17p Deletion Research Articles

A Phase 2 Study of Acalabrutinib, Umbralisib, and Ublituximab (AU2) in Treatment-Naïve and Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL)

Background Targeting the B-cell receptor (BCR) signaling pathway is highly effective for the treatment of CLL. However, attainment of undetectable minimal residual disease (uMRD) is infrequent with current BCR targeting strategies. Here, we report results of a phase 2 study testing time-limited dual BCR inhibitor therapy with a BTK inhibitor acalabrutinib and a PI3Kδ inhibitor umbralisib. With the goal of enhancing depth of remission, a glycoengineered anti-CD20 antibody ublituximab was initiated during Cycle (C) 7. Methods In this investigator-sponsored trial, eligible patients with treatment-naïve (TN) or relapsed or refractory (RR) CLL received up to 24 cycles of AU2 (NCT04624633). Each cycle was 28 days. Daily oral doses of acalabrutinib (100mg BID) and umbralisib (800mg QD) began on C1 Day (D) 1. Ublituximab IV began on C7 for 6 cycles (D1, 2, 8, and 15 of C7, D1 of C8-12). Treatment was stopped after C12 if patients achieved complete response (CR) by IWCLL criteria, while all others continued acalabrutinib and umbralisib until C24. In March 2023, umbralisib was withdrawn from drug development, and those who were receiving study therapy at the time continued acalabrutinib alone until C24. The primary endpoint was the CR rate after C24. MRD was assessed in peripheral blood (PB) and bone marrow (BM) using multi-color flow cytometry at 10 -4. Results The study enrolled 29 patients from December 2020 to January 2022. The median age at enrollment was 63 years (range 40-77). Most patients were male (62%), had TN CLL (72%), and had unmutated IGHV (79%). RR CLL patients had a median of 1 prior line of therapy, most frequently (88%) with chemoimmunotherapy. Genetic alterations were commonly observed at study enrollment, including deletion 17p or TP53 mutation (21%), deletion 11q or ATM mutation (38%), trisomy 12 (24%), and mutations of NOTCH1(45%), SF3B1 (24%), and RAS/RAF pathway (21%). 7% had complex karyotype defined as ≥3 abnormalities. At a median follow-up of 23 months (range 2-29), with 7 patients still receiving therapy prior to reaching C24, there were no deaths, Richter's transformations, or CLL progression events on the study. The rates of CR, BM uMRD, and PB uMRD at best response were 30%, 44%, and 67%, respectively (Table 1). There were no statistically significant differences in CR and uMRD rates between TN and RR groups at best response. CR and BM uMRD rates improved during the second year of therapy (CR rate: 11% after C12, 28% after C24; BM uMRD rate: 22% after C12, 39% after C24). PB uMRD occurred earlier than BM uMRD, and the rate (44%) remained unchanged between C12 and C24. After completion of study therapy, PB MRD was monitored every 3 months. Five patients had MRD conversion at the end of therapy or shortly after treatment cessation. Median time from the last uMRD to detectable MRD (dMRD) in PB was 3 months (range 3-6). Most (80%, n=4/5) patients with MRD conversion had partial response or dMRD in BM at best response. Two patients stopped treatment early (due to the diagnosis of second cancer and G4 transaminitis, in 1 patient each). In addition, 7 (24%) patients stopped umbralisib between C15-19 due to discontinuation of drug supply; these patients remained on acalabrutinib monotherapy until C24. All others completed up to 24 cycles of treatment including 3 (10%) patients who stopped therapy per protocol after C12 once CR was achieved. Dose reduction of umbralisib (38%) was more common than acalabrutinib (7%) and was most frequently due to diarrhea (21%) and transaminitis (14%). Only 2 (7%) patients required temporary dose reduction of acalabrutinib to avoid drug-drug interaction. Notable adverse events included diarrhea or colitis (76%, 10% Grade 3 or higher [G≥3]), bruising (69%, 0% G≥3), transaminitis (62%, 17% G≥3), COVID-19 infection (55%, 7% G≥3), hypertension (52%, 10% G≥3), rash (24%, 0% G≥3), and pneumonitis (7%, 3% G≥3). One (3%) patient had Pneumocystis jirovecii pneumonia while being off of mandatory prophylaxis due to non-compliance. None had atrial fibrillation. Conclusions Dual inhibition of the BCR signaling pathway with up to 2 years of AU2 provided a time-limited treatment option for TN and RR CLL patients. Immune-related adverse events were frequently observed and required dose reduction of umbralisib in 38% of the patients but most were then able to stay on therapy. Longer follow-up is required to determine durability of remission off therapy.

Primary and Secondary Plasma Cell Leukemia Risk Stratification By Circulating Plasma Cells

Plasma cell leukemia (PCL) is a rare plasma cell dyscrasia associated with poor outcomes. PCL can present de novo as primary PCL (pPCL) or from preceding multiple myeloma as secondary PCL (sPCL). The International Myeloma Working Group (IMWG) in 2021 updated the diagnostic criteria for pPCL using circulating plasma cells (CPC) to ≥5% instead of ≥20% as both carry similar survival. In this single center retrospective analysis, we compared clinical features and overall survival (OS) for 104 patients: with pPCL ≥5% CPC ( N = 35), sPCL 5-19% CPC (sPCL-A) ( N = 11), sPCL ≥20% CPC (sPCL-B) ( N = 38), and spillover group 1-4% CPC regardless of newly diagnosed or secondary status ( N = 20). Mean ages were 57 - 59 years old for all groups (range: 25 - 84) with no difference in age between groups ( p = 0.90). There was a slight predominance of females (54.8%) over males and blacks (53.9%) over non-blacks. More patients with pPCL were black (65.7%) compared to sPCL and spillover groups, although this difference was not statistically significant ( p = 0.35). pPCL patients presented with the highest mean WBC at diagnosis (23.4 x10 9/L) compared to sPCL-A (5.5 x10 9/L), sPCL-B (12.2 x10 9/L), and spillover (7.2 x10 9/L) groups ( p <0.001). Patients in all groups were anemic (hemoglobin 7.8 - 9.3 g/dL), thrombocytopenic (platelets 60.2 - 101.2 x 10 9/L), and had abnormal renal function (creatinine 1.4 - 2.7 mg/dL), elevated LDH (918.7 - 1515.5 units/L, highest in sPCL-A), and elevated uric acid levels (5.9 - 8.2 mg/dL). sPCL patients and spillover group had more lytic lesions on skeletal survey ( p = 0.009) and positron emission tomography (PET) ( p = 0.02) compared to patients with pPCL. sPCL-A patients had higher incidences of lymphadenopathy ( N = 5/11, 62.5%, p = 0.02) and central nervous system invasion ( N = 4/11, 36.4%, p = 0.02) compared to other groups. There was no difference in incidence of extramedullary disease ( p = 0.29) between groups. Over 90% of patients with PCL had high-risk cytogenetics with 63% carrying a complex karyotype. Hypodiploidy was seen more often in patients with pPCL and sPCL-B compared to sPCL-A and spillover ( p = 0.005). There was no difference between groups in incidence of deletion 17p ( p = 0.53). All patients benefited in terms of OS from daratumumab (HR 0.44, 95% CI 0.25 - 0.76; p = 0.004), IMiDs (HR 0.13, 95% CI 0.08 - 0.23; p <0.001), PIs (HR 0.43, 95% CI 0.25 - 0.75; p = 0.003), chemotherapy (HR 0.56, 95% CI 0.32 - 0.98; p = 0.04), and autologous hematopoietic stem cell transplantation (HR 0.33, 95% CI 0.16 - 0.71; p = 0.004). DVT-PACE type regimens (dexamethasone-bortezomib-thalidomide-cisplatin-doxorubicin-cyclophosphamide-etoposide) are used for pPCL and may be less effective in sPCL and spillover patients who have been heavily pretreated. We noted no benefit in OS for all patients including pPCL, sPCL-A, sPCL-B, and spillover groups who received DVT-PACE (HR 0.97, 95% CI 0.62 - 1.50, p = 0.88). The lack of OS benefit in pPCL patients receiving DVT-PACE persisted on multivariate analysis when adjusting for PCL group alone (HR 0.79, 95% CI 0.50 -1.26; p = 0.33) and PCL group, age, platelet count, and presence of high-risk cytogenetics (HR 1.11, 95% CI 0.63 - 1.94; p = 0.72). After median follow-up of 6.70 months, median OS was 7.14 months for the entire cohort with 92.3% ( N = 96) deceased. On univariate analysis, male sex, age >58 years old, high-risk cytogenetics, LDH >604 units/L, creatinine >1.40 mg/dL, hemoglobin <8.3 g/dL, platelets <53.5 x10 9/L, lytic lesions on skeletal survey and on PET, extramedullary disease on PET, and plasmacytoma were associated with inferior median OS (Table 1). pPCL had higher median OS (13.1 months) compared to sPCL-A (3.1 months, p = 0.03) and sPCL-B (3.5 months, p = 0.003) (Figure 1). There was no statistically significant difference in median OS between pPCL (13.1 months) and spillover patients (10 months) ( p = 0.48) as well as sPCL-A (3.1 months) and sPCL-B (3.5 months) ( p = 0.80). Although the spillover group was associated with a trend towards better OS compared to sPCL-A and sPCL-B groups, this was not statistically significant, which may be due to low N which is a limitation of the study. Further research is required to better understand the clinical features, treatment responses, and outcomes of pPCL, sPCL, and spillover group (1-4% CPC).

Inhibition of SIRT6 aggravates p53-mediated ferroptosis in acute lung injury in mice

Although studies have shown that protein 53 (p53)-mediated ferroptosis is involved in acute lung injury (ALI), the mechanism of its regulation remains unclear. The protective effects of Sirtuin 6 (SIRT6), a histone deacetylase, have been demonstrated in multiple diseases; however, further studies are needed to elucidate the role of SIRT6 in ALI. In the present study, we hypothesize that SIRT6 protects against lipopolysaccharide (LPS)-induced ALI by regulating p53-mediated ferroptosis. We observed that the inhibition of ferroptosis prevented LPS-induced ALI. The knockout of p53 blocked LPS-induced ferroptosis and ALI, suggesting that p53 facilitated ALI by promoting ferroptosis. In addition, the inhibition of SIRT6 aggravated LPS-induced ferroptosis and ALI, while the depression of ferroptosis blocked the exacerbation of lung injury induced by SIRT6 inhibition. The results suggest that SIRT6 protects against ALI by regulating ferroptosis. Furthermore, the inhibition of SIRT6 reinforced the p53 acetylation and the deletion of p53 rescued the exacerbation of ferroptosis induced by SIRT6 inhibition. The findings indicate that SIRT6 regulates the acetylation of p53 and prevents p53-mediated ferroptosis. In conclusion, our results indicate that SIRT6 protects against LPS-induced ALI by regulating p53-mediated ferroptosis, thereby demonstrating that SIRT6 holds great promise as a therapeutic target for ALI.

Chromosome 1p36 candidate gene ZNF436 predicts the prognosis of neuroblastoma: a bioinformatic analysis

BackgroundGenetic 1p deletion is reported in 30% of all neuroblastoma and is associated with the unfavorable prognosis of neuroblastoma. The expressions and prognosis of 1p candidate genes in neuroblastoma are unclear.MethodsPublic neuroblastoma cohorts were obtained for secondary analysis. The prognosis of 1p candidate genes in neuroblastoma was determined using Kaplan-Meier and cox regression analysis. The prediction of the nomogram model was determined using timeROC.ResultsFirst, we confirmed the bad prognosis of 1p deletion in neuroblastoma. Moreover, zinc finger protein 436 (ZNF436) located at 1p36 region was down-regulated in 1p deleted neuroblastoma and higher ZNF436 expression was associated with the longer event free survival and overall survival of neuroblastoma. The expression levels of ZNF436 were lower in neuroblastoma patients with MYCN amplification or age at diagnosis ≥ 18months, or with stage 4 neuroblastoma. ZNF436 had robust predictive values of MYCN amplification and overall survival of neuroblastoma. Furthermore, the prognostic significance of ZNF436 in neuroblastoma was independent of MYCN amplification and age of diagnosis. Combinations of ZNF436 with MYCN amplification or age of diagnosis achieved better prognosis. At last, we constructed a nomogram risk model based on age, MYCN amplification and ZNF436. The nomogram model could predict the overall survival of neuroblastoma with high specificity and sensitivity.ConclusionsChromosome 1p36 candidate gene ZNF436 was a prognostic maker of neuroblastoma.

Radiogenomics Provides Insights into Gliomas Demonstrating Single-Arm 1p or 19q Deletion.

IDH-mutant gliomas are further divided on the basis of 1p/19q status: oligodendroglioma, IDH-mutant and 1p/19q-codeleted, and astrocytoma, IDH-mutant (without codeletion). Occasionally, testing may reveal single-arm 1p or 19q deletion (unideletion), which remains within the diagnosis of astrocytoma. Molecular assessment has some limitations, however, raising the possibility that some unideleted tumors could actually be codeleted. This study assessed whether unideleted tumors had MR imaging features and survival more consistent with astrocytomas or oligodendrogliomas. One hundred twenty-one IDH-mutant grade 2-3 gliomas with 1p/19q results were identified. Two neuroradiologists assessed the T2-FLAIR mismatch sign and calcifications, as differentiators of astrocytomas and oligodendrogliomas. MR imaging features and survival were compared among the unideleted tumors, codeleted tumors, and those without 1p or 19q deletion. The cohort comprised 65 tumors without 1p or 19q deletion, 12 unideleted tumors, and 44 codeleted. The proportion of unideleted tumors demonstrating the T2-FLAIR mismatch sign (33%) was similar to that in tumors without deletion (49%; P = .39), but significantly higher than codeleted tumors (0%; P = .001). Calcifications were less frequent in unideleted tumors (0%) than in codeleted tumors (25%), but this difference did not reach statistical significance (P = .097). The median survival of patients with unideleted tumors was 7.8 years, which was similar to that in tumors without deletion (8.5 years; P = .72) but significantly shorter than that in codeleted tumors (not reaching median survival after 12 years; P = .013). IDH-mutant gliomas with single-arm 1p or 19q deletion have MR imaging appearance and survival that are similar to those of astrocytomas without 1p or 19q deletion and significantly different from those of 1p/19q-codeleted oligodendrogliomas.

Accurate interpretation of p53 immunohistochemical patterns is a surrogate biomarker for TP53 alterations in large B-cell lymphoma

BackgroundTo clarify the relationship between p53 immunohistochemistry (IHC) staining and TP53 alterations (including mutations and deletions) in large B-cell lymphomas (LBCLs) and to explore the possibility of p53 IHC expression patterns as surrogate markers for TP53 alterations.MethodsA total of 95 patients diagnosed with LBCLs were selected, and paraffin samples were taken for TP53 gene sequencing, fluorescence in situ hybridization and p53 IHC staining. The results were interpreted by experienced pathologists and molecular pathologists.ResultsForty-three nonsynonymous TP53 mutations and p53 deletions were detected in 40 cases, whereas the remaining 55 cases had wild-type TP53 genes. The majority of TP53 mutations (34/43, 79.1%) occurred in exons 4-8, and R248Q was the most common mutation codon (4/43, 9.3%). The highest frequency single nucleotide variant was C > T (43.6%). p53 expression was interpreted as follows: Pattern A: p53 staining was positive in 0%-3% of tumor cells, Pattern B: p53 staining was positive in 4-65% of tumor cells, Pattern C: more than 65% of tumor cells were stained positive for p53. The p53 IHC expression patterns were associated with TP53 alterations. Gain of function variants and wild-type TP53 tended to exhibit type C and B p53 expression patterns, but loss of function variants were exclusively seen in type A cases. Additionally, interpretation of the staining by various observers produced significant reproducibility.ConclusionsThe p53 IHC expression patterns can be used to predict TP53 alterations and are reliable for diverse alteration types, making them possible surrogate biomarkers for TP53 alterations in LBCLs.

Targeting sphingosine kinase 1 in p53KO thymic lymphoma.

Sphingosine kinase 1 (SK1) is a key sphingolipid enzyme that is upregulated in several types of cancer, including lymphoma which is a heterogenous group of malignancies. Treatment for lymphoma has improved significantly by the introduction of new therapies; however, subtypes with tumor protein P53 (p53) mutations or deletion have poor prognosis, making it critical to explore new therapeutic strategies in this context. SK1 has been proposed as a therapeutic target in different types of cancer; however, the effect of targeting SK1 in cancers with p53 deletion has not been evaluated yet. Previous work from our group suggests that loss of SK1 is a key event in mediating the tumor suppressive effect of p53. Employing both genetic and pharmacological approaches to inhibit SK1 function in Trp53KO mice, we show that targeting SK1 decreases tumor growth of established p53KO thymic lymphoma. Inducible deletion of Sphk1 or its pharmacological inhibition drive increased cell death in tumors which is accompanied by selective accumulation of sphingosine levels. These results demonstrate the relevance of SK1 in the growth and maintenance of lymphoma in the absence of p53 function, positioning this enzyme as a potential therapeutic target for the treatment of tumors that lack functional p53.

OR17-05 Lysine Demethylase KDM1A And Ectopic Expression Of GIP-Receptor In Somatotropinomas Of Patients With Paradoxical Response To Oral Glucose

Abstract Disclosure: F. Chasseloup: None. L. Tosca: None. D. Regazzo: None. A. Proust: None. M. Hage: None. E. Kuhn: None. C. Jublanc: None. K. Mokhtari: None. S. Salenave: None. S. Gaillard: None. F. Parker: None. A. Boch: None. G. Tachdjian: None. P. Chanson: None. J. Bouligand: None. G. Occhi: None. P. Kamenicky: None. Introduction: Paradoxical increase of GH following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing’s syndrome. Most patients also displayed a deletion of chromosome 1p, including the KDM1A locus in their adrenal tissues, resulting in complete loss of KDM1A expression. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism. The aim of our study was to search for genetic abnormalities of KDM1A in somatotroph pituitary adenomas. Methods: We collected somatotropinoma specimens from acromegalic patients followed in two tertiary endocrine centers in France and one in Italy. Somatic DNA was studied by targeted exome NGS and array-CGH. GIPR and KDM1A expression was quantified in the tumors using digital droplet PCR. Results: We included 186 patients: 108 patients (70.6 %) had a classic pathological GH response after oral glucose load, whereas 45 patients (29.4%) displayed a paradoxical rise of GH concentrations. Patients with a paradoxical response displayed higher IGF-1 levels (360 ± 111.8 % above ULN vs. 309.8 ± 107.3 %, p= 0.0130) and less invasive and smaller tumors (14.5 ± 5.58 mm vs. 18.5 ± 8.73 m, p= 0.0066). Amongst the 146 somatotropinoma specimens analyzed by targeted-NGS, no tumor harbored a KDM1A pathogenic variant, and 35 tumors harbored GNAS mutations (6 with and 29 without paradoxical GH responses). We identified a recurrent 1p deletion encompassing the KDM1A locus in 26 tumors, which were more frequently but not exclusively found in patients with paradoxical GH response compared to those with classic GH response. Somatic deletion of one KMD1A allele was associated with lower KDM1A expression (p=4.5e-5) and higher GIPR expression (p=0.0005). Discussion: Unlike in GIP-dependent PBMAH, we did not identify KDM1A genetic variants in a large cohort of acromegalic patients, independently of their GH response pattern to oral glucose loading. We identified recurrent 1p deletion in some tumors. Pituitary adenomas with a loss of one KDM1A copy due to chromosome 1p deletion harbored higher levels of GIPR transcripts than adenomas diploid for the KDM1A locus. If KDM1A haploinsufficiency leads to partial transcriptional derepression at the GIPR locus and a paradoxical rise of GH after glucose load warrants further investigations. Presentation: Saturday, June 17, 2023

Contribution of BAP1 loss and p16 (CDKN2A) deletion analysis to the definitive diagnosis of mesothelioma in effusion cytology.

The cytological diagnosis of mesothelioma is a controversial issue, and definitive diagnosis often requires ancillary tests. The aim of this study was to investigate the contribution of BRCA1-associated protein (1) (BAP1) loss and p16 (CDKN2A) homozygous deletion (HD) on the early diagnosis of mesothelioma in effusion fluids. Between 2019-2022, 21 pleural and peritoneal fluid samples diagnosed with atypical mesothelial proliferation in our institution were included in the study. The slides of the cases that underwent BAP1 immunohistochemistry (IHC) were retrieved from the archive and re-examined. Homozygous deletion (HD) of p16 (CDKN2A) was investigated by the fluorescence in situ hybridization (FISH) method in cell blocks of cytology samples. At least 100 atypical mesothelial cells were counted in each case, and the HD threshold value was >10%. The mean age of the cases was 63.47 years (34-90 years), female/male ratio was 3/1. Of the pleural mesothelioma cases, 16 were epithelioid, 2 were biphasic, and 1 were sarcomatoid. Two cases were diagnosed with peritoneal well-differentiated papillary mesothelioma (WDPM). BAP1 loss was observed in 11 (69%) of 16 cases. HD deletion of p16 (CDKN2A) was seen in 11 (58%) patients with FISH. The HD threshold value was 10-20% in 6 of the cases, 30-50% in 3 cases, and above 90% in 2 cases. While HD deletion was observed in p16 (CDKN2A) in all biphasic and sarcomatoid cases (n=3), no deletion was observed in peritoneal WDPM (n=2). Positivity was observed with at least one method in 12 (86%) of 14 pleural mesotheliomas who underwent both BAP1 IHC and p16 (CDKN2A) FISH. Due to technical reasons, the FISH signal could not be obtained in two cell blocks, so no results could be obtained. Asbestos exposure in areas where mesothelioma is endemic and/or the presence of proliferating mesothelial cells in cytological examination are important clues for diagnosis. In controversial cases, BAP1 IHC should be the first step in an ancillary test. Although the FISH method applied to cell blocks has cytology-specific limitations and difficulties, investigating the p16 (CDKN2A) deletion with FISH in selected cases will contribute to the diagnosis.

The p53 Family of Transcription Factors Represses the Alpha- fetoprotein Gene Expression in Hepatocellular Carcinoma.

p53 deletion and mutation as well as upregulation of alpha-fetoprotein (AFP) are hallmarks of hepatocarcinogenesis. p63 and p73 belong to the family of p53-related transcription factors expressing a variety of isoforms. The expression of dominant negative (ΔN) p73 is related to the reduced survival of patients with hepatocellular carcinoma (HCC). In this study, we characterized the interaction between p53 family-dependent signaling pathways and the regulation of AFP at the gene and protein levels as essential determinants of therapeutic response and prognosis in HCC. Putative p53-, p63- and p73-binding sites within the AFP gene were identified in silico. Hep3B cells were transfected with plasmids encoding for p53, p63 and p73 to analyze the interplay of the p53 family with AFP. AFP transcription was determined by RT-qPCR. Protein levels of AFP, p53, p63 and p73 were analyzed by Western blot. Underlining the importance of the crosstalk between the p53 family-dependent pathways and AFP regulation we identified eight novel putative binding sites for the members of the p53 family within the introns 1, 2, 3, 4, 7, 8, 11, and 12 of the AFP gene. Accordingly, full-length isoforms of p53, p63 and p73 efficiently downregulated AFP both on mRNA and protein level. Thus, the p53 family members were identified to be major regulators of AFP repression. Of note, p63 was characterized as a novel and p73 as the most efficient repressor of AFP. p53 mutation and upregulation of AFP are essential oncogenic events in the development of HCC. Here we show that AFP gene regulation occurs via a combined action of the p53 family members p53, p63 and p73. All three tumor suppressors reduce AFP gene and protein expression. Thus, our findings reveal a novel interaction of p53 family-dependent signaling pathways and AFP regulation at the gene and protein levels in HCC.

Application of R2-ISS risk stratification to patients with multiple myeloma treated with autologous stem cell transplants at UAMS

AbstractThe Second Revision of the International Staging System (R2-ISS) was published in 2022 and has been validated in several cohorts of patients with multiple myeloma (MM). In this study, we investigated a total of 860 patients with MM who received an upfront autologous stem cell transplantation between 2001 and 2021. The median age of the patients was 60 years, with a median overall survival (OS) of 123 months and median progression-free survival (PFS) of 70 months. We collected the variables included in the ISS, R-ISS, and R2-ISS systems as well as additional standard variables. Our analyses demonstrated that all 3 ISS series systems (ISS, R-ISS, and R2-ISS) exhibited robust discrimination in terms of both OS and PFS among our study cohort. The ISS system effectively stratified patients into 3 risk groups, whereas the R-ISS system accurately identified patients at extremely high or low risk. The R2-ISS system further refined risk stratification by dividing patients into 4 more balanced risk groups. Furthermore, we specifically focused on identifying variables that distinguished patients with OS < 3 years and OS > 10 years within the low-risk R2-ISS stages (I and II) and high-risk R2-ISS stages (III and IV). Our findings revealed that age, hemoglobin, and 1p deletion significantly influenced the classification of patients in the low-risk R2-ISS stage. Additionally, serum light chain, platelet count, age, and the presence of the t(14;16) translocation were found to affect high-risk classification.

Transcription factor E4F1 dictates spermatogonial stem cell fate decisions by regulating mitochondrial functions and cell cycle progression

BackgroundSpermatogonial stem cells (SSCs) provide a foundation for robust and continual spermatogenesis in mammals. SSCs self-renew to maintain a functional stem cell pool and differentiate to supply committed progenitors. Metabolism acts as a crucial determinant of stem cell fates; however, factors linking metabolic programs to SSC development and maintenance are poorly understood.ResultsWe analyzed the chromatin accessibility of undifferentiated spermatogonia at the single-cell level and identified 37 positive TF regulators that may have potential roles in dictating SSC fates. The transcription factor E4F1 is expressed in spermatogonia, and its conditional deletion in mouse germ cells results in progressive loss of the entire undifferentiated spermatogonial pool. Single-cell RNA-seq analysis of control and E4f1-deficient spermatogonia revealed that E4F1 acts as a key regulator of mitochondrial function. E4F1 binds to promotors of genes that encode components of the mitochondrial respiratory chain, including Ndufs5, Cox7a2, Cox6c, and Dnajc19. Loss of E4f1 function caused abnormal mitochondrial morphology and defects in fatty acid metabolism; as a result, undifferentiated spermatogonia were gradually lost due to cell cycle arrest and elevated apoptosis. Deletion of p53 in E4f1-deficient germ cells only temporarily prevented spermatogonial loss but did not rescue the defects in SSC maintenance.ConclusionsEmerging evidence indicates that metabolic signals dictate stem cell fate decisions. In this study, we identified a list of transcription regulators that have potential roles in the fate transitions of undifferentiated spermatogonia in mice. Functional experiments demonstrated that the E4F1-mediated transcription program is a crucial regulator of metabolism and SSC fate decisions in mammals.Graphical

Bladder Neoplasia in Pediatric Patients-A Single-Center Experience Including a Case Series.

Objective: Bladder lesions like urothelial carcinoma are rare in the first two decades of life. A biopsy of the bladder or urinary cytological examination is seldom required. Gross painless hematuria is the most relevant clinical syndrome. Methods: A retrospective analysis of surgical pathology records collected between 1984 and 2014 at our institution was performed in a search for cases of urothelial neoplasms originating within the urinary bladder in pediatric patients. Diagnoses were confirmed based on pathologic examination using the 2004 World Health Organization (WHO) classification system. We selected keywords such as bladder neoplasia, bladder lesion, urothelial neoplasia, rhabdomyosarcoma, and children. In addition, we describe clinical presentation and diagnostic procedures as well as treatment and follow-up of two patients. A review of the literature was performed to analyze recommendations concerning diagnostic staging, treatment, and follow-up examinations as well as surveillance of urothelial tumors in the pediatric population. Results: Screening the pathology database of the Institute of Medical Genetics and Pathology of the University Hospital Basel between 1988 and 2014 yielded 287 samples involving the urinary bladder, 110 autopsies, 135 biopsies, and 42 cytology specimens. Of these, most samples originated from malformations and inflammation. Only five were tumors: two were urothelial tumors and three were rhabdomyosarcomas. The majority of specimens comprised resections of the diverticula or distal ureter. Our case reports include two patients with a urothelial tumor. Among the urothelial tumors, one was a papillary urothelial neoplasm of low malignant potential (PUNLMP). Painless hematuria was the directing clinical symptom. The tumor was investigated by FISH, and a 9p21 deletion was found. The second tumor-like lesion was a fibroepithelial polyp arising from the bladder neck. Conclusions: Bladder tumors in children are rare and mostly consist of urothelial and mesenchymal neoplasms. Rhabdomyosarcoma is the most common malignant bladder tumor in childhood. Similar to adult urothelial neoplasms, the loss of 9p21 is also implicated in urothelial neoplasms in childhood. Despite an increasing number of case reports and small series published within the last 2 decades, general treatment protocols including recommendations for staging, tumor markers, and follow-up examinations are still not yet available for this tumor entity in the pediatric population.

In Vivo Intra-Uterine Delivery of TAT-Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53-Deficient Endometrial Cancers.

Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans-activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT-Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high-grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.

Optical Genome Mapping Reveals the Complex Genetic Landscape of Myeloma.

Fluorescence in situ hybridization (FISH) on enriched CD138 plasma cells is the standard method for identification of clinically relevant genetic abnormalities in multiple myeloma. However, FISH is a targeted analysis that can be challenging due to the genetic complexity of myeloma. The aim of this study was to evaluate the potential of optical genome mapping (OGM) to detect clinically significant cytogenetic abnormalities in myeloma and to provide larger pangenomic information. OGM and FISH analyses were performed on CD138-purified cells of 20 myeloma patients. OGM successfully detected structural variants (SVs) (IGH and MYC rearrangements), copy number variants (CNVs) (17p/TP53 deletion, 1p deletion and 1q gain/amplification) and aneuploidy (gains of odd-numbered chromosomes, monosomy 13) classically expected with myeloma and led to a 30% increase in prognosis yield at our institution when compared to FISH. Despite challenges in the interpretation of OGM calls for CNV and aneuploidy losses in non-diploid genomes, OGM has the potential to replace FISH as the standard of care analysis in clinical settings and to efficiently change how we identify prognostic and predictive markers for therapies in the future. To our knowledge, this is the first study highlighting the feasibility and clinical utility of OGM in myeloma.

P16.01.A THE CASE OF AGGRESSIVE DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR IN ADULT TREATED WITH CRANIOSPINAL IRRADIATION AND TEMOZOLOMIDE

Abstract BACKGROUND Diffuse leptomeningeal glioneuronal tumor (DLGNT) is a rare tumor, which was introduced as a separate entity in the 2016 World Health Organization classification of central nervous system tumors. It is most common in pediatric age, but very rare in adults. Literature demonstrates limited reports regarding treatment strategy in adult. We present a case of young female affected by DLGNT with aggressive behavior. MATERIAL AND METHODS A 28-year-old woman presented with severe headache and vomiting. Brain MRI showed diffuse leptomeningeal enhancement with hydrocephalus. At first, she was under suspicion of tuberculosis meningitis with mild increased protein level in CSF study. Her symptom rapidly improved after ventriculoperitoneal shunt and she started anti-tuberculosis medication. Despite 8-month of medication, her neurological status gradually deteriorated with paraparesis and follow-up MR images showed progression of disease. She underwent open biopsy of superficial brain lesions. RESULTS Intraoperatively, multiple nodular masses were attached inner side of dura mater and diffuse adhesion on subarachnoid space of brain surface. Histological examination revealed small round cells with oligodendroglial-like aspects. Immunohistochemistry showed positivity for Olig2, synaptophysin and S100. Chromosome 1p deletion was confirmed by FISH. Next-generation sequencing profile disclosed mutation of H3K28M, FGFR1 and ATRX. Diagnosis was consistent with DLGNT. She received craniospinal irradiation of 36 Gy in 20 fractions, then started chemotherapy with temozolomide (TMZ) at a dose 200mg/m2/day for 5 days in 4 weeks. After 4 cycles of TMZ, MR images demonstrated stable disease. CONCLUSION DLGNT is a very rare tumor in adult. Diagnosis needed high level of suspicion and histological confirmation. We present a very rare adult DLGNT patient with aggressive clinical features who showed stable state treated with craniospinal irradiation and TMZ chemotherapy.

Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF-RIP3 pathway was dependent on p53.

Heatstroke is a life-threatening disease. Present study was aimed to investigate the mechanism in heat induced intestinal epithelial cell death. Heat stress in vitro model was established on IEC cells with 42℃ for 2h. Caspase-8 inhibitor, Caspase-3 inhibitor, RIP3 inhibitor, TLR3 agonist, poly(I:C) and p53 knockdown were used to determine the signaling pathway. Heatstroke in vivo model was established on C57BL/6 mice, with a temperature of 35.5℃±0.5℃ and a relative humidity of 60% ± 5%. The intestine necroptosis and inflammatory cytokines were measured. Pifithrin α (3mg/kg) and p53 knockout mice were used to evaluate the role of p53. Heat stress-induced reduction of cell viability was remarkable reversed by RIP3 inhibitor. Heat stress induced upregulation of TLR3 and facilitate the formation of TRIF-RIP3 complex. The heat stress induced upregulation of RIP3 and p-RIP3 were normalized by the deletion of p53. Meanwhile, p53 knockout decreased TLR3 expression and blocked the formation of TLR3-TRIF complex. The deletion of p53 blocked the decreased cell viability and restored the activation of RIP3-MLKL signaling after heat stress, however, which were abolished by re-expression of p53 via Tp53 OE. Increased the expression of TLR3 in the p53-deficient cells could not affect the heat stress induced necrotic cell death, which suggests that heat stress induced necroptosis via TLR3-TRIF-RIP3 signaling pathway is dependent on p53. Heat stress promoted p53 phosphorylation, then upregulated TLR3 and enhanced the interaction of TRIF-RIP3, which would activate the RIP3-MLKL signaling pathway to mediate necroptosis in intestinal epithelial cells.

17p Deletion in Patients with De Novo Acute Myeloid Leukemia and their Clinico-Haematologic Features

Objective: To determine the frequency of 17p deletion in patients with Acute Myeloid Leukemia and their clinical-hematologic features using fluorescence in situ hybridization.&#x0D; Study Design: Cross-sectional study.&#x0D; Place &amp; Duration of Study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Dec 2018 to Dec 2019.&#x0D; Methodology: All diagnosed cases of Acute Myeloid Leukemia of all ages and genders were included. Interphase FISH testing was performed using blood or bone marrow specimens using 10μL of the Meta systems XL p53 probe, and a total of 500 nuclei per assay were analyzed using a fluorescent microscope. Del 17p positivity and negativity were noted. Clinico-haematologic features of the patients with and without del 17p were also noted.&#x0D; Results: In our targeted population of 84 patients, there were 25(29.8%) females and 59(70.2%) males. The mean age of presentation was 36.3±1.6 years. The mean total leucocyte count(TLC)was 31.2±4.2×109/L, and the mean platelet count was 69.3±4.5×109/L. Del 17p was detected in a total of 8(9.5%) patients. The median age of patients with del 17p at diagnosis was 33 years, the mean TLC was 12.32±10.36x109/L, and the mean platelet count was 80±26.73*109/L. Prominent clinical features among patients with del 17p included fever and pallor.&#x0D; Conclusion: Our study suggests a relatively low frequency of del 17p in AML (9.5%), which is consistent with international data.

Fluorescence In Situ Hybridization (FISH) for Detection of c-MYC Amplifications in AML Patients: A Potential Marker for Risk Stratification

Abstract Background c-MYC is a transcription factor (located on 8q24) which regulates genes involved in proliferation, cell growth, differentiation, and apoptosis. The c-MYC amplification is related to a rapid proliferation of the affected cell clone in patients with myeloid neoplasms. Gene amplification (amp) is one of the basic mechanisms connected with over-expression of oncogenes. c-MYC is routinely analyzed in mature lymphoid neoplasms especially in Burkitt’s Lymphoma while not assessed for acute myeloid leukemia (AML).Despite the fact the c- MYC oncogene is commonly over-expressed in AML it has remained an elusive target. Routine assessment for c- MYC in AML may pave the way for targeting the c-MYC in treatment of AML patients. Several experiments are ongoing investigating both direct and indirect c-MYC targeting therapies and further establishing cMYC as an oncogene of central importance to AML pathogenesis and treatment. Aim of the Work In this study we aimed to define the prognostic value of c-MYC amplification in adult AML patients, and to relate it to other prognostic factors in AML. Patients and Methods The study was a randomized control study, conducted at the Clinical Pathology Department, Ain-Shams University Hospitals during the period from January 2019 till June 2020. It was carried on 50 adult AML patients (30 newly diagnosed and 20 on Therapy) and 10 healthy age-matched volunteers as control. All subjects underwent full history taking, thorough clinical examination, laboratory investigations including; CBC, Examination of Leishman’s stained peripheral blood films, Bone marrow aspiration and examination of Leishman’s stained bone marrow smears, Cytochemical studies using Myeloperoxidase stain, Immunophenotyping of bone marrow or peripheral blood samples and Fluorescence in situ hybridization analysis (FISH). Results we had 9 patients with c-MYC amplification (6 in the newly diagnosed group and 3 in the on therapy group). So the percentage of c-MYC amplification in our patients was 9/50 (18%). To further elucidate the effect of c-MYC amplification on our patients’ clinical, laboratory and prognostic factors, an analytical comparison was done between negative and positive patients for cMYC amplification as regard-studied parameters in each group of patients. Both group of patients showed a statistical significant association between c-MYC amplification and outcome where the majority of patients with positive c-MYC amplification achieved incomplete remission. In addition, c-MYC amplification showed a statically significant association with Trisomy 8 in both patients’ groups and with 17p deletion and hepatosplenomegaly in on therapy group. Univariate logistic regression analysis for the association between c-MYC amplification and outcome showed statistically significant association between c-MYC amplification and incomplete remission in newly diagnosed AML patients with P-value = 0.013 and odds ratio (95% CI) of 14.0. Conclusion Our study has shown that c-MYC amplification is associated with adverse prognosis, poor treatment outcome and it might be involved in the process of AML progression. Targeting cMYC may be of value in the development of new-targeted AML therapeutics.

New Prognostic Indicators in CLL: Relation to Disease Burden

Abstract Background Identifying prognostic markers is important for the clinical and pathological course of chronic lymphocytic leukemia (CLL). Cluster of differentiation CD49d expression and percentage of smudge cells in CLL patients have recently been reported as newer prognostic factor. Aim of the Study To estimate CD49d expression as an independent prognostic marker in CLL patients and to investigate its relation to the known prognostic factors Methods Thirty adults with CLL were enrolled. Smudge cells percentage was calculated by microscopic evaluation of blood smears. Testing of cluster of differentiation CD49d expression was done by flowcytometry and detection of ataxia telangiectasia mutated (ATM) gene, protein 53(P53) gene deletions and trisomy 12 were performed using fluorescent in situ hybridization (FISH). Results CD49d expression was associated with lymphadenopathy, splenomegaly, advanced staging, high total leucocytic count, immunophenotyping scoring, CD38 positivity, short LDT, low smear cell percentage, and presence of p53 deletion. Low percentage of Smear cell (&amp;lt;30%) showed a significantly higher incidence of lymphadenopathy and organomegaly. Additionally, it was associated with a high total leucocytic count, low platelet count, high atypical lymphocytic count and typical IPT scoring, shorter LDT, positive CD38 expression and CD49d expression and ATM and P53 deletions. Conclusion CD49d expression and low smudge cell percent were consistently associated with poor prognostic indicators, suggesting that they could be considered as adverse predictors and they should be merged together to improve the power of the prognostic panels of CLL in the clinical setting.