Citations

e20044 Background: Primary Cutaneous Diffuse Large B-cell Lymphoma, Leg-type (PCDLBCL) is a rare lymphoma comprising 4% of all cutaneous lymphomas with an aggressive clinical course. We investigated disease characteristics and outcomes among patients PCDLBCL. Methods: Following IRB approval, we identified patients retrospectively with PCDLBCL treated at the Mayo Clinic Cancer Center 1998-2019. Results: 45 pts were identified with a median age of 71 years (Range- 48-95) and 28 (62%) were female. Disease locations were legs (62%), arms (22%), head (11%), trunk (2%) and multiple sites (2%). At diagnosis, (51%) had a single lesion, (29%) had multiple lesions in 1-2 contiguous regions, (16%) had multiple lesions in non-contiguous regions or > 2 regions and data was not available (4%). Immunohistochemistry showed positive staining for BCL-2 (92%), BCL-6 (76%), c-MYC (62%), MUM1 (95%), CD10 (11%), and cytoplasmic IgM (8/9- 89%). By Han’s criteria, (95%) had activated B cell lymphoma (ABC). Median Ki-67 was 90% (range, 50,100%). 9/12 (75%) were positive for MYD88 L265P mutation. 13/21 (62%) were double expressor and 3/22 (14%) had double hit lymphoma. 37/45 (82%) had ECOG < 2. LDH was elevated in 13/37 (35%). The median number of therapies was 1 (range; 1-11). The initial treatments were chemo-radiation (30%), chemo alone (38%), rituximab alone (11%), radiation (RT) alone (16%), rituximab-RT (2.5%), and surgical (2.5%). The median follow-up time for the whole cohort was 80 months (95% CI; 32, 169). The median progression free survival and overall survival (OS) were 20 months (95% CI; 12, 34) and 80 months (95% CI; 48, 119). 22/45 (49%) are alive. The cause of death was disease-related in 11/23 (48%). The median number of relapses was 1 (range; 0, 11) with (60%) relapsed after first line. 4/45 pts had autologous stem cell transplant after relapse. 7/45 (16%) had a systemic relapse at a median time of 35 months from diagnosis (range; 16, 112). Age above 60 years was associated with worse OS; 70 months (95% CI; 30, 99) vs NR (95% CI; 135, NR), p = 0.025. ECOG performance status < 2 was associated with better OS, NR months (95% CI; 80, NR) vs 48 months (95% CI; 3.3, 88),p = 0.0003. 1 pt received Ibrutinib on 11th relapse and had 3 years of PFS. Conclusions: PCDLBCL is predominantly ABC type, affects elderly pts and is characterized by multiple cutaneous relapses followed in some cases by systemic relapses. Increased age and poor performance status had a negative impact on OS. High incidence of MYD88 L265P mutation is observed in PCDLBCL similar to immune-privileged site lymphomas.

e20036 Background: Hepatosplenic T cell lymphoma (HSTL) is a rare subtype of mature T cell lymphomas accounting for less than 1 percent of non-Hodgkin lymphomas. it has been associated with poor prognosis. It typically involves sinusoids in the spleen, liver, and bone marrow. Methods: Following IRB approval, we retrospectively evaluated patients with HSTL treated at the Mayo Clinic Cancer Center 1996-2019. Kaplan-Meier survival analysis and univariate analysis to identify prognostic factors were performed. We investigated clinical characteristics and outcomes among patients with HSTL. Results: 22 patients (15 males) with median age of 45.5 years (range- 15.5-80.6) were identified. The majority of patients had B symptoms (12/22), cyotpenias (20/22), massive splenomegaly (16/22), liver involvement (13/22), and/or bone marrow involvement (14/22). Additionally, 42% tested positive for peripheral blood involvement and 8/22 (36%) were on chronic immunosuppression related to solid organ transplant (2), inflammatory bowel disease (2), and rheumatologic disorders (4). Phenotypically, the majority of patients were positive for gamma/delta (18/22), isochromosome 7q (8/13), and 4/9 (44%) tested positive for trisomy 8. Initial therapies included CHOP/CHOEP (14), ICE (2), EPOCH (1), pentostatin (1), and radiation (1). The median follow-up was 33 months (range 2,137). Progression free survival and overall survival (OS) were 9.5 months (95% CI; 1.8, 16.3) and 12 months (95% CI; 4.9, 18.5) respectively. 10/22 (45%) underwent splenectomy with one patient receiving splenectomy alone as treatment. 8/22 (36%) had stem cell transplant (SCT- 5 allogeneic and 3 autologous). Out of the 22 patients 4 (18%) were long-term survivors with 55, 74, 95, and 137 months; 3/4 had splenectomy at diagnosis and 2/4 had allogeneic SCT. The longest survivor had splenectomy only. Liver involvement was associated with worse OS; 10 months (95% CI; 1.6, 18) vs 35 months (95% CI; 4, NR), p = 0.036. Immune suppression at diagnosis was associated with worse OS; 5 months (95% CI; 1.3, 18) vs 18 months (95% CI; 9, 74), p = 0.036. A trend for better OS was seen with splenectomy and SCT. Conclusions: HSTL is a rare lymphoma with poor prognosis. Long-term survival was seen in 18% only. Liver involvement and immune suppression had a negative impact on OS. A larger cohort is needed to better evaluate the impact of different treatment options.

e19543 Background: Langerhans cell sarcoma (LCS) is a very rare cancer with the reported incidence of 2 per 100,000,000. It originates from antigen presenting cells belonging to the dendritic cell/macrophage family. Methods: Following IRB approval, we identified LCS patients treated at the Mayo Clinic Cancer Center. Medical charts were reviewed to extract clinical data. Results: Our cohort consists of 5 patients with confirmed diagnosis of LCS. The median age at diagnosis was 34 years (range, 11-75) with 60% (3/5) females. One female was an African American and the rest were Caucasians. ECOG performance status at the time of diagnosis was 0 in 4 patients and 3 in 1 patient. Four patients had disseminated disease at diagnosis. 3/5 patients had moderate splenomegaly at diagnosis. 2/5 patients presented with a bulky mediastinal tumor and 1/5 patients presented with bulky intraabdominal disease. Predominant sites of disease are summarized in the table. BRAF mutation was negative in all 3 tested patients. The median follow up for the whole cohort was 25 months (range; 5, 41). Three patients relapsed after the initial treatment. Patient # 4 received allogeneic stem cell transplantation after the 4th relapse and is in CR 14 months later. Patient # 5 had PD-L1 expression > 95% and was started on pembrolizumab with sustained response for 1 year. Conclusions: LCS is extremely rare and is associated with unfavorable outcomes after currently available therapeutic interventions. More studies on the genetic and molecular basis of LCS are necessary to develop targeted therapies. [Table: see text]

Abstract Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. Significance: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery. This article is highlighted in the In This Issue feature, p. 1241

Abstract Background: Maturity onset diabetes in young 3 (MODY 3) is caused by mutation of the hepatic nuclear factor 1 alpha (HNF-1A) gene. Craniofacial macrosomia (CFM) is associated with an abnormal development of craniofacial structures during the embryonic period. Maternal diabetes and genetic predisposition have been associated with CFM1. There are rare reports about an association of MODY 3 and CFM. Clinical case: An 11-year-old male patient presented with right side CFM (mild mandibular hypoplasia, internal auditory canal absence, severe pinna hypoplasia, abnormal orbital size and location, O3.M0.E3.N0.S02) noted at 8 months of age. Preoperative examination revealed A1c at 10.9%. After short term intensive insulin therapy, he had standard bread meal test: fasting glucose 8.11 mmol/L, insulin 13.9 mIU/L (3-25), C-peptide 1.25 ng/ml (0.81-3.85); 1 hour glucose 10.05 mmol/L, insulin 27 mIU/L, C-peptide 2.42 ng/ml; 2 hour glucose 8.17 mmol/L, insulin 16.09 mIU/L, C-peptide 2.11 ng/ml. GADA, IAA and ICA were negative. The mother was diagnosed diabetes at age 27years, when the patient was 8-month-old, and received insulin therapy. The mother was blind by age 35years due to diabetic retinopathy and died of DKA at 38-years-old. The patient’s 16-year-old brother had left side CFM (O2.M1.E2.N0.S0) and his OGTT was normal. The father was diagnosed with impaired glucose tolerance. The family had whole genome sequencing by Sanger technique, and resequenced the mutation with side primers. The CGA to CAA mutation was present at the 686 loci of exon 3 of HNF1A gene in the patient and mother. The HNF1A exon 3 mutation of CGA to CAA resulted in the change of arginine to glutamine which by the HGMA database is recognized as a reported MODY3 gene mutation. There was a mutation of G to A in the 4 loci of exon 1 of the insulin coding region in chromosome 11 in both the patient and elder brother. Neither elder brother nor father had the CGA mutation of HNF1A. Conclusion: There has not been a previous report of a relationship between HNF1A and CFM. In this case, the elder brother had CFM without a HNF1A mutation which does not support a connection between CFM and HNF1A. The two brothers both had CFM and insulin coding gene mutations which would represent a new association not previously described. Further testing is needed to confirm a relationship between the two. Reference: 1. Chen Q, Zhao Y, Shen G, Dai J. Etiology and Pathogenesis of Hemifacial Microsomia. J Dent Res 2018; 97(12): 1297-305. 2. Gougoutas AJ, Singh DJ, Low DW, Bartlett SP. Hemifacial microsomia: clinical features and pictographic representations of the OMENS classification system. Plast Reconstr Surg 2007; 120(7): 112e-20e.

Abstract Background: Previous studies have proposed combination of various factors that predict poor mobilization after GCSF stimulation. However, there is no single clinical or laboratory test that reliably correlates with bone marrow reserve and PBPC mobilization. Peripheral blood CD34+ cell count (pCD34) reliably correlates with PBPC mobilization and yield but only after GCSF administration. Methods: We have prospectively followed 36 autologous PBPC transplant candidates, from the time of initial evaluation to transplant, to evaluate factors the correlate with bone marrow reserve and autologous PBPC mobilization. Results: The patients median age was 63 (range 26 – 76). 21 of the 36 patients were diagnosed with dysproteinemia, primarily multiple myeloma, 10 patients had non-Hodgkin’s lymphoma while the remaining 5 had other form of hematological malignancies. Our evaluation shows baseline platelet count prior to growth factor administration significantly correlate with total CD34+ cell yield (spearman: r = 0.64, p &lt;0.001, Figure 1). In addition, daily platelet count during PBPC harvest correlate with CD34+ cell yield for the day (spearman: r = 0.6, p &lt;0.001). Using multiple linear regression model (F = 19.1, p &lt;0.001, R2 = 0.64), we have determined patients’ age (p = 0.03) and type of disease (p &lt;0.001), in addition to platelet count (p&lt;0.001), significantly correlate with total CD34+ cell yield. Further analysis showed low baseline platelet count predicts poor PBPC mobilization. Using Receiver Operating Curve (ROC), we have determined that platelet count of 174,000/ul was the best cut-off for collecting at least 2 million/Kg CD34+ cells with sensitivity of 81% and specificity of 78% and estimated correct classification of 81%. Even though pCD34 counts were slightly more sensitive and were better at identifying poor mobilizers in our cohort (best cut-off 10/ul, sensitivity 96%, specificity 67%, correct classification 89%), their clinical utility were diminished since the values were only known after GCSF administration. Conclusion: Baseline platelet count is a sensitive predictor of bone marrow reserve and can be used prior to growth factor administration to predict poor mobilization. Figure 1: Correlation of baseline platelet count and total CD34+ cell yield Figure 1:. Correlation of baseline platelet count and total CD34+ cell yield

INTRODUCTION Patients with peripheral artery disease (PAD) are at a heightened risk of cardiovascular events and have markedly reduced vascular function and exercise tolerance. Heat therapy (HT) has emerged as a practical treatment to improve cardiovascular health in young individuals as well in patients with overt cardiovascular disease. We recently reported that a single 90 min session of HT applied to the legs of patients with PAD using water‐circulating trousers increases popliteal artery blood flow and reduces blood pressure and the circulating levels of the potent endogenous vasoconstrictor endothelin‐1 (ET‐1). The objective of the present study was to test the hypothesis that the repeated exposure to leg HT would lower blood pressure and ET‐1 levels and improve walking performance and conduit artery and cutaneous vascular function in patients with symptomatic PAD. METHODS Thirty‐two patients with symptomatic PAD (Age: 68± 8 years; Ankle‐brachial index: 0.67 ± 0.1) were assigned using a balanced randomized design to undergo 90 min of HT (48°C) or a thermoneutral treatment (33°C) 3 times per week for 6 consecutive weeks. The following outcomes were assessed at baseline and 3 and 6 weeks after the intervention: 1) resting ankle‐brachial index, 2) post‐occlusive reactive hyperemia in the popliteal artery (phase contrast magnetic resonance imaging), 3) leg cutaneous thermal hyperemia (laser Doppler flowmetry), 4) resting blood pressure every 5 min for 70 min, 5) 6‐min walk distance and 6) serum levels of ET‐1 and total nitrate (NOx). RESULTS Participants completed 17.7 ± 0.6 treatment sessions, resulting in a compliance rate of 98.7%. In the group exposed to HT, systolic blood pressure (SBP) decreased from baseline by ~3 mmHg at 6 weeks, while in the control group SBP increased by ~1 mmHg. A significant group by time interaction was observed for the changes in SBP from baseline to 6 weeks ( p=0.0049) with significant group differences at 20 min (Change in SBP: Control: 6.27 ± 15.5 vs. HT: −7.60 ± 13.6 mmHg, p=0.015 ) and 60 min (Change in SBP: Control: 4.13 ± 15.1 vs. HT: −7.33 ± 13.7 mmHg, p=0.0386 ). Similarly, serum ET‐1 levels decreased by ~15% from baseline to week 6 following exposure to HT, while a ~6% increase in this parameter occurred in the control group (Change in ET‐1: Control: 0.16 ± 0.6 vs. HT: −0.30 ± 0.4 pg/mL, p=0.035 – one outlier removed). Resting ABI, diastolic BP, 6MWT distance, hyperemic peak flow and cutaneous thermal hyperemia were not different between groups. CONCLUSION Exposure to repeated HT reduces systolic blood pressure and ET‐1 levels in patients with symptomatic PAD. These findings indicate that HT may be a practical and accessible nonpharmacological tool to reduce cardiovascular risk in PAD patients. Support or Funding Information American Heart Association Scientist Development grant (16SDG27600003).

Cardiac troponin I (cTnI) is a key regulator of cardiomyocyte contraction. Phosphorylation of cTnI at Ser22/23 is mediated by protein kinase A, C, and G, and is associated with reduced Ca 2+ sensitivity of myocardial contraction. In previous in vivo and in vitro studies, we showed that hypothermia/rewarming (rewarming shock) reduces the Ca 2+ sensitivity of myocardial contraction due to cTnI Ser22/23 phosphorylation. Rewarming shock was also found in isolated cardiomyocytes; however, demonstrating a relationship with cTnI phosphorylation is complicated by varying cardiomyocyte viability after isolation. Using a modified Langendorff technique, the cardiomyocyte viability gradually declines over a 4‐h period from ~80% to ~40%. This decline in cell viability is not accounted for in typical Western blot measurements and, therefore, assessments of cTnI phosphorylation are highly subject to misrepresenting actual physiological changes in cTnI phosphorylation. To address this issue, we developed a novel quantitative technique to measure cTnI phosphorylation at Ser22/23 in viable cardiomyocytes using confocal microscopy. We demonstrated that cardiomyocytes with a rod‐shaped morphology and uniform striation patterns display robust transient contractile and cytosolic Ca 2+ responses to electrical stimulation. All of these rod‐shaped cardiomyocytes exclude trypan blue, while a disproportionate fraction of non‐rod‐shaped cardiomyocytes are non‐viable. Separately, using 2D gel electrophoresis, we validated the specificity of an antibody that targets phosphorylated cTnI. Using this antibody, we verified that the extent of cTnI phosphorylation in rod‐shaped cardiomyocytes increased with isoproterenol in a dose dependent fashion. This novel methodology allows a more accurate measurement of cTnI phosphorylation in isolated primary cardiomyocytes that is not affected as cell viability declines over time. Support or Funding Information This publication was made possible by Grant Number T32 HL 105355 from the National Heart, Lung and Blood Institute (NHLBI), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIH.

Inflammation and pro‐inflammatory cytokines (e.g., TNFα, IL‐13) induce airway smooth muscle (ASM) hyper‐reactivity and remodeling characteristic of asthma. We hypothesized that exposure to TNFα (and IL‐13) increases isometric force generation in porcine ASM via several underlying mechanisms: 1) increasing sensitivity to ACh stimulation; 2) enhancing the cytosolic Ca 2+ ([Ca 2+ ] cyt ) response to ACh; 3) increasing the Ca 2+ sensitivity of force generation; 4) enhancing the Ca 2+ ‐dependent phosphorylation of myosin regulator light chain (rMLC); 5) promoting expression of non‐muscle rMLC; 6) increasing actin and myosin heavy chain (MyHC) content (i.e., the number of contractile units); and/or 7) promoting cytoskeletal remodeling (actin polymerization). Studies were performed on intact ASM strips (~1 mm wide × ~5 mm long) dissected from porcine tracheas obtained from a local abattoir. The ASM strips were placed in PSS with and without TNFα (100 ng/ml) for 24 h. The ACh concentration dependence of force generation ASM was determined (ACh range from 10 nM to 1mM), and the EC50 (i.e., the ACh concentration that produces 50% of maximal ASM force) was determined. In control, untreated ASM, the EC50 for ACh was 2.6 μM compared to 1.3 μM following TNFα treatment. We found that across all ACh concentrations, ASM force increased after TNFα treatment with an ~50% increase in maximum ASM force. The [Ca 2+ ] cyt response induced by the EC50 [ACh] was similar in control and TNFα treated ASM. Thus, with an increase in force and no effect on [Ca 2+ ] cyt , there was an apparent increase in Ca 2+ sensitivity of force after TNFα treatment. However, in both control and TNFα treated ASM, the extent of rMLC phosphorylation induced by ACh stimulation was not affected by TNFα treatment. Importantly, the extent of rMLC phosphorylation at the ACh EC50 was comparable between control and TNFα treatment indicating no change in Ca 2+ sensitivity. The expression of non‐muscle rMLC (determined by 2D gel electrophoresis) was also unaffected by TNFα treatment. Total actin and MyHC concentrations increased by ~45% following TNFα treatment; thus, the number of contractile units contributing to force increased. This was reflected by a marked increase in the Ca 2+ activation of ATP consumption (cross‐bridge cycling) of ASM following TNFα treatment. Cytoskeletal remodeling was evaluated by ACh‐induced actin polymerization (F‐/G‐actin ratio) but was found to be unaffected by TNFa treatment. Together, our results show that two primary mechanisms underlie the marked increase in ASM force generation induced by 24‐h TNFα exposure: 1) an increase in ACh sensitivity, and 2) an increase in actin and MyHC concentrations (number of contractile units). Support or Funding Information Supported by NIH grant HL126451 (GCS) and HL150890 (GCS)