery of T cell counts was delayed during and after maintenance, and patients treated with BR had an increased risk of adverse events of grade G3–G5, including secondary neoplasms and infections, when compared to patients treated with CHOP or CVP (Hiddemann et al., 2018). Although bendamustine appears to play the pivotal role in this context, some data suggest that rituximab maintenance may prolong CD4+ cell lymphopenia induced by BR (Yutaka et al., 2015). The contribution of rituximab-induced B-cell depletion to PML risk is unlikely to be mediated by a deficit in the humoral response to JC virus; on the contrary, it may be related to the immunomodulatory effect of B cells on T cell subsets in maintaining CD4+ and CD8+ T-cell homeostasis and promoting expansion in response to a viral infection (Durali et al., 2015). Our three cases of PML occurred in older adults. This raises the question whether age-related immunosenescence could be an additional contributing risk factor. Aging was shown to impact the PML-risk in patients with multiple sclerosis who were treated with disease-modifying therapies (Mills & Mao-Draayer, 2018). Studies considering the effect of age on T cell recovery after bendamustine are necessary to address this question. Our report is intended to raise awareness about the risk of PML in patients undergoing highly immunosuppressive therapy with BR followed by rituximab maintenance. A multidisciplinary interaction between neurologists, hematologists and neuroradiologists is essential for the appropriate identification of such a fatal disease. The occurrence of unexplained neurological symptoms in a patient treated with BR should prompt diagnostic work-up with MRI and study of CSF for the presence of JC virus by PCR. In our limited real-life experience, death was more often due to other causes than lymphoma itself in the older adults with FL aged more than 70 years who were treated with BR and rituximab maintenance. Considering alternative chemotherapy regimens before rituximab maintenance, or avoiding rituximab maintenance following BR, could be strategies to reduce the prolonged and profound immunosuppression, particularly in older adults. The authors thank all the staff at the Hematology, Neurology, Radiology and Nuclear Medicine Units of the "Fondazione Policlinico Universitario Agostino Gemelli IRCCS" in Rome who were involved in the management of patients with lymphoma and their treatment-related complications. The authors declare no conflicts of interest. F.D. and S.H. designed the research study, analysed data and wrote the paper; R.M. collected data, revised the paper and finally approved it; F.P., S.B., A.C., E.M., A.M., L.L. and G.M. contributed to patient management and data collection, revised the paper and finally approved it; N.M. collected data, revised the paper and finally approved it; V.D. revised the paper and finally approved it.