This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic ''safe harbor'' sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS AAVS1, hROSA26, and CCR5 was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by in vitro and in vivo cleavage-sensitivity tests, and analyzed for populationlevel and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and-independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introducing a large (17 kb) gRNA-specified deletion in the PAX3/FOXO1 fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor MYF5 in human rhabdomyosarcoma cells. An engineering ''toolkit'' was developed to enable easy use of the most extensively characterized of these new human sites, SHS231, located on the proximal long arm of chromosome 4. The target sites identified here have the potential to serve as additional human SHS to enable basic and clinical gene editing and genome-engineering applications.

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BackgroundThe advent of the immunomodulatory imide drugs (IMiDs) lenalidomide and thalidomide for the treatment of patients with plasma cell myeloma (PCM), has contributed to more than a doubling of the overall survival of these individuals. As a result, PCM patients join survivors of other malignancies such as breast and prostate cancer with a relatively new clinical problem – second primary malignancies (SPMs) – many of which are a result of the treatment of the initial cancer. PCM patients have a statistically significant increased risk for acute myeloid leukemia (AML) and Kaposi sarcoma. IMiD treatment has also been associated with an increased risk of myelodysplastic syndrome (MDS), AML, and squamous cell carcinoma of the skin. However, within these overlapping groups, acute lymphoblastic leukemia (ALL) is much less common.Case presentationHerein, we describe an elderly man with PCM and a 14-year cumulative history of IMiD therapy who developed persistent pancytopenia and was diagnosed with B-cell acute lymphoblastic leukemia (B-ALL). He joins a group of 17 other patients documented in the literature who have followed a similar sequence of events starting with worsening cytopenias while on IMiD maintenance for PCM. These PCM patients were diagnosed with B-ALL after a median time of 36 months after starting IMiD therapy and at a median age of 61.5 years old.ConclusionsPCM patients with subsequent B-ALL have a poorer prognosis than their de novo B-ALL counterparts, however, the very low prevalence rate of subsequent B-ALL and high efficacy of IMiD maintenance therapy in PCM should not alter physicians’ current practice. Instead, there should be a low threshold for bone marrow biopsy for unexplained cytopenias.

3001 Background: FOR46, a fully human antibody (ab) conjugated to monomethyl auristatin E (MMAE), targets a tumor selective epitope of CD46, which is highly expressed in mCRPC and treatment-emergent small cell neuroendocrine cancer (t-SCNC). CD46 is enriched in tumor cells upon treatment with androgen signaling inhibitors (ASI). Following dose escalation (Phase 1a), dose expansion was undertaken in 2 cohorts (Phase 1b): 1) Pts with de novo or t-SCNC and 2) pts with mCRPC without a t-SCNC component. Pts with adenocarcinoma enrolled in dose escalation and expansion are included in this analysis. Methods: Eligible pts had mCRPC, with progression on at least 1 ASI, with no prior chemotherapy for CRPC. Phase 1a pts received FOR46 0.1-3.0 mg/kg IV Q3 weeks (wks). The primary objectives in phase 1a were to assess adverse effects (AEs) and select the phase 1b dose; and in phase 1b to assess efficacy. For phase 1b, tumor biopsy in the CRPC setting for assignment to the 2 cohorts was required. CD46 expression was not required for inclusion in the expansion cohort, but was evaluated using a non-epitope specific CD46 polyclonal ab. Histology and CD46 expression were centrally reviewed. Results: Thirty-three pts were enrolled in phase 1a and 10 in phase 1b (including 6 treated in ph1a at the expansion dose or higher). Overall, 36 pts were treated at doses > 1.2 mg/kg. Following excess toxicity in pts with body mass indices > 30 (3 of 3 with Gr 4 neutropenia and 1 of 3 with Gr 3 fatigue at 2.4 mg/kg), further dosing was calculated using adjusted body weight (AJBW) rather than actual weight, allowing escalation to 3.0 mg/kg. The 2.7 mg/kg dose by AJBW was determined to be the MTD and phase 1b dose. The most common AEs at the 2.7 mg/kg dose were neutropenia (77% Gr 3 or 4), infusion reactions (37%, all < Gr 2), fatigue (31%, all < Gr 2) and peripheral neuropathy (24%, all < Gr 2)). Fourteen of 31 evaluable pts (45.2%) at > 1.2 mg/kg achieved a PSA50 response with 10 (32.3%) confirmed. Five pts were not evaluable for PSA response; 3 had no post-baseline PSA and 2 had baseline PSA < 1 ng/mL. The median duration of confirmed PSA50 response is >16 wks (range 6-48+ wks, with 4 ongoing at 12, 24, 25 and 48 wks). 18 pts had measurable lesions; 8 of 18 (44.4%) had tumor regression, with 4 (22.2%) confirmed partial responses (PR). The median duration of response is > 14 wks (range 9 -31+ weeks with 2 ongoing at 13 and 31 wks). Eight pts were evaluable for CD46 expression with a median H-score of 245 (range 0-300). Two pts with PRs had H-scores of 15 and 300; 4 with confirmed PSA50 had H-scores of 10, 15, 40 and 300. Conclusions: FOR46, a novel ADC targeting CD46, demonstrates clinical activity in mCRPC pts, with an acceptable safety profile, similar to other MMAE-containing ADCs. FOR46 merits further investigation in pts with mCRPC, alone and in combination with agents that enhance CD46 expression. Clinical trial information: NCT03575819.

292 words) Safe Harbor Sites (SHS) are genomic locations where new genes or genetic elements can be introduced without disrupting the expression or regulation of adjacent genes. We have identified 35 potential new human SHS in order to substantially expand SHS options beyond the three widely used canonical human SHS, AAVS1 , CCR5 and hROSA26 . All 35 potential new human SHS and the three canonical sites were assessed for SHS potential using 9 different criteria weighted to emphasize safety that were broader and more genomics-based than previous efforts to assess SHS potential. We then systematically compared and rank-ordered our 35 new sites and the widely used human AAVS1 , hROSA26 and CCR5 sites, then experimentally validated a subset of the highly ranked new SHS together versus the canonical AAVS1 site. These characterizations included in vitro and in vivo cleavage-sensitivity tests; the assessment of population-level sequence variants that might confound SHS targeting or use for genome engineering; homology-dependent and -independent, SHS-targeted transgene integration in different human cell lines; and comparative transgene integration efficiencies at two new SHS versus the canonical AAVS1 site. Stable expression and function of new SHS-integrated transgenes were demonstrated for transgene-encoded fluorescent proteins, selection cassettes andCas9 variants including a transcription transactivator protein that were shown to drive large deletions in a PAX3/FOXO1 fusion oncogene and induce expression of the MYF5 gene that is normally silent in human rhabdomyosarcoma cells. We also developed a SHS genome engineering 'toolkit' to enable facile use of the most extensively characterized of our new human SHS located on chromosome 4p.We anticipate our newly identified human SHS, located on 16 chromosomes including both arms of the human X chromosome, will be useful in enabling a wide range of basic and more clinically-oriented human gene editing and engineering.

The spleen is among the most common extranodal sites for Hodgkin and non-Hodgkin lymphomas (NHLs); however, among lymphomas arising from the spleen, primary splenic lymphomas (PSLs) are rare. The group of PSLs includes primary splenic diffuse large B-cell lymphoma (PS-DLBCL), splenic red pulp small B-cell lymphoma, splenic marginal zone lymphoma (SMZL), and a splenic hairy cell leukemia variant. Delineating between the PSL variants can be challenging, especially as fine-needle aspirate and core needle biopsy of the spleen are not routinely offered at most medical centers. Herein, we describe the clinical course of 2 representative patients who presented with non-specific gastrointestinal symptoms, the first who was diagnosed with PS-DLBCL and the second who was diagnosed with SMZL. We review and contrast the clinical presentations, imaging techniques, and laboratory findings of these discrete lymphoma variants and offer strategies on how to delineate between these varied splenic processes. We also examine the use of splenectomy and splenic needle biopsy as diagnostics and, in the case of splenectomy, a therapeutic tool. Finally, we also briefly review treatment options for these varied lymphoma sub-types while acknowledging that randomized trials to guide best practices for PSLs are lacking.

In 2016, the World Health Organization provisionally classified Epstein-Barr virus (EBV)-positive mucocutaneous ulcer (EBVMCU) as a lymphoid neoplasm under the subcategory of mature B-cell neoplasms. Here we describe a 59-year-old Chinese woman with a long history of recurrent sinus and oropharyngeal infections and was diagnosed initially with EBVMCU and later, diffuse large B-cell lymphoma (DLBCL). She had sought medical attention after a particularly long bout of increased facial pressure and nasal obstruction. As a young adult she had undergone a tonsillectomy, adenoidectomy, and received innumerable treatments for bronchiectasis and recurrent sinopulmonary infections. A sinus computerized tomography (CT) scan showed mucosal thickening and swellings of the left frontal posterior sinus. A subsequent 18fludeoxyglucose (18FDG) positron emission tomography (PET)-CT scan showed a large hypermetabolic mass (standardized uptake value [SUV] 30.6) centered in the left half of the nasopharynx and sinus processes and extending across the mid-line. She underwent an endoscopic turbinate reduction with removal of a polypoid soft-tissue mass. Immunohistochemical studies indicated large atypical lymphoid cells that stained positively for CD20, CD30, EBER-1, MUM-1, OCT-2, and PAX-5 and variably for BCL-6, CD15, CD45, and CD79a. The Ki-67 proliferation index was 100% in the atypical cells. Tissue blocks were reviewed locally and at the National Institute of Health and were felt to be most consistent with EBVMCU. She received 4000 cGy involved-field external-beam radiation therapy over 20 fractions. At completion of treatment, no residual abnormalities were identified and no additional adjuvant therapy was pursued. Three years later she presented to medical attention with shortness of breath and a non-productive cough. A PET-CT scan showed whiteout of the left lung and intense 18FDG uptake in a left perihilar nodule (SUV of 18). Bronchoscopy revealed many reactive polypoid bronchial wall masses and a complete collapse of the lower left lobe due to an obstructing mass. The left lower lobe intraluminal mass biopsies however showed EBV-positive DLBCL of non-germinal center phenotype. She received six cycles of conventional R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine, and prednisone) and 36 months later she remains cancer free but with persistent bronchial inflammatory nodules and recurrent infections. Using the key words "EBV mucocutaneous ulcers," "EBV-associated lymphoproliferative disorders," and "lymphoproliferative disorders," we identified 100 cases of EBVMCU in the literature. EBVMCU is manifested as a well-circumscribed ulcer typically unifocal (90%) and of the oropharynx (58%), gastrointestinal (GI) tract (20%), and skin (20%). Immunohistochemical studies of the ulcers reveal monoclonal B-immunoblasts staining positively for CD20, CD30, EBER-1, MUM-1, OCT-2, and PAX-5 and staining variably for BCL-6, CD15, CD45, and CD79a. EBVMCU is most commonly associated with medication-induced (iatrogenic) immunosuppression (65%), advanced age-associated immunosenescence (27%), and primary (3%) and acquired (3%) immunodeficiencies. The median age for all EBVMCU cases is 68.5 (range, 16 -101) years. Of the 65 cases where medication-induced immunosuppression contributed to EBVMCU, 45 (69%) were in patients greater than 60 years-old. EBVMCU is a predominantly indolent disease reminiscent of post-transplant lymphoproliferative disorders in which conservative strategies are primarily employed with potential escalation to B-cell targeted therapy with or without cytotoxic chemotherapy, resection, and/or radiotherapy. Surveillance for EBVMCU should follow B-cell directed therapies and should be considered in the differential diagnosis in cases of suspected lymphoma relapse. Additionally, other LPDs, including lymphomas, should be monitored after resolution of EBVMCU. Due to overlap of the immunohistochemical profiles and risk factors associated with EBVMCU and other ulcerative LPDs of the oral and sinus cavities, nasopharynx, or GI tract, EBVMCU should be considered in the differential diagnosis to prevent under- or over-diagnosis and their associated potential repercussions. Disclosures No relevant conflicts of interest to declare.

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