Jo-Anne Vergilio

Bactericidal/Permeability-Increasing Protein (rBPI21) and Fluoroquinolone Mitigate Radiation-Induced Bone Marrow Aplasia and Death

Even when given 24 hours after lethal radiation, a fragment of an endotoxin-neutralizing protein plus a fluoroquinolone antibiotic improves survival and hematopoietic recovery in mice. Reducing Risks of Radiation Radiation—intentional or not—kills dividing cells, dealing a double whammy to the body. The demise of dividing intestinal cells renders the gut leaky, letting microbes in and shuttling down the immune cell production line in the bone marrow, leaving the body extra susceptible to the invading microbes. In a new approach to treating these lethal effects of radiation, Guinan et al. deliver a double whammy of their own by combining an antibiotic and an inhibitor of dangerous microbial endotoxin to prevent death by radiation in mice. The dual drugs are effective when given 24 hours after the radiation, a boon for use in a disaster where immediate treatment might not be possible. The authors found a clue for this new approach to radiation mitigation by studying patients irradiated in preparation for a bone marrow transplant. Among the responses was a drop in serum concentrations of BPI (bactericidal/permeability-increasing protein), a protective protein that binds to lipopolysaccharide, which is shed by microbes and can cause severe lethal reactions in patients. They reasoned that replacing the declining BPI could be beneficial and tested this idea in mice. Although BPI alone did not help irradiated mice survive, when BPI was given together with an antibiotic, about 70 to 80% of the animals lived, whereas almost none of the untreated animals were alive after 20 days. The combined BPI/antibiotic therapy also caused a rebound in the number of cells in the bone marrow after their radiation-induced depletion. What makes this combo treatment particularly appealing is the fact that both BPI and the antibiotic have previously been used safely in humans, an important point when calculating the risk-benefit ratio of treating individuals whose exact dose of radiation may not be clear. With nontoxic agents, one can more comfortably treat someone who may have received a small dose. This advantage, plus the fact that, at least in mice, the BPI/antibiotic can be administered a full day after exposure and still be effective, suggests that this double-drug approach should be tested for use in humans. Identification of safe, effective treatments to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the U.S. Food and Drug Administration has yet to approve any agent for a mass-casualty radiation disaster. Because patients undergoing hematopoietic stem cell transplantation undergo radiation treatment that produces toxicities similar to radiation-disaster exposure, we studied patients early after such treatment to identify new approaches to this problem. Patients rapidly developed endotoxemia and reduced plasma bactericidal/permeability-increasing protein (BPI), a potent endotoxin-neutralizing protein, in association with neutropenia. We hypothesized that a treatment supplying similar endotoxin-neutralizing activity might replace the BPI deficit and mitigate radiation toxicity and tested this idea in mice. A single 7-Gy radiation dose, which killed 95% of the mice by 30 days, was followed 24 hours later by twice-daily, subcutaneous injections of the recombinant BPI fragment rBPI21 or vehicle alone for 14 or 30 days, with or without an oral fluoroquinolone antibiotic with broad-spectrum antibacterial activity, including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle plus fluoroquinolone, the combined rBPI21 plus fluoroquinolone treatment improved survival, accelerated hematopoietic recovery, and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 plus fluoroquinolone initiated 24 hours after lethal irradiation, combined with their established favorable bioactivity and safety profiles in critically ill humans, suggests the potential clinical use of this radiation mitigation strategy and supports its further evaluation.

Comprehensive genomic profiling of anal squamous cell carcinoma reveals distinct genomically defined classes

BACKGROUND Squamous cell cancers of the anal canal (ASCC) are increasing in frequency and lack effective therapies for advanced disease. Although an association with human papillomavirus (HPV) has been established, little is known about the molecular characterization of ASCC. A comprehensive genomic analysis of ASCC was undertaken to identify novel genomic alterations (GAs) that will inform therapeutic choices for patients with advanced disease. PATIENTS AND METHODS Hybrid-capture-based next-generation sequencing of exons from 236 cancer-related genes and intronic regions from 19 genes commonly rearranged in cancer was performed on 70 patients with ASCC. HPV status was assessed by aligning tumor sequencing reads to HPV viral genomes. GAs were identified using an established algorithm and correlated with HPV status. RESULTS Sixty-one samples (87%) were HPV-positive. A mean of 3.5 GAs per sample was identified. Recurrent alterations in phosphoinositol-3-kinase pathway (PI3K/AKT/mTOR) genes including amplifications and homozygous deletions were present in 63% of cases. Clinically relevant GAs in genes involved in DNA repair, chromatin remodeling, or receptor tyrosine kinase signaling were observed in 30% of cases. Loss-of-function mutations in TP53 and CDKN2A were significantly enhanced in HPV-negative cases (P < 0.0001). CONCLUSIONS This is the first comprehensive genomic analysis of ASCC, and the results suggest new therapeutic approaches. Differing genomic profiles between HPV-associated and HPV-negative ASCC warrants further investigation and may require novel therapeutic and preventive strategies.

Comprehensive Genomic Profiling of 282 Pediatric Low‐ and High‐Grade Gliomas Reveals Genomic Drivers, Tumor Mutational Burden, and Hypermutation Signatures

This study highlights the value of comprehensive genomic profiling in the largest known cohort of pediatric glioma patients and explores the most common alterations across diagnosis and anatomic location. Tumor mutational burden and associated genetic factors that may predispose patients to developing a hypermutator phenotype are also discussed.

Comprehensive genomic profiles of metastatic and relapsed salivary gland carcinomas are associated with tumor type and reveal new routes to targeted therapies

Abstract Background Relapsed/metastatic salivary gland carcinomas (SGCs) have a wide diversity of histologic subtypes associated with variable clinical aggressiveness and response to local and systemic therapies. We queried whether comprehensive genomic profiling could define the tumor subtypes and uncover clinically relevant genomic alterations, revealing new routes to targeted therapies for patients with relapsed and metastatic disease. Patients and methods From a series of 85 686 clinical cases, DNA was extracted from 40 µm of formalin-fixed paraffin embedded (FFPE) sections for 623 consecutive SGC. CGP was carried out on hybridization-captured, adaptor ligation-based libraries (mean coverage depth, >500×) for up to 315 cancer-related genes. Tumor mutational burden was determined on 1.1 Mb of sequenced DNA. All classes of alterations, base substitutions, short insertions/deletions, copy number changes, and rearrangements/fusions were determined simultaneously. Results The clinically more indolent SGC including adenoid cystic carcinoma, acinic cell carcinoma, polymorphous low-grade adenocarcinoma, mammary analog secretory carcinoma, and epithelial–myoepithelial carcinomas have significantly fewer genomic alterations, TP53 mutations, and lower tumor mutational burden than the typically more aggressive SGCs including mucoepidermoid carcinoma, salivary duct carcinoma, adenocarcinoma, not otherwise specified, carcinoma NOS, and carcinoma ex pleomorphic adenoma. The more aggressive SGCs are commonly driven by ERBB2 PI3K pathway genomic alterations. Additional targetable GAs are frequently seen. Conclusions Genomic profiling of SGCs demonstrates important differences between traditionally indolent and aggressive cancers. These differences may provide therapeutic options in the future.

Unique genomic features in adolescent and young adult, as compared to older adult, non-Hodgkin lymphoma and potential therapeutic targets.

Non-Hodgkin lymphoma (NHL) can occur at any age, and is common in adolescents and young adults (AYA, age 15–39 years). The clinical outcome of AYA NHL can differ compared to younger and older populations. Treatment challenges include choosing between the standard paediatric versus the adult treatment strategies and a low rate of patient enrolment in clinical trials. In addition, there is very limited information on age-related genomic profiles of NHLs to facilitate the clinical investigation of new treatment therapies specific for the AYA population. Next generation sequencing was performed to identify genomic alterations in 195 NHL patients including 75 AYA and 120 older adults (OA) across 11 types of NHL. The details of disease types and clinical characteristics are shown in Table I. Sequence data were assessed for base substitutions, short insertions/deletions, copy number alterations and gene fusions/rearrangements. The genomic profiling of AYA and OA groups revealed both similarities and differences. There were 4 5 genomic alterations (GA) identified per case in AYA compared to 5 1 GA in OA. The top two most commonly altered genes for both groups were the same, including IGH/IGK/IGL rearrangements (35% AYA vs. 33% OA) and TP53 (27% in both). TNFAIP3 was altered in 17% of AYA and 14% of OA. In diffuse large B cell lymphoma (DLBCL) IGH/IGK/IGL rearrangements were the most frequent alterations in both age groups (42% AYA vs. 36% OA). CDKN2A mutations were more common in OA DLBCL (36% OA vs. 7% AYA). Genes that were more frequently altered in AYA DLBCLs compared with OA DLBCLs were B2M (39% AYA vs. 14% OA), TNFAIP3 (36% AYA vs. 14% OA), CD58 (29% AYA vs. 9% OA), CIITA (19% AYA vs. 2% OA) and PDCD1LG2 (19% AYA vs. 0% OA). Genes that were more frequently altered in OA DLBCLs included KMT2D (also termed MLL2; 31% OA vs. 19% AYA), CDKN2B (26% OA vs. 3% AYA), MYD88 (21% OA vs. 3% AYA), CREBBP (19% OA vs. 7% AYA) and CD79B (16% OA vs. 0% AYA). Clinically relevant genomic alterations, defined as GA associated with approved drugs or novel therapies under evaluation in registered clinical trials, were identified in 59% of AYAs and 70% of OAs. ALK fusion and histone modification (HM) genomic alterations displayed different patterns overall and were identified in various NHL subtypes (Table I). Genomic alterations of many other genes and frequencies are shown in Figures S1–S4, and detailed data are shown in Tables S1–S3. ALK fusions were more frequently seen in AYA (7/75; 9%) compared to OA (1/120; <1%) with a significant Pvalue of 0 006. Further division of the age groups showed even more difference in the rate of ALK fusions. Anaplastic large cell lymphoma (ALCL) is more common in AYA and 4/5 of AYA ALCL patients harboured NPM1-ALK fusions (80%). ALK fusions were not identified in the 2 OA ALCLs (0%) (P = 0 14). Three DLBCL AYA patients (10%) had CLTC-ALK or SEC31A-ALK fusions, and only one single NPM1-ALK was found in 58 OA DLBCLs (2%). All ALK fusions have been previously observed in large B-cell lymphomas (Gascoyne et al, 2003; Van Roosbroeck et al, 2010). In a study of 11 ALK-positive NHL patients, 9 of who were AYA, all demonstrated response or stable disease upon treatment with crizotinib (Gambacorti Passerini et al, 2014). Alterations in HM genes have been commonly reported in many cancer types. In the present study, we observed that these alterations were predominantly seen in OA and much less commonly in AYA. The difference in the alteration frequency of HM genes between AYA (13/75; 17%) and OA (47/ 120; 39%) was highly significant (P = 0 001), and included alterations in ASXL1, CREBBP, EP300, EZH2, KDM5A, KMT2D, KMT2C (also termed MLL3), SETD2, and WHSC1 (also termed MMSET) (Fig 1). The rate of HM gene mutation varied with a further breakdown of age groups. HM gene

Next-Generation Sequencing Reveals Pathway Activations and New Routes to Targeted Therapies in Cutaneous Metastatic Melanoma.

Background: Comprehensive genomic profiling of clinical samples by next-generation sequencing (NGS) can identify one or more therapy targets for the treatment of metastatic melanoma (MM) with a single diagnostic test. Methods: NGS was performed on hybridization-captured, adaptor ligation–based libraries using DNA extracted from 4 formalin-fixed paraffin-embedded sections cut at 10 microns from 30 MM cases. The exons of 182 cancer-related genes were fully sequenced using the Illumina HiSeq 2000 at an average sequencing depth of 1098X and evaluated for genomic alterations (GAs) including point mutations, insertions, deletions, copy number alterations, and select gene fusions/rearrangements. Clinically relevant GAs (CRGAs) were defined as those identifying commercially available targeted therapeutics or therapies in registered clinical trials. Results: The 30 American Joint Committee on Cancer Stage IV MM included 17 (57%) male and 13 (43%) female patients with a mean age of 59.5 years (range 41–83 years). All MM samples had at least 1 GA, and an average of 2.7 GA/sample (range 1–7) was identified. The mean number of GA did not differ based on age or sex; however, on average, significantly more GAs were identified in amelanotic and poorly differentiated MM. GAs were most commonly identified in BRAF (12 cases, 40%), CDKN2A (6 cases, 20%), NF1 (8 cases, 26.7%), and NRAS (6 cases, 20%). CRGAs were identified in all patients, and represented 77% of the GA (64/83) detected. The median and mean CRGAs per tumor were 2 and 2.1, respectively (range 1–7). Conclusion: Comprehensive genomic profiling of MM, using a single diagnostic test, uncovers an unexpectedly high number of CRGA that would not be identified by standard of care testing. Moreover, NGS has the potential to influence therapy selection and can direct patients to enter relevant clinical trials evaluating promising targeted therapies.

Genomic Characterization of Testicular Germ Cell Tumors Relapsing After Chemotherapy

BACKGROUND Although both seminomatous and nonseminomatous testicular germ cell tumors (TGCTs) have favorable outcomes with chemotherapy, a subset is chemorefractory, and novel therapeutic options are needed. OBJECTIVE To molecularly characterize chemotherapy-refractory TGCTs. DESIGN, SETTING, AND PARTICIPANTS Archival tissues from 107 chemotherapy-treated and relapsed TGCT patients (23 seminomas; 84 nonseminomas) underwent hybrid-capture-based genomic profiling to evaluate four classes of genomic alterations (GAs). Tumor mutational burden (TMB) and microsatellite instability (MSI) were also measured. INTERVENTION Genomic profiling on tumor samples from chemotherapy-refractory TGCTs. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Descriptive analyses and differences between seminoma and nonseminoma subgroups were reported. RESULTS AND LIMITATIONS The mean GA/tumor was 2.9 for seminomas and 4.0 for nonseminomas (p=0.04). KRAS alterations (mainly amplifications) were the most common GAs at the single-gene level (47.8% of seminomas and 51.2% of nonseminomas). RAS-RAF pathway (56.5% vs 52.3%) and cell-cycle pathway (52.2% vs 56.0%) were the most common GA classes in seminomas and nonseminomas, respectively. Receptor tyrosine kinase pathway and PI3K pathway GAs were more frequent in seminomas (p=0.02). Median TMB was 1.8 mutations/Mb for seminomas and 2.7 mutations/Mb for nonseminomas (p=0.098), and MSI-high status was found in one nonseminoma only (1.2%). A lack of clinical outcome correlation is a limitation of the present analyses. CONCLUSIONS In chemotherapy-refractory TGCTs, trials with agents targeting the KRAS pathway may be pursued due to the high frequency of KRAS GAs. Overall, the GAs found in refractory seminomas and nonseminomas differ significantly. Considering the frequency of high TMB or MSI-high status, immunotherapy may benefit a small subset of nonseminomas. PATIENT SUMMARY Testicular cancers that are resistant to or relapse after standard chemotherapy may harbor genomic alterations that are potentially druggable, particularly in the clinical trial setting, and genomic profiling can guide clinical research and disclose therapeutic opportunities for these patients.

General paucity of genomic alteration and low tumor mutation burden in refractory and metastatic hepatoblastoma: comprehensive genomic profiling study

Hepatoblastoma (HBL) is a hepatic malignancy of infants and young children, which is often cured by combinations of surgery and chemotherapy. Management of refractory and metastatic HBL is challenging. Comprehensive genomic profiling was performed on 31 refractory and metastatic HBL using a hybrid-capture, adaptor ligation-based next-generation sequencing assay. Tumor mutation burden (TMB) was calculated from a minimum of 1.11 Mb of sequenced DNA and reported as mutations/Mb. The results were analyzed for all classes of genomic alterations (GA). Activating CTNNB1 mutation was the most frequent GA seen in 19 (61%) of cases. All 3 (100%) mixed epithelial and mesenchymal HBL harbored CTNNB1 mutation. The small cell undifferentiated subtype showed SMARCB1 loss in both cases. There was no significant further correlation of GA with histologic subtype. In addition to the potential targeting of CTNNB1, other rarely identified possible targetable GA included ERBB4 (6%) and FBXW7, SRC and BRCA2 (each at 3%). The mean TMB was 3.5 mut/Mb, the median was 1.7 mut/Mb. There were 2 HBL with ≥10 mut/Mb. No alterations in TP53 were identified, and alterations in the DNA repair pathways were rare. Refractory and metastatic HBL is characterized by a general paucity of GA and is dominated by frequent CTNNB1 mutation and overall low TMB. Although potentially targetable GA are seen on occasion in HBL and a small number of cases have high TMB with potential to respond to immune checkpoint inhibitors, advanced HBL will remain a treatment challenge.

Comparative Genomic Profiling of Refractory/Metastatic Penile and non-Penile Cutaneous Squamous Cell Carcinoma: Implications for Selection of Systemic Therapy

Purpose: Metastatic penile squamous cell carcinoma is an aggressive malignancy with limited treatment options. We compared the potential therapy impacting genomic alterations between metastatic penile squamous cell carcinoma and nonpenile metastatic cutaneous squamous cell carcinoma. Materials and Methods: DNA was extracted from 40 &mgr; of formalin fixed, paraffin embedded samples from 78 cases of metastatic penile squamous cell carcinoma and 338 of metastatic cutaneous squamous cell carcinoma. Comprehensive genomic profiling was performed using a hybrid capture, adaptor ligation based, next generation sequencing assay to a mean coverage depth of greater than 500×. The tumor mutational burden was determined on 1.1 Mbp of sequenced DNA and microsatellite instability was determined on 114 loci. Results: Potential targeted therapy opportunities in metastatic penile squamous cell carcinoma cases included alterations in the MTOR pathway (NF1 genomic alterations in 7% and PTEN genomic alterations in 4%) and in the DNA repair pathway (BRCA2 and ATM genomic alterations in 7% each) and tyrosine kinase (EGFR genomic alterations in 6%, and FGFR3 and ERBB2 genomic alterations in 4% each). The tumor mutational burden was significantly higher in predominantly ultraviolet light exposed metastatic squamous cell carcinoma than in metastatic penile squamous cell carcinoma, making metastatic squamous cell carcinoma potentially more responsive to immunotherapies than metastatic penile squamous cell carcinoma. Microsatellite high status was extremely rare for metastatic penile and metastatic cutaneous squamous cell carcinoma. CD274 (PD-L1) amplification was also rare in both tumor types. Conclusions: Metastatic penile squamous cell carcinoma is a unique subtype of squamous cell carcinoma with distinctive genomic features which contrast with those identified in metastatic cutaneous squamous cell carcinoma of nonpenile ultraviolet light exposed skin. Although not rich in predictors of the response to immunotherapy (the tumor mutational burden and microsatellite instability are low), more than a quarter of metastatic penile squamous cell carcinoma cases may potentially benefit from existing and available therapies targeting MTOR, DNA repair and tyrosine kinase pathways.PURPOSE Metastatic penile squamous cell carcinoma is an aggressive malignancy with limited treatment options. We compared the potential therapy impacting genomic alterations between metastatic penile squamous cell carcinoma and nonpenile metastatic cutaneous squamous cell carcinoma. MATERIALS AND METHODS DNA was extracted from 40 μ of formalin fixed, paraffin embedded samples from 78 cases of metastatic penile squamous cell carcinoma and 338 of metastatic cutaneous squamous cell carcinoma. Comprehensive genomic profiling was performed using a hybrid capture, adaptor ligation based, next generation sequencing assay to a mean coverage depth of greater than 500×. The tumor mutational burden was determined on 1.1 Mbp of sequenced DNA and microsatellite instability was determined on 114 loci. RESULTS Potential targeted therapy opportunities in metastatic penile squamous cell carcinoma cases included alterations in the MTOR pathway (NF1 genomic alterations in 7% and PTEN genomic alterations in 4%) and in the DNA repair pathway (BRCA2 and ATM genomic alterations in 7% each) and tyrosine kinase (EGFR genomic alterations in 6%, and FGFR3 and ERBB2 genomic alterations in 4% each). The tumor mutational burden was significantly higher in predominantly ultraviolet light exposed metastatic squamous cell carcinoma than in metastatic penile squamous cell carcinoma, making metastatic squamous cell carcinoma potentially more responsive to immunotherapies than metastatic penile squamous cell carcinoma. Microsatellite high status was extremely rare for metastatic penile and metastatic cutaneous squamous cell carcinoma. CD274 (PD-L1) amplification was also rare in both tumor types. CONCLUSIONS Metastatic penile squamous cell carcinoma is a unique subtype of squamous cell carcinoma with distinctive genomic features which contrast with those identified in metastatic cutaneous squamous cell carcinoma of nonpenile ultraviolet light exposed skin. Although not rich in predictors of the response to immunotherapy (the tumor mutational burden and microsatellite instability are low), more than a quarter of metastatic penile squamous cell carcinoma cases may potentially benefit from existing and available therapies targeting MTOR, DNA repair and tyrosine kinase pathways.

PD46-02 CARCINOMAS OF THE RENAL MEDULLA: A COMPREHENSIVE GENOMIC PROFILING (CGP) STUDY

INTRODUCTION AND OBJECTIVES: BAP1 is a ubiquitin carboxy-terminal hydrolaseenzyme (UCH)whichalongwith theASXL1/2 protein forms the PR-DUB, chromatin associated complex which removes monoubiquitin from H2A in nucleosomes regulating gene expression.BAP1 is a tumor suppressor genecommonlymutatedgene in clear cell renal cell carcinoma (ccRCC) and other cancers such as uveal melanoma (UM) and mesothelioma. In tumors, BAP1 mutations occur throughout the protein well outside the N-terminal UCH catalytic region while C-terminal BAP1 truncations/mutations in the ULD domain also inactivate BAP. The aim of this study was to investigate the potential mechanisms whereby these highly spatially distinct mutations in BAP1, many of which occur outside the UCH catalytic domain, serve to completely inactivate the enzyme function. We focused on BAP1s obligate partner protein ASXL1/2 which binds the BAP1 ULD domain and may exert a global regulatory function in the context of PR-DUB in chromatin. A mechanistic understanding of these events might provide insight towards restoration of normal BAP1 functions in tumors leading to therapeutic benefit. METHODS: BAP1 mutations derived from multiple tumor types were identified, including UM, Mesothelioma and ccRCC and introduced into expression vectors and the proteins expressed as recombinants in e.coli and purified to homogeneity. BAP1 and ASXL1/2 protein complexes were evaluated for protein interaction and UCH enzyme activity using GST-interaction studies, Western blot and gel filtration analysis. Molecular modeling of the complex was performed by Blast analysis, molecular docking methods and refined using insights from our mutagenic study. RESULTS: This structure-function study revealed that the BAP1 and ASXL1/2 is direct, high affinity and that multiple contact points between BAP1, ASXL1/2 exist that stabilize the native catalytic structure the The domain structure of BAP1 is unique, multi-lobed and seems to be highly allosteric with respect to enzyme activation. ASXL1/ 2 is an obligate partner for BAP1 and the enzyme is dead with out ASXL1/2 binding which occurs in the BAP1 ULD domain: many tumor derived mutants in the BAP1 ULD domain disrupt ASXL1/2 binding and abolish BAP1 activity. We also discovered a highly conserved domain in ASXL1/2 as minimally essential for BAP1 binding and activation. Mutations in multiple tumors at widely scattered locations decreased ubiquitin hydrolase activity in re-constituted protein complexes and destabilized binding of BAP1 and ASXL1/2 upon modeling. CONCLUSIONS: Different mutations derived from multiple tumors occur throughout the sequence of BAP1 and apparently act allosterically to alter the ability of ASXL1/2 to both bind and/or interact unproductively with the BAP1 UCH region which leads to loss of enzymatic activity and loss of tumor suppression. This analysis suggests that small molecule approaches might be used to reactivate the latent UCH activity in some tumors and restore wild type function of BAP1.