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Oncocytic histologic features can be seen in a variety of salivary gland carcinomas. We performed a comprehensive molecular profiling of 15 salivary gland malignancies with oncocytic features (diagnosed as oncocytic carcinoma, carcinoma NOS with oncocytic features, or salivary duct carcinoma with oncocytic features). We reveal multiple novel molecular alterations that have not been previously described in other salivary gland malignancies, including, but not limited to, KEL amplification (13.3%, 2/15), PARP1 amplification (13.3%, 2/15), and EPHB4 amplification (13.3%, 2/15). Alterations in KMT2C (13.3%, 2/15), ERBB3 (13.3%, 2/15), CTNNA1 (13.3%, 2/15), and SMAD4 (20%, 3/15) were also found in this series and have been reported in other salivary gland malignancies. Alterations that have been reported in salivary duct carcinoma were also identified, including TP53 (40%, 6/15) , ERBB2 mutations (13.3%, 2/15) , ERBB2 amplification (13.3%, 2/15), PIK3CA (26.7%, 4/15) , PTEN (20%, 3/15), BRCA2 (20%, 3/15), BRAF (20%, 3/15), CDKN2A/B (20%, 3/15), CDH1 (13.3%, 2/15), and HRAS (13.3%, 2/15). Oncocytic salivary gland malignancies are a molecularly heterogenous group of tumors with partial overlap with salivary duct carcinoma subtypes.

Background In the current study, we examined the real-world prevalence of highly pigmented advanced melanomas (HPMel) and the clinicopathologic, genomic, and ICPI biomarker signatures of this class of tumors. Materials and Methods Our case archive of clinical melanoma samples for which the ordering physician requested testing for both PD-L1 immunohistochemistry (IHC) and comprehensive genomic profiling (CGP) was screened for HPMel cases, as well as for non-pigmented or lightly pigmented advanced melanoma cases (LPMel). Results Of the 1268 consecutive melanoma biopsies in our archive that had been submitted for PD-L1 IHC, 13.0% (165/1268) were HPMel and 87.0% (1103/1268) were LPMel. In the HPMel cohort, we saw a significantly lower tumor mutational burden (TMB, median 8.8 mutations/Mb) than in the LPMel group (11.4 mut/Mb), although there was substantial overlap. In examining characteristic secondary genomic alterations (GA), we found that the frequencies of GA in TERTp, CDKN2A, TP53, and PTEN were significantly lower in the HPMel cases than in LPMel. A higher rate of GA in CTNNB1, APC, PRKAR1A, and KIT was identified in the HPMel cohort compared with LPMel. Conclusions In this study, we quantified the failure rates of melanoma samples for PD-L1 testing due to high melanin pigmentation and showed that CGP can be used in these patients to identify biomarkers that can guide treatment decisions for HPMel patients. Using this practical clinical definition for tumor pigmentation, our results indicate that HPMel are frequent at 13% of melanoma samples, and in general appear molecularly less developed, with a lower TMB and less frequent secondary GA of melanoma progression.

BACKGROUND AND AIMS Columnar cell variant (CCV) is a rare papillary thyroid carcinoma subtype. The majority of CCV occur in older patients and are large, invasive tumours that pursue an aggressive clinical course. Rare well-circumscribed CCV occur in younger female patients and are comparatively indolent. METHODS AND RESULTS We retrospectively identified CCV with material available to perform targeted next-generation sequencing and correlated molecular results with clinicopathological features and outcome. Our cohort was comprised of nine CCV. Nearly all were aggressive tumours; however, one was predominantly well-circumscribed and arose in a thyroglossal duct cyst of a 26-year-old woman who had no evidence of disease at last follow-up. Seven (78%) cases demonstrated activating oncogenic driver alterations in BRAF, including BRAF V600E, an activating N486_P490del deletion, and BRAF-AGK fusions. Activating RAS mutations were seen in two (22%) cases. Additionally, three (33%) cases had TERT promoter mutations, four (44%) had loss of the tumour suppressor CDKN2A and one (11%) case had a loss of function TP53 mutation. Most cases (89%) also demonstrated copy number alterations, including recurrent gain of chromosome 1q (five cases) and losses of chromosome 9p (three cases) and 22q (four cases). The one case without secondary pathogenic mutations or copy number alterations was the tumour in the 26-year-old woman. CONCLUSIONS We found that CCV is primarily a BRAF-driven tumour, with most also harbouring secondary oncogenic mutations and multiple chromosomal gains and losses. Moreover, our findings suggest that molecular analysis could potentially be used to help risk stratify CCV.

Understanding the paleoecology of species is fundamental to reconstructions of paleoecological communities, analyses of changing paleoenvironments, and the evolutionary history of many lineages. One method of establishing paleoecology is through comparing the morphology of extant analogs to extinct species; this method has been applied to many vertebrate groups using predictive linear models, but has been rarely applied using invertebrate taxa or non-linear frameworks. The ant fossil record, which frequently preserves specimens in three-dimensional fidelity, chronicles putative faunal turnover during the Late Cretaceous and into Cenozoic. The earliest fossil species comprise enigmatic stem-groups that underwent extinction concomitant with crown ant diversification. Here, we apply a wide-ranging extant ecomorphological dataset to demonstrate the utility of Random Forest machine learning classification in predicting the ecology of the stem-group hell ants. We reconstruct a predicted ecomorphological assemblage of this phenotypically aberrant group of extinct ants, and compare predicted hell ant ecologies to the ecological occupations of their closest living analogs, lineages of solitary predators with highly specialized mandibular morphology. In contrast to previous hypotheses, we find that hell ants were primarily leaf litter or ground-nesting and foraging taxa, and that the ecological breadth of this unusual lineage mirrored that of living groups. Results suggest ecological coherence between the Mesozoic and modern communities, even as the earliest occupants of predatory niches were phylogenetically and morphologically distinct.

BACKGROUND: Pathogenic POLE exonuclease domain mutations (pPOLE) undermine mismatch error correction during DNA replication, causing somatic ultramutation and response to immunotherapy. We examined the pan-cancer landscape of POLE mutations and applied a POLE-specific variant classification model. METHODS: Comprehensive genomic profiling was performed during clinical care. Mutational signature calling was performed via decomposition using the 96-feature single-base substitution COSMIC reference signatures. A POLE-specific classification model encompassing mutation position in the exonuclease domain, TMB, presence of POLE signature, absence of other signatures, germline frequency, and other features was applied to identify pPOLE mutations causative of ultramutation. RESULTS: POLE mutation status was evaluated in 458,437 samples (425,520 tissue biopsies (TB) and 32,917 liquid biopsies (LB)). One or more POLE alterations, including pathogenic alterations and variants of unknown significance (VUS), were detected in 3.8% of samples. 19,470 total alterations were identified, 84.8% of which were missense substitutions. Application of the POLE-specific classification model identified 35 unique pathogenic variants, many of which were VUS prior to this study. 749 samples harbored a pPOLE, more than half (56.6%) of which were either p.P286R (n=245) or p.V411L (n=179). pPOLE were found in 1.4% (199/13,688) of endometrial cancers (EC) and 0.5% (270/55,981) of colorectal cancers (CRC) and were rarer in a long tail of other malignancies. The overall pPOLE rate was significantly lower in LB than TB (0.02% vs 0.17%, P CONCLUSIONS: pPOLE were seen in both TB and LB across cancer types. The high rate of passenger mutations underscores the utility of this POLE-specific variant classification model. Because TMB can be underestimated when tumor purity is near the limit of detection for the assay, accurate detection and classification of pPOLE is critical for identifying patients who may benefit from immunotherapy. Citation Format: Rachel B. Keller, James Haberberger, Tyler Janovitz, Alexa B. Schrock, Hanna Tukachinsky, Lei Zhong, Douglas A. Mata, Lyle V. Lopez, Zoe Fleischmann, Radwa Sharaf, Ethan S. Sokol, Garrett M. Frampton, Nimesh R. Patel, Douglas I. Lin, Geoff R. Oxnard, Erik A. Williams, Julia A. Elvin, Brennan Decker. POLE-specific variant classification strategy is critical for identifying patients who may benefit from immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 305.

Activation of the tyrosine kinase receptor IGF1R is targetable with existing tyrosine kinase inhibitors (TKIs) and monoclonal antibodies, but mutations in IGF1R have not been systematically characterized. Pan-cancer analysis of 326,911 tumors identified two distinct, activating non-frameshift insertion hotspots in IGF1R, which were significantly enriched in adenoid cystic carcinomas (ACCs). IGF1R alterations from 326,911 subjects were analyzed by variant effect prediction class, position within the gene, and cancer type. 6502 (2.0%) samples harbored one or more alterations in IGF1R. Two regions were enriched for non-frameshift insertions: codons 663-666 at the hinge region of the fibronectin type 3 domain and codons 1034-1049 in the tyrosine kinase domain. Hotspot insertions were highly enriched in ACCs (27.3-fold higher than in the remainder of the pan-cancer dataset; P = 2.3 × 10

Context.— Multiple procedural techniques can be used to obtain tissue to create a formalin-fixed, paraffin-embedded specimen for comprehensive genomic profiling (CGP) in lung cancer. The literature is mixed on whether the procedure affects CGP success. Objective.— To examine whether biopsy procedure affects lung cancer CGP success. Design.— This was a cross-sectional study of all patients with lung cancer whose specimens were submitted for CGP between January and February 2020. Multiple quality control metrics were used to determine whether cases were successfully profiled. Results.— In all, 3312 samples were identified. Overall, 67.5% (2236 of 3312) of samples were obtained from biopsies, 13.0% (432 of 3312) from fine-needle aspirations (FNAs), 9.7% (321 of 3312) from resections, 5.3% (174 of 3312) from fluid cytology cell blocks, and 4.5% (149 of 3312) from bone biopsies. Overall, 70.1% (2321 of 3312) of cases passed CGP, 15.4% (510 of 3312) of cases were released as qualified reports, and 14.5% (481 of 3312) of cases failed CGP. Resection samples were the most likely to be successfully sequenced, failing in only 2.8% (9 of 321) of instances, while fluid cytology specimens were the least likely, failing in 23.0% (40 of 174) of instances. Biopsy (14.5% [324 of 2236]), FNA (18.5% [80 of 432]), and bone biopsy (18.8% [28 of 149]) specimens failed at intermediate frequencies. On multivariate logistic regression analysis of CGP success on specimen type, fluid cytology (odds ratio [OR], 0.08; 95% CI, 0.03–0.19), biopsy (OR, 0.25; 95% CI, 0.11–0.52), FNA (OR, 0.14; 95% CI, 0.06–0.32), and bone biopsy (OR, 0.07; 95% CI, 0.03–0.17) specimens had decreased odds of CGP success relative to resection samples. Among patients with successfully sequenced samples, 48.0% were eligible for at least 1 therapy, based on a companion diagnostic or National Comprehensive Cancer Network biomarker. Conclusions.— The method of tissue acquisition was an important preanalytic factor that determined whether a sample would be successfully sequenced and whether a clinically actionable genomic alteration would be detected.

Endometrial serous carcinoma (EMSC) is an aggressive variant of uterine cancer with limited therapeutic options. We sought to define distinct clinicopathologic and genomic EMSC subgroups.We retrospectively analyzed 2159 EMSC and 2346 endometrioid-type endometrial carcinomas (EEC) tissue specimens that had undergone comprehensive genomic profiling (CGP) via the FoundationOne CDx assay during routine clinical care. High tumor mutational burden (TMB) was defined as ≥10mut/Mb using the FDA-approved CDx cutoff for pembrolizumab. Microsatellite instability (MSI) was determined on 95 loci. Evidence of homologous recombination deficiency (HRD) was determined via genomic loss of heterozygosity (gLOH), a validated HRD detection method for predicting PARP inhibitor effectiveness in ovarian carcinoma. High gLOH was defined as ≥16%.A genomic analysis of 2159 EMSCs revealed a predominance of TP53 mutations, microsatellite stability, low tumor mutational burden (TMB), and recurrent alterations of PIK3CA, PPP2R1A, ERBB2, CCNE1, FBXW7 and MYC. Evidence of HRD via high gLOH was identified in 22% of EMSCs. BRCA1 and BRCA2 alterations, as well as unique SET (solid, pseudo-endometrioid, and transitional cell-like) variant morphology, were enriched in HRD-EMSC. There was an increased frequency of CCNE1 amplification, a lower prevalence of PIK3CA and PPP2R1A alterations, and no differences in HRD, MSI or TMB biomarker frequencies in patients of predicted African ancestry. EMSC exhibited distinct gene mutation frequencies and MSI, TMB and gLOH biomarker signatures compared to a cohort 2346 EEC.Molecularly defined subgroups provide a framework to test the susceptibility of EMSC to targeted therapies in specific genetic settings (e.g. HRD, PIK3CA, PPP2R1A, ERBB2, MYC, CCNE1).

BACKGROUND The DNA repair gene ATM is remarkable for its roles across the spectrum of neoplastic biology, including inherited risk of multiple malignancies, somatic tumor biology, and clonal hematopoiesis (CH). We hypothesized that comparison of liquid biopsy (LB) versus tumor tissue (TT) from germline ATM pathogenic mutation carriers (gATM+) might offer additional insights into the role of ATM in CH. METHODS 34,825 LB samples and 384,847 TT specimens were sequenced for up to 324 genes during routine clinical care and analyzed for all classes of genomic alterations (Frampton 2013, Woodhouse 2020). Individual variants were assessed for germline versus somatic origin using the validated somatic-germline zygosity algorithm (Sun 2018). RESULTS Germline ATM mutations were observed in 0.9% (113/12,217) of LB and 0.8% (3,029/384,847) of TT (P CONCLUSION The finding of numerous ATM mutations under positive selection in gATM+ subjects was specific to LB, increased in incidence with age, and co-mutations tended to have low VAF. All these features are consistent with polyclonal hematopoiesis (polyCH) involving ATM co-mutations in gATM+ subjects. Additional investigation is needed to extend our understanding of this phenomenon. Citation Format: Brennan Decker, Angela A. Kou, Tyler Janovitz, Douglas A. Mata, Ethan S. Sokol, Dexter X. Jin, Hanna Tukachinsky, Jo-Anne Vergilio, Julia A. Elvin, Geoffrey R. Oxnard. Patients with germline ATM mutations develop clonal hematopoiesis characterized by co-occurrence of multiple somatic ATM alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 57.

Key Points Question Can deep learning systems perform hematoxylin and eosin (H&E) staining and destaining and are the virtual core biopsy samples generated by them as valid and interpretable as their real-life unstained and H&E dye–stained counterparts? Findings In this cross-sectional study, deep learning models were trained using nonstained prostate core biopsy images to generate computationally H&E stained images, and core biopsy images were extracted from each whole slide, consisting of approximately 87 000 registered patch pairs of 1024 × 1024 × 3 pixels each. Comprehensive analyses of virtually stained images vs H&E dye–stained images confirmed successful computational staining. Meaning The findings of this study suggest that whole slide nonstained microscopic images of prostate core biopsy, instead of tissue samples, could be integrated with deep learning algorithms to perform computational H&E staining and destaining for rapid and accurate tumor diagnosis., This cross-sectional study describes the development of deep learning algorithms to virtually dye prostate tissue samples and evaluates the effectiveness of images generated using this method vs hematoxylin and eosin–stained images for histopathological diagnosis of tumors., Importance Histopathological diagnoses of tumors from tissue biopsy after hematoxylin and eosin (H&E) dye staining is the criterion standard for oncological care, but H&E staining requires trained operators, dyes and reagents, and precious tissue samples that cannot be reused. Objectives To use deep learning algorithms to develop models that perform accurate computational H&E staining of native nonstained prostate core biopsy images and to develop methods for interpretation of H&E staining deep learning models and analysis of computationally stained images by computer vision and clinical approaches. Design, Setting, and Participants This cross-sectional study used hundreds of thousands of native nonstained RGB (red, green, and blue channel) whole slide image (WSI) patches of prostate core tissue biopsies obtained from excess tissue material from prostate core biopsies performed in the course of routine clinical care between January 7, 2014, and January 7, 2017, at Brigham and Women’s Hospital, Boston, Massachusetts. Biopsies were registered with their H&E-stained versions. Conditional generative adversarial neural networks (cGANs) that automate conversion of native nonstained RGB WSI to computational H&E-stained images were then trained. Deidentified whole slide images of prostate core biopsy and medical record data were transferred to Massachusetts Institute of Technology, Cambridge, for computational research. Results were shared with physicians for clinical evaluations. Data were analyzed from July 2018 to February 2019. Main Outcomes and Measures Methods for detailed computer vision image analytics, visualization of trained cGAN model outputs, and clinical evaluation of virtually stained images were developed. The main outcome was interpretable deep learning models and computational H&E-stained images that achieved high performance in these metrics. Results Among 38 patients who provided samples, single core biopsy images were extracted from each whole slide, resulting in 102 individual nonstained and H&E dye–stained image pairs that were compared with matched computationally stained and unstained images. Calculations showed high similarities between computationally and H&E dye–stained images, with a mean (SD) structural similarity index (SSIM) of 0.902 (0.026), Pearson correlation coefficient (PCC) of 0.962 (0.096), and peak signal to noise ratio (PSNR) of 22.821 (1.232) dB. A second cGAN performed accurate computational destaining of H&E-stained images back to their native nonstained form, with a mean (SD) SSIM of 0.900 (0.030), PCC of 0.963 (0.011), and PSNR of 25.646 (1.943) dB compared with native nonstained images. A single blind prospective study computed approximately 95% pixel-by-pixel overlap among prostate tumor annotations provided by 5 board certified pathologists on computationally stained images, compared with those on H&E dye–stained images. This study also used the first visualization and explanation of neural network kernel activation maps during H&E staining and destaining of RGB images by cGANs. High similarities between kernel activation maps of computationally and H&E-stained images (mean-squared errors

604 Background: MTAP loss is represented across a wide variety of cancer types including PDAC and is an emerging target for synthetic lethality-based cancer therapies. Preclinically, MTAP loss leads to the accumulation of 2-methylthioadenosine, reduced protein arginine N-methyltransferase 5 (PRMT5) methylation activity and increased vulnerability to targeting of the methionine adenosyltransferase IIα (MAT2A)/ PRMT5 axis. In addition, 9p21 loss, homozygous co-deletion of MTAP/CDKN2A or homozygous deletion of either gene have been associated with an immunologically “cold” tumor microenvironment, primary resistance to anti PD(L)1 immunotherapy (IO) and poor prognosis phenotype (Han G, Nat Commun 2021). We investigated concurrent mutations and immune biomarkers in clinical PDAC samples with MTAP-loss versus -intact status. Methods: From a series of 177705 consecutive cases, we performed comprehensive genomic profiling on 9423 cases of PDAC using an FDA-approved assay (F1CDx) to evaluate all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. PD-L1 expression was determined by immunohistochemistry (Dako 22C3). Furthermore, we correlated pertinent findings within a database of 16558 cases of clinically advanced cancer with MTAP loss. Results: 2003 (21.3%) of 9423 PDAC demonstrated MTAP-loss. Similar gender, age and number of GA per tumor were observed between MTAP-loss and -intact groups. Frequencies of TP53, CDKN2A/B, SMAD4, PTEN and ARID1A were significantly higher in MTAP-loss PDAC. However, previously-described biomarkers of IO efficacy (MSI, TMB, CD274 amplification and PD-L1 expression) and resistance ( STK11, KEAP1 and MDM2) were infrequent and similar in both groups. The frequencies of other potentially targetable GA including BRCA1/2, ATM, KRAS G12C, ERBB2, BRAF, FGFR1, NF1 and PIK3CA were also infrequent and similar in both groups of PDAC patients. Amongst a database of 16558 cases of clinically advanced cancer with MTAP loss, 1538 (9.3%) featured co-alterations in MTAP and SMAD4. 52% of the MTAP/SMAD4 co-altered cases were PDAC. Conclusions: MTAP loss is associated with a distinctive concurrent genomic profile in PDAC and represents a potential new synthetic lethality-based opportunity for treatment with PRMT5 and MAT2A inhibitors. Furthermore, MTAP loss may represent an independent negative predictive biomarker for immune checkpoint inhibition in PDAC.[Table: see text]

8555 Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy with limited therapeutic options. Immunotherapy has emerged as an effective therapeutic strategy, particularly in the non-epithelioid MPM subgroup. An improved understanding of the molecular profile of MPM may inform targeted therapeutic approaches and provide insights into factors underlying differential sensitivity to therapies. Methods: We performed comprehensive genomic profiling of MPMs from 980 patients, with histologic subtyping on a subset (n = 340; n = 235 epithelioid, n = 48 sarcomatoid, n = 57 biphasic). Analyses included quantifying tumor mutational burden (TMB), assessing microsatellite instability (MSI), and evaluating PD-L1 expression (Dako 22C3). Results: Median TMB of MPM was 1.74 (0.87-2.61) mutations per megabase (mut/mb). Median TMB was comparable for non-epithelioid (biphasic, sarcomatoid) vs epithelioid MPM (1.25 mut/mb vs 1.25 mut/mb, p = 0.43). In the overall cohort, inactivating alterations in the following genes were most prevalent: CDKN2A (49%), BAP1 (44%), CDKN2B (42%), MTAP (35%), and NF2 (33%). MTAP loss co-occurred with CDKN2A and CDKN2B loss in > 99% and 94% of cases, respectively. MTAP loss was observed in 28% of epithelioid vs 46% of non-epithelioid MPMs (p = 0.03). BAP1 alterations were detected in 51% of epithelioid vs 36% of non-epithelioid MPMs (p = 0.81). PD-L1 expression was assessed in 308 MPMs, among which 153 (49%) had PD-L1 ≥1%, including 35 (11%) with PD-L1 ≥50%. Among 164 specimens that underwent histologic subtyping and PD-L1 evaluation, PD-L1 was expressed in 46 (71%) non-epithelioid MPMs compared to 49 (51%) epithelioid MPMs (p = 0.03). Given the high prevalence of alterations involving the two genes, we evaluated impact of BAP1 and MTAP status on PD-L1 and TMB. TMB (1.74 mut/mb vs 1.74 mut/mb) and PD-L1 expression (50% vs 54%) were comparable in BAP1-altered vs wildtype specimens. Although TMB was comparable in MTAP-deficient and MTAP-intact tumors (1.25 mut/mb vs 1.74 mut/mb), more tumors with MTAP loss expressed PD-L1 (68% vs 44%, p = 0.0004). Conclusions: Compared to epithelioid MPM, non-epithelioid tumors demonstrate comparable TMB, higher PD-L1 expression, and enrichment for MTAP loss. As MTAP is frequently altered in MPM, further study is indicated to explore the relationship between MTAP status and MPM biology, including sensitivity to therapeutics such as immunotherapy and synthetic lethal approaches.

Although differentiated thyroid carcinomas typically pursue an indolent clinical course, it is important to identify the subset of tumors that are most likely to behave aggressively so that patients with these tumors are counseled and treated appropriately. Extent of disease is fundamental to the prognostication for differentiated thyroid carcinoma; however, there are additional histologic features of the tumor separate from extent of disease that have been shown to affect clinical course. This review will start with a discussion of aggressive variants of papillary thyroid carcinoma, move to the prognostic significance of vascular invasion in follicular thyroid carcinoma, and finish with a discussion of Hurthle cell carcinoma, with an emphasis on why it is not considered a subtype of follicular thyroid carcinoma in the 2017 WHO Classification of Tumors of Endocrine Organs.

The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein (NS1), a DNA binding endonuclease, and the Rep proteins of Adeno-Associated Virus (AAV) led us to hypothesize that NS1 may facilitate targeted nicking of the human genome and B19 vDNA integration. We adapted an integration capture sequencing protocol (IC-Seq) to screen B19V infected human CD36+ EPCs for viral integrants, and discovered 40,000 unique B19V integration events distributed throughout the human genome. Computational analysis of integration patterns revealed strong correlations with gene intronic regions, H3K9me3 sites, and the identification of 41 base pair consensus sequence with an octanucleotide core motif. The octanucleotide core has homology to a single region of B19V, adjacent to the P6 promoter TATA box. We present the first direct evidence that B19V infection of erythroid progenitor cells disrupts the human genome and facilitates viral DNA integration.

Adeno-associated virus serotype 5 (AAV-5) is a human parvovirus that infects a high percentage of the population. It is the most divergent AAV, the DNA sequence cleaved by the viral endonuclease is distinct from all other described serotypes and, uniquely, AAV-5 does not cross-complement the replication of other serotypes. In contrast to the well-characterized integration of AAV-2, no published studies have investigated the genomic integration of AAV-5. In this study, we analyzed more than 660,000 AAV-5 integration junctions using high-throughput integrant capture sequencing of infected human cells. The integration activity of AAV-5 was 99.7% distinct from AAV-2 and favored intronic sequences. Genome-wide integration was highly correlated with viral replication protein binding and endonuclease sites, and a 39-bp consensus integration motif was revealed that included these features. Algorithmic scanning identified 126 AAV-5 hot spots, the largest of which encompassed 3.3% of all integration events. The unique aspects of AAV-5 integration may provide novel tools for biotechnology and gene therapy. IMPORTANCE Viral integration into the host genome is an important aspect of virus host cell biology. Genomic integration studies of the small single-stranded AAVs have largely focused on site preferential integration of AAV-2, which depends on the viral replication protein (Rep). We have now established the first genome wide integration profile of the highly divergent AAV-5 serotype. Using integrant capture sequencing, more than 600,000 AAV-5 integration junctions in human cells were analyzed. AAV-5 integration hot spots were 99.7% distinct from AAV-2. Integration favored intronic sequences, occurred on all chromosomes, and integration hot spot distribution was correlated with human genomic GAGC repeats and transcriptional activity. These features support expansion of AAV-5 based vectors for gene transfer considerations.

Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics.

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)beta at day 7 of differentiation increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Using an Id1-YFP hESC reporter line, we showed that TGFbeta inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

Background Adeno-associated virus (AAV) serotype 2 prevalently infects humans and is the only described eukaryotic virus that integrates site-preferentially. In a recent high throughput study, the genome wide distribution of AAV-2 integrants was determined using Integrant Capture Sequencing (IC-Seq). Additional insight regarding the integration of AAV-2 into human genomic DNA could be gleaned by low-throughput sequencing of complete viral-chromosomal junctions. Findings In this study, 140 clones derived from Integrant-Capture Sequencing were sequenced. 100 met sequence inclusion criteria, and of these 39 contained validated junction sequences. These unique sequences were analyzed to investigate the structure and location of viral-chromosomal junctions. Conclusions Overall the low-throughput analysis confirmed the genome wide distribution profile gathered through the IC-Seq analysis. We found no unidentifiable sequence inserted at AAV-2 chromosomal junctions. Assessing both left and right ends of the AAV genome, viral breakpoints predominantly occurred in one hairpin of the inverted terminal repeat and AAV genomes were preferentially integrated as single copies.