Predictive factors of endometriosis progression into ovarian cancer

Background In recent years, the endometriosis has overcome a noteworthy renaissance in the recognition of its potential. In certain patients, a demonstrable malignant progression of ectopic foci leading to development of ovarian cancer is seen. The knowledge of endometriosis overthrow background into endometriosis associated ovarian cancer is of paramount importance for selection of patients at risk. The goal of the presented study was to review a malignant potential of the endometriosis and to specify predictive factors of endometriosis progression into ovarian cancer. Altogether 189 patients were included in the study. Conventional cytogenetics as well as measurement of transcriptional activity of CTNNB1 (β-catenin) and HIF1A (HIF1-α) genes were prospectively studied in 60 endometriosis patients and 50 control group patients. The retrospective histopathological analysis was performed in 19 endometriosis associated ovarian cancer patients and 60 patients with histologically confirmed endometriosis. Results Five endometriosis patients showed a deviation from normal cytogenetics finding without affecting of their phenotype. In 6 cases of endometriosis associated ovarian cancer ectopic endometrium was not confirmed. The remaining 13 cases demonstrated either benign or atypical endometriosis or even structures of borderline carcinoma. Atypical endometriosis was histologically confirmed in 20% of 60 endometriosis patients. Determination of gene expression (CTNNB1, HIF1A) formed two subgroups. Transcriptionally incipient endometriosis subgroup with insignificant genes expression compared to control group. In transcriptionally evident endometriosis subgroup were genes expressions significantly higher compared to control group (p < 0.01) as well as transcriptionally incipient endometriosis subgroup (p < 0.05). Conclusions Significant structural abnormalities of chromosomes are not included in genetic rigging of endometriosis patients. Atypical endometriosis represents a histopathologically detectable intermediate of endometriosis progression. Determination of genes expression CTNNB1 and HIF1A helps to allocate risk patients with endometriosis where more precise management is needed.

A Novel Machine Learning-Derived Molecular Classification Scheme with Prognostic Significance

The gold standard for the diagnosis of MDS relies on morphologic alterations hampered by a great deal of subjectivity. Cytogenetic and clinical features allow for clinical classifications predictive of survival. However, with a few exceptions (SF3B1MT, del5q, and certain balanced translocations), neither classic histo-morphology nor prognostic scoring systems (e.g., IPSS-R) are reflective of pathogenic underpinnings. To date supervised analyses of mutational data did not succeed to produce profiles specific or predictive of traditional disease sub-entities. Large cohorts with clinical annotation and a sufficient follow-up allow for innovative biostatistical approaches to subgroup patients according to molecular profiles. Objective operator-independent subcategorization may be congruent with common pathogenic links, rational applications of targeted therapeutics and better prognostications. We hypothesized that machine-learning (ML) strategies used for analysis of mutational/cytogenetic profiles will enable recognition of invariant disease subcategories according to their molecular configurations. Herein, we compiled a meta-analytic database (our cohorts and publicly available sources) of 3,011 MDS (median age 71yrs) and 6,788 pAML/sAML. Results of deep targeted sequencing of a panel of 55 myeloid mutations were collected together with cytogenetics. We then performed unsupervised analysis of MDS and AML patients using Bayesian Latent Class Analysis (BLCA). A consensus matrix was then clustered using Ward's criteria to generate final cluster assignment based on the highest silhouette value. To identify genomic signatures, we used Random Forest classification and extracted mutations with highest global importance indicated by mean decrease in accuracy. Using BLCA we identified 5 unique genomic clusters (GCs) with 3 distinct prognostic outcomes [low risk (LR), intermediate risk (Int), and high risk (HR)] that were validated by survival analysis (Fig.1A,B). The LR group included GC-1 and was characterized by the highest prevalence of normal cytogenetics (100%) and SF3B1 MT (25%) with co-occurring DNMT3A MT (14%), and absence of ASXL1 MT, ETV6 MT, STAG2 MT, TP53 MT, and complex/abnormal cytogenetics. Int group included GC-2 and GC-4. GC-2 was characterized by a higher percentage of abnormal cytogenetics cases than LR group and absence of STAG2 MT, SRSF2 MT, ASXL1 MT, TP53 MT, and normal/complex cytogenetics. GC-4 had the highest frequency of SRSF2 MT (52%) with co-occurring ASXL1 MT (59%), TET2 MT (40%), normal karyotype, and absence of complex/abnormal cytogenetics. Finally, HR included GC-3 and GC-5. GC-3 included ASXL1 MT (67%) with co-occurring SRSF2 MT (47%), TET2 MT (37%), STAG2 MT (22%), and absence of normal cytogenetics. GC- 5 had the highest frequency of -5/del(5q) (50%), -7/del(7q) (43%), -17/del(17p) (16%) and the highest odds of complex karyotype (92%) as well as TP53 MT (48%). Paralleling the genomic ML-based clustering, the clinical relevance of these subgroups was reflected in significantly different survivals [median (95% CI)]: i) GC-1 [69 (59-80)], ii) GC-2 [35 (29-41)], iii) GC-3 [12 (10-16)], GC-4 [27 (22-34)], and GC-5 [9 (7-11)] months (Fig.1C). We then classified the MDS cohort according to the recently published and validated AML GCs (Awada et al Blood 2021) to investigate overlapping genomic features. Overall, 90% of MDS GC-1 and 67% of MDS GC-2 had the same molecular architecture of AML GC-2 and 70% of MDS GC-5 had the same molecular features of AML GC-4. In addition, 98% of MDS GC-3 and 92% of MDS GC-4 had the same features of AML GC-3 (Fig.1D). In sum, we propose a novel objective molecular classification of MDS and related diseases that allows subgrouping of patients according to shared pathogenesis for a better prognostic resolution without errors derived from subjectivity. The model was then internally and externally validated using a cohort of 200 cases. Results of a validation cohort and online URL site of molecular clustering will be presented at the meeting. Figure 1 Figure 1. Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Patel: Alexion: Consultancy, Other: educational talks, Speakers Bureau; Apellis: Consultancy, Other: educational talks, Speakers Bureau. Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astex: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

Clinical Characteristics and Prognosis of Thirty-Three Patients with Myeloid Neoplasms and DDX41 Mutation: Mayo Clinic Experience

Background: The DEAD-box helicase 41 (DDX41), an RNA helicase, have been described as a component of the RNA spliceosome (Cheah et al. International Journal of Hematology 2017). Although DDX41 mutations predispose to late-onset higher grade myeloid neoplasms (MN), these patients may have a trend toward favorable prognosis and outcomes. In this work, we describe the clinical characteristics and survival outcomes of isolated and co-mutated DDX41 patients (pts). Methods: We retrospectively analyzed 4,524 consecutive pts who underwent next-generation sequencing (NGS) (OncoHeme 42 genes panel, Mayo Clinic) testing and included 32 pts harboring pathogenic DDX41 mutation and one pt with proven DDX41 germline variant of unknown significance (VUS). Chart review of DDX41-mutated (m) cases between 2009 and 2021 was conducted after IRB approval. We compared overall survival (OS) of unmatched 27 t(8;21)AML and 40 inv(16) AML pts with 10 m DDX41-AML pts. JMP® Pro 14.1.0 Software was used for statistical analysis. Results: DDX41 mutations characteristics: Our cohort included 19 (58%) myelodysplastic syndromes (MDS), 10 (30%) acute myelogenous leukemia (AML), 2 (6%) myeloproliferative neoplasms (MPN), one clonal cytopenia of undetermined significance (CCUS) (3%) and one (3%) germline carrier. Germline testing was carried out in 10 pts, 9 of whom (90%) were confirmed to be germline). The start-loss variant (p.M1I) was the most common mutation type (N=10, 31%). Other types were frameshift (N=9, 28%), missense mutation (N=8, 25%), nonsense (N=3, 9%), and splice site mutation (N=2, 2%). Twenty-one (65.6%) DDX41 mutations clustered in the N-terminus (NT), 7 (22%) in the helicase-C domain (HCD), and 4 (12.5%) in the DEAD-box domain. Compared to NT mutations, patients with HCD mutation had no family history of solid tumors and were more likely to have an accompanying additional DDX41-VUS (0% vs 70%; p=.001) and (N=6, 86% vs. N=2,10%; p=.0001); respectively. I solated vs. co-mutated DDX41: Twenty (60%) pts were isolated-DDX41 and 13 (40%) were co-mutated. The median DDX41-VAF was 48% vs. 45% (p= .2) in the isolated compared to the co-mutated cases, respectively. The median number of co-mutations in the 13 co-mutated cases was 1 (range,1-3) with DNMT3A (38%), ASXL1 (30%), JAK2 (N=3, 23%), and EZH2 (N=2, 15%) were the most common co-mutations detected. Isolated DDX41 had more males (85% vs. 54%, p=.05), the p.M1I variant (47% vs. 8%, p=.02), normal cytogenetics (100% vs. 91%, p= .02), and less family history of solid tumors (77% vs. 33%, p= .02) compared to their co-mutated counterparts. However, there was no difference in OS (p=.99). Comparison of clinical characteristics and hematological features of isolated and co-mutated DDX41 pts are reported in (Table 1). Treatment and survival outcomes in DDX41-MDS/AML : Twenty-three (80%) patients were treated, MDS pts received hypomethylating agents (HMA) (N=10, 71%), HMA plus Venetoclax (HMA+VEN) (N=1, 7%), erythropoiesis-stimulating agents (N=2, 14%) and lenalidomide (N=1 ,7%). AML pts were treated with induction chemotherapy (N=6, 67%) and HMA+VEN (N=3, 33%). Overall response rate of MDS/AML patients was 77% and 100% of AML pts achieved complete remission (CR) when treated with induction chemotherapy or HMA+VEN regimen. There was no significant difference in OS between responders vs. non-responders 2-yr-OS (90% vs. 50%; p=.38) and treated vs. untreated 2-yr-OS (83% vs. 100%; p=.52). Comparing m DDX41-AML vs. core binding factor-AML: After a median follow-up of 33.3 months, all m DDX41-AML patients were alive. There was a significantly better OS in mDDX41-AML patients compared to pts with t(8;21) AML with 2-yr-OS (100% vs. 51%; p=.024) and a trend of better survival when compared to inv(16) AML 2-yr-OS (100% vs. 84%; p=.2). Conclusion We describe the characteristics and outcomes of m DDX41 patients. We demonstrated that isolated and co-mutated m DDX41 patients have different features. Isolated DDX41 patients had male predominance, more p.M1I variant, normal cytogenetics and less family history of solid tumors. In this study we found that m DDX41 AML has high response to treatment and has comparable (if not possibly better) OS compared to other "favorable risk" AML. This study, although limited by the small number of patients, supports the universal testing for DDX41 mutation in adults with MN diagnosis. Figure 1 Figure 1. Disclosures Al-Kali: Astex: Other: Research support to institution; Novartis: Research Funding. Foran: abbvie: Research Funding; OncLive: Honoraria; boehringer ingelheim: Research Funding; trillium: Research Funding; pfizer: Honoraria; takeda: Research Funding; revolution medicine: Honoraria; bms: Honoraria; gamida: Honoraria; actinium: Research Funding; aptose: Research Funding; novartis: Honoraria; servier: Honoraria; taiho: Honoraria; syros: Honoraria; sanofi aventis: Honoraria; certara: Honoraria; kura: Research Funding; h3bioscience: Research Funding; aprea: Research Funding; sellas: Research Funding; stemline: Research Funding. Badar: Pfizer Hematology-Oncology: Membership on an entity's Board of Directors or advisory committees. Salama: Mayo Clinic: Current Employment, Other: Mayo Clinic had the contractual work for the central pathology review for this study and I was one of the reviewing pathologists; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Litzow: Astellas: Research Funding; Biosight: Other: Data monitoring committee; Amgen: Research Funding; AbbVie: Research Funding; Actinium: Research Funding; Pluristem: Research Funding; Jazz: Other: Advisory Board; Omeros: Other: Advisory Board. Patnaik: Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Research Funding; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Activity of Limited-Schedule Bendamustine and Methylprednisolone in Chronic Lymphocytic Leukemia

Bendamustine was approved by the United States Food and Drug Administration in 2008 for the treatment of Chronic Lymphocytic Leukemia (CLL). Corticosteroids have been administered in regimens with chemotherapy for CLL for more than 40 years. Despite this, the combination of Bendamustine and Methylprednisolone has not been explored in CLL. We describe our experience with Bendamustine 100 mg/m2 and Methylprednisolone 125 mg IV on days 1 and 2 and Peg-filgastrim 6 mg subcutaneously on day 3 every 28 days for 3-4 cycles in 12 patients with CLL. Cycles have been limited to attempt to prevent cumulative marrow toxicity due to Bendamustine. Patient characteristics: 7 males/5 females, median age-58, Binet stage B (8), C (2), A (2); ZAP70/CD38+ (5), Trisomy 12 (3), 13q- (2), normal cytogenetics (2); median ECOG status 1, no prior therapy in 8. Median number of cycles received = 3 (two received 4 cycles). Most common toxicities: nausea/emesis (5), fever/flu-like symptoms (4), arthralgia/myalgia (4). There have been no treatment-related deaths. One patient was briefly hospitalized for pneumonia. Complete responses have been seen in 6 patients (median duration = 50.7 months; range: 12.1+ to 87.3 months). Partial responses have been seen in 4 patients (median duration = 17.3+ months; range: 3.3 to 26.9+ months). The combination of Bendamustine and Methylprednisolone given in a limited schedule is well-tolerated and is active in CLL. Disclosures No relevant conflicts of interest to declare.

Genetic features and clinical outcomes of patients with isolated and comutated DDX41-mutated myeloid neoplasms

DDX41 mutations (germline and somatic) are associated with late onset myelodysplastic syndromes/acute myeloid leukemia (MDS/AML). Myeloid neoplasms (MN) with germline predisposition was identified as a distinct category in the 2016 WHO classification revision, including MN with germline DDX41 mutation. We retrospectively analyzed the molecular findings and clinical characteristics of thirty-three DDX41-mutated (mDDX41) patients at our institution. We identified 14 distinct pathogenic DDX41 variants in 32 patients and 8 DDX41 variants of unknown significance (VUS) in 9 patients. Five (16%) patients had a second DDX41 somatic mutation p.R525H and 13 (40%) had at least one additional oncogenic co-mutation in other genes. The median age at the time of diagnosis was 66 years, with male predominance (72%) and the majority of patients had normal cytogenetics (91%). Two-year overall survival (OS) was 86% and 6 (21%) MDS/AML patients with relatively preserved hematopoietic function were observed without further intervention. In comparison to AML patients with prognostically more favorable subtypes [t(8;21), n=27 and inv(16), n=40], mDDX41 patients in our cohort showed similarly favorable OS. Our study highlights that mDDX41-MN patients often have an indolent course and mDDX41-AML has comparable OS to favorable-risk AML.

PAX5 P80R mutated acute leukemia followed by genetically-related histiocytic proliferation with clonal IGH gene rearrangements

Introduction/Objective PAX5 is a transcription factor critical for B-cell development and its genetic alterations are common in B lymphoblastic leukemia/lymphoma (B-ALL). We report a case of PAX5 P80R mutated acute leukemia with predominantly monocytic immunophenotype followed by a genetically-related histiocytic proliferation. Methods/Case Report A 27-year-old male presented with pancytopenia, epistaxis, and blurry vision. Bone marrow exam showed 95% blasts with nuclear indentations, occasional prominent nucleoli and basophilic cytoplasm. Blasts were positive for bright CD33, HLA-DR, CD14, bright CD64, partial CD123 and partial CD19. In addition, a minute population of B lymphoblasts positive for CD19, CD20, and dim TdT was seen. The AML FISH panel showed markedly aneuploid population with all probes demonstrating 3 to 5 signals. PAX5 P80R and KRAS G13D mutations were detected by NGS. Post induction bone marrow showed no evidence of acute leukemia with normal cytogenetics and FISH results. Subsequent two bone marrow exams performed due to progressive cytopenias demonstrated a prominent histiocytic proliferation with sheets of mature appearing histiocytes with abundant cytoplasm, oval to folded nuclei and occasional hemophagocytosis. This population was positive for CD163, CD14, CD68R, CD68, cyclin D1 with variable OCT2 expression and low proliferative activity. PAX5 P80R and KRAS G13D were detected. AML FISH panel showed aneuploidy in histiocytic appearing cells and IgH gene rearrangement studies by PCR showed prominent clonal population. The patient remained pancytopenic and died of disseminated fungal infection. Results (if a Case Study enter NA) NA Conclusion PAX5 P80R mutation has been primarily reported in B-ALL and is exceedingly rare in acute myeloid leukemias. This alteration has been linked to downregulation of B-cell lineage genes affecting B-cell maturation, and not surprisingly a proportion of PAX5 P80R mutated B-ALL cases show a switch to a monocytic lineage. The reported case demonstrates diagnostic caveats including unusual features of acute leukemia at the time of initial diagnosis and subsequent genetically-related histiocytic proliferation.

Intermediate between Idiopathic Hypereosinophilia and Chronic Eosinophilic Leukemia: A Report of Two Hypereosinophilic Cases with Possible Novel Molecular Mutations

To distinguish a reactive eosinophilia from its malignant counterpart is challenging. Establishing clonality of the eosinophils is crucial and considered the determining factor for establishing a diagnosis. Cases of hypereosinophilia without clear reactive etiologies, no evidence of end-organ damage, normal cytogenetics, and no molecular mutations are termed as “Idiopathic Hypereosinophilia (IHE).” For cases which lie between the spectrum of chronic eosinophilic leukemia (CEL) and IHE, identification of underlying molecular abnormalities might be helpful in better understanding the disease process and prognosis. Here, we report two cases of hypereosinophilia in which five possible novel molecular mutations were identified by targeted next-generation sequencing (NGS) analysis. They were FBXW7, KM2A, TCF3, ERBB4, and MET. With multiple genetic mutations, these cases could be classified as chronic eosinophilic leukemia. Both these young patients responded well to steroid therapy. While targeted NGS is a useful tool in identifying new molecular mutation associated with hypereosinophilia, our cases raise the question of further investigating this entity and if there is a possibility of an intermediate category lying between the spectrum of CEL and IHE. Defining hypereosinophilia with clonal molecular abnormality as a malignant process may need to be revisited. Even though attempts are being made to identify mutations in IHE, it might be more significant clinically to differentiate them based on response to steroid therapy and prognosis.

Impact of p53 and BCL2 Expression on Prognosis in Patients with Acute Myeloid Leukaemia

Introduction Acute myeloid leukaemia (AML) is a heterogenous disorder that arises from clonal expansion of malignant hematopoietic precursor cells. The tumor suppressor p53 gene plays an important role in regulation of the cell cycle and apoptosis. Somatic mutations of the p53 gene have been reported in 5-10% of all patients with AML, although the rate is higher in therapy-related disease and elderly patients. Alteration or loss of p53 is one of the most powerful independent indicators of poor outcome. BCL2 positivity in AML and its impact on prognosis is less well described. Methods This is a single-centre, retrospective analysis of AML patients treated over a ten year period (2006-2016). Patients were identified through our pathology database. P53 expression (as a surrogate marker for tp53 mutation) and BCL2 expression were analysed by immunohistochemical (IHC) analysis of marrow biopsies (greater than 30% was considered positive). We analyzed AML presentation, management and overall survival (OS), and correlated these with presence of p53 mutation and BCL2 expression. Results We identified 48 patients; the majority were elderly (median age 68.5 years). Thirty-four patients (71%) haddenovopresentation of AML, 6 had underlying Myelodysplastic Syndrome (13%), 4(8%) progressed to AML from underlying Myelofibrosis, and 8% had therapy-associated AML. Patient and disease characteristics are outlined inTable 1. Median blast count by IHC of bone marrow biopsies was 70%. The results of cytogenetic analysis were available for 38 patients (79%). Fourteen patients (37%) had normal cytogenetics, but a wide range of genetic abnormalities were identified(Table 1). NPM1 (for 22 patients) and FLT3 mutation analysis (for 33 patients) were available, and reported as positive in 4(18%) and 5(15%) respectively. p53 overexpression was identified in 6 patients (12.5%). 50% of these had secondary AML, and the remainder weredenovopresentations. Four of these patients (66%) had complex cytogenetics, one had deletion 5q and cytogenetic analysis was not available for the remaining patient. Median age of patients who were p53 positive was 60 years, compared to 70 years in those without p53 overexpression. BCL2 expression analysis showed a median value of 60% (range 5-100%). BCL2 positivity was seen in 39 patients (81.2%); half of this cohort (15 patients) had normal cytogenetics and only five patients (16%) had abnormalities that indicated poor risk cytogenetics (complex, deletion 7q, etc.). Thirty-nine patients (83%) received active treatment and nine received supportive care only due either to frailty or patient choice. Twenty-two patients (60%) had induction chemotherapy, 7(19%) received Azacitadine, and a further 7 patients had low-dose cytarabine. Nineteen patients (51%) had relapsed/refractory disease. Five patients (13.5%) with BCL2 positive disease received Venetoclax as a single agent for relapsed or refractory disease. Median OS was 11.5 months (range 0-72); 39 patients (81%) had died at the time of analysis. 26 died from disease progression (79%), and 7(19%) of sepsis. One patient was lost to follow up. OS in those with wild-type p53 was 11.5 months, which compared favorably to 7.5 months in those with p53 mutation. BCL2 overexpression also conferred a poorer prognosis; median OS was 10 months compared to 17 months in BCL2 negative disease. Conclusion This study reflects the potential use of p53 and BCL2 in real-life practice in assessing prognosis in AML patients outside of a clinical trial setting. We confirmed the previously reported poor prognosis of p53 alterations in AML (OS 7.5 months), and the study also indicates a poorer prognosis in patients with BCL2 positivity. The widespread availability of IHC techniques for trephine biopsies and the potential for rapid turn-around times in routine clinical laboratories suggests that further studies of the prognostic impact of such gene alterations are warranted. Disclosures No relevant conflicts of interest to declare.