(1) Background: Clinical and molecular data on patients with unexplained erythrocyto-sis is sparse. We aimed to analyze the clinical and molecular features of patients with congenital erythrocytosis in our tertiary reference center. (2) Methods: In 34 patients with unexplained erythrocytosis, a 13-gene Next-Generation Sequencing erythrocytosis panel developed at our center was conducted. (3) Results: In 6/34 (18%) patients, eight different heterozygous gene variants were found. These patients were, therefore, diagnosed with congenital erythrocytosis. Two patients had two different gene variants each. All variants were characterized as variants of unknown significance as they had not previously been described in the literature. The rest of the patients (28/34, 82%) had no detected gene variants. (4) Conclusions: Our experience shows that the NGS panel can be helpful in determining the reasons for persistent, unexplained erythrocytosis. In our cohort of patients with erythrocytosis, we identified some, thus far unknown, gene variants which may explain the clinical picture. However, further investigations are needed to determine the relationship between the molecular findings and the phenotype.

(1) Background: Polycythaemia is defined by an increase in haemoglobin (Hb) concentration, haematocrit (Hct) or red blood cell (RBC) count above the reference range adjusted to age, sex and living altitude. JAK2 unmutated polycythaemia is frequent but under-investigated in original publications. In this retrospective cohort study, we investigated the clinical and laboratory data, underlying causes, management and outcomes of JAK2 unmutated polycythaemia patients. (2) Methods: The hospital database was searched to identify JAK2 unmutated patients fulfilling WHO 2016 Hb/Hct criteria for PV (Hb >16.5 g/dL in men and >16 g/dL in women, or Hct > 49% in men and >48% in women, or RBC mass > 25% above mean normal predicted value) between 2008 and 2019. Clinical and laboratory data were collected and analysed. (3) Results: From 727,731 screened patients, 294 (0.04%) were included, the median follow-up time was 47 months. Epo and P50 showed no clear pattern in differentiating causes of polycythaemia. In 30%, the cause remained idiopathic, despite extensive work-up. Sleep apnoea was the primary cause, also in patients under 30. Around 20% had received treatment at any time, half of whom had ongoing treatment at the end of follow-up. During follow-up, 17.2% developed a thromboembolic event, of which 8.5% were venous and 8.8% arterial. The mortality was around 3%. (4) Conclusions: Testing for Epo and P50 did not significantly facilitate identification of underlying causes. The frequency of sleep apnoea stresses the need to investigate this condition. Idiopathic forms are common. A diagnostic flowchart based on our data is proposed here. NGS testing should be considered in young patients with persisting polycythaemia, irrespective of Epo and P50 levels.

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(1) Background: Thrombophilia testing utility has remained controversial since its clinical introduction, because data on its influence on treatment decisions are limited. (2) Methods: We conducted a single-center retrospective cohort study of 3550 unselected patients referred for thrombophilia consultation at the Bern University Hospital in Switzerland from January 2010 to October 2020. We studied the influence of thrombophilia testing results on treatment decisions and evaluated the association between thrombophilia and thromboembolic and pregnancy-related morbidity events after testing up to 03/2021. (3) Results: In 1192/3550 patients (34%), at least one case of thrombophilia was found and 366 (10%) had high-risk thrombophilia. A total of 211/3550 (6%) work-ups (111/826 (13%) with low-risk thrombophilia and 100/366 (27%) with high-risk thrombophilia) led to an appropriate decision to extend or initiate anticoagulation, and 189 (5%) negative results led to the withholding of anticoagulation therapy inappropriately. A total of 2492 patients (69%) were followed up for >30 days, with a median follow-up of 49 months (range, 1–183 months). Patients with high-risk thrombophilia had a higher risk of subsequent venous thromboembolic events and pregnancy-related morbidity compared to those without thrombophilia. (4) Conclusions: Our study demonstrated the limited usefulness of thrombophilia work-up in clinical decision-making. High-risk thrombophilia was associated with subsequent venous thromboembolism and pregnancy-related morbidity.

Background: Natural killer (NK) cell-based immunotherapies are emerging as a new, cutting-edge, promising cancer treatment. NK cells have been used to generate autologous as well as allogenic chimeric antigen receptor NK (CAR-NK) cells, and are currently being tested in multiple clinical trials. The advantage of these cells over CAR-T cells is that they show a superior toxicity profile, less on-target/off-tumor effects, and their toxicity mechanism could be independent of CAR. Additionally, allogenic CAR-NK cells are less immunogenic and cause less graft versus host disease in comparison to CAR-T cells. NK-cell collection during apheresis is the first step in the production of CAR-NK-cells. Efficient collection of NK-cells is paramount to successful manufacture of a CAR-NK cell product. Here, we present our single-center experience on individualized high-flow autologous lymphocyte apheresis in heavily pre-treated patients who qualified for a CAR-T treatment. In this abstract, we demonstrate data on NK-cell collections that derived from our research on T-cell collections. Study design and methods: In this retrospective, descriptive study, we compared collection efficiencies (CE%), absolute efficiencies (AE; absolute yield) and relative efficiencies (RE; AE/total number of absolute circulating pre-apheresis cells) for NK- and T-cell collections. Recruitment of cells was assumed if RE>1. The analysis was performed for 9 heavily pre-treated diffuse large B-cell lymphoma patients who underwent autologous T-cell collections for CAR-T treatment between October 2018 and November 2019. These 9 patients were chosen because the data on pre-apheresis NK- and T-cell concentrations, and harvest NK- and T-cell yield, were available for them. For calculation of CE% a standard formula was used (Figure 1, A). Results: A total of 9 high-flow lymphocyte collections achieved the cell yield of 1x10 9 cells for T- cells and 88% (n=8) achieved the cell yield of 1x10 9 cells for NK-cells. The median pre-apheresis cell concentration for NK-cells was 0.21 x10 9/L (range 0.05 - 0.29) and for T-cells was 0.69 x10 9/L (0.11 - 2.09). The median average blood flow was 77 ml/min (49 - 132), the median processed blood volume was 13.51L (7.35 - 31.17), while the median ratio of processed blood volume to total blood volume was 2.57 (1.46 - 6.67). The median AE for NK-cells with 1.58x10 9 (0.55 - 5.9) was markedly lower but not statistically significant (p=0.14) than that of T-cells with 5.5x10 9 (1.88 - 18.41). The CE of NK-collections was median 89% (29 - 125), while the CE of T-cell collections was median 68% (31 - 93) and they were not statistically different (p=0.605). The same was observed for RE which was 3.04 (0.63 - 4.31) for NK-cells and 1.91 (0.68 - 4.52) for T-cells with no statistically significant difference (Independent-Samples Mann-Whitney U Test, p=0.836). Figure 1 presents the differences in AE (B), CE (C) and RE (D) between NK- and T-cell collections. Conclusions: During high-flow lymphocyte apheresis, NK- as well as T-cells are recruited, as in the majority of collections the cell yield is greater than the absolute circulating pre-apheresis cell count (when RE was > 1). Thus, the origin and immunophenotype of these NK- and T-cells should be subject of further investigation. CE for NK- and T-cells are similar and high enough to perform efficient cell collections of both cell types for immunotherapy. Figure 1 Figure 1. Disclosures Jalowiec: Kite Pharma Gilead Sciences': Honoraria, Speakers Bureau. Daskalakis: Amgen: Other: Travel grant; Roche: Other: Travel grant; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; Celgene/BMS: Consultancy, Other: Travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; NovoNordisk: Other: Travel grant.

Background: Real life data of underlying causes of JAK2-negative polycythemia is sparse. We aimed to analyze clinical and laboratory data of patients at our tertiary referral hospital, in order to identify the most frequent underlying causes of JAK2-negative polycythemia. Particularly, we intended to analyze the prevalence and causes of hereditary erythrocytosis. Methods: The hospital database was searched to find patients ≥15 years of age with polycythemia (inpatients and outpatients) between 1 st Oct 2008 and 31 st Jul 2019. Using a stepwise process, we focused on patients in whom a JAK2 result was available. For the diagnosis of polycythemia, including reactive and relative, the diagnostic criteria of the 2016 WHO classification for polycythemia vera were used: hemoglobin >165 g/L in men and >160 g/L in women, or hematocrit >49% in men and >48% in women. Results: Files of 727,731 patients were screened, and in 4,391 polycythemia was mentioned as a diagnosis on the electronic record. Of these, polycythemia was confirmed in 1,483 based on laboratory values. From them, 391 were tested for JAK2 mutation, and 294 were negative, thus representing our study cohort. The median age at polycythemia diagnosis was 46 years (r 15 - 89), and the majority of patients were males (N=242, 82%). The median Hb and Hct value were 172 g/L (r 157 - 224) and 51% (r 45 - 68) respectively. The patients were followed up for a median time of 4 years (r 12 days - 21 years). In 61% of patients (179/294) reactive polycythemia was diagnosed, while in 4.8% (14/294) relative polycythemia was present. In 30% of patients (89/294) the cause of polycythemia was undetermined, whereas in 70% (205/294) an underlying cause explaining polycythemia was found (Table 1). In 12 patients (4.1%), a congenital cause was identified. Among them, hemoglobinopathy or high-oxygen affinity Hb (N=8, 2.7%) were the most common (Table 1). Sleep apnea confirmed through a polysomnography (N=55, 18.7%), followed by smoking (N=51, 17.3%), increased HbCO>5% (N=14, 4.8%), respiratory diseases (N=13, 4.4%), and non-cancer kidney disease, for instance renal cysts (N=12, 4.1%), were the most common associated causes. Polysomnographies were performed on 60 patients, of whom sleep apnea was confirmed in 92% (N=55), and a significant proportion of these patients were younger than 40 years old (N=18, 33%). From all patients with undetermined causes (N=89), we focused on those with persistent polycythemia for further investigations. Accordingly, 25 patients were identified, all male and 72% (N=18) younger than 40 years of age. In 12/25 patients a congenital erythrocytosis NGS panel was performed, and in 3/12 patients 4 different heterozygous mutations were found (two of which were in one patient). All mutations were characterized as variants of unknown significance (VUS). None of these mutations were previously described in the literature, and their finding in patients with polycythemia suggests a pathogenic likelihood (Table 2). Conclusion: In one third of JAK2-negative patients with polycythemia, despite extensive workup, no underlying cause was found. Sleep apnea was the main cause of secondary forms, even in young patients, underlining the importance of performing a polysomnography in the workup of such patients. The NGS erythrocytosis panel offers easy and accessible characterization of some idiopathic forms, however there is still a majority of these patients unable to be characterized. In this cohort the investigation with NGS showed 4 mutations which have not been reported in the literature. The presence of these in patients with polycythemia suggests the probability of a relationship in its pathogenesis. Figure 1 Figure 1. Disclosures Jalowiec: Kite Pharma Gilead Sciences': Honoraria, Speakers Bureau. Rovo: AG Alexion: Research Funding; CSL Behring: Research Funding; Novartis: Research Funding; AG Alexion: Honoraria; BMS: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria; OrPhaSwiss GmbH: Honoraria; Swedish Orphan Biovitrum AG: Honoraria; Amgen: Other: Financial support for congresses and conference travel; AstraZeneca: Other; BMS: Other; Sanofi: Other; Roche: Other.

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