Selective Outgrowth Research Articles

Important Role for Glutamine in Human and Mouse TME Models of CLL ; Towards Development of a PET Tracer As a Novel Diagnostic Tool

The amino acid transporter ASCT2, encoded by the gene SLC1A5, is the main glutamine importer in proliferating human and mouse cells. We recently showed that glutamine is the main fuel of the tricarboxylic acid (TCA) cycle in cells from chronic lymphocytic leukemia (CLL) patients, and that blocking its import attenuated CD40/BCR-induced venetoclax (VEN) resistance 1. In the current study, we have investigated the role of ASCT2 and the consequences of its inhibition in human and mouse CLL cells and in healthy lymphocytes by flow cytometry, gene expression analysis, extracellular flux analysis, metabolomics, CRISPR/Cas9 gene editing and adoptive-cell transfer experiments. To enable in vivo assessment of ASCT2 in the future with Positron Emission Tomography (PET), we have explored the potential use of 18F-labelelled ( 2S,2R)-4-fluoroglutamine (4- 18F]FGln) 2 as PET tracer. Increased expression of ASCT2 upon PMA/ionomycin stimulation was observed in healthy B and T cells. In BCR/CD40-stimulated CLL cells, the increased expression was irrespective of IGHV status. We dissected the main routes of glutamine usage in CD40- and BCR-stimulated CLL cells and found that in addition to its role in the TCA cycle, glutamine import is linked to mTOR signaling. The ASCT2 inhibitor V9302 impaired mTOR signaling and decreased translation of new proteins, including the anti-apoptotic proteins Bcl-XL and Mcl-1. Of relevance for therapeutic approaches, the effector and proliferative capacity of human T cells was preserved upon V9302 treatment. This suggests that blocking ASCT2 will not affect immune responses. The SLC1A5 gene was deleted by CRISPR/Cas9 technique in murine TCL1 cells 3. Adoptive cell transfer experiments of SLC1A5-KO and WT TCL1 cells injected at 50-50% ratio in recipient mice showed selective outgrowth of SLC1A5-WT cells, with no detection of the SLC1A5-KO in the spleen of overt leukemic mice. This indicates that TCL1 leukemic cells rely on SLC1A5/ASCT2 for growth. Finally, we explored the high expression of ASCT2 and glutamine addiction in CLL from the diagnostic perspective. With the aim of improving the currently suboptimal [ 18F]FDG PET for imaging of CLL LN sites 4, we successfully synthesized 4-[ 18F]FGln. We observed uptake in CD40-stimulated CLL cells, which was decreased in the presence of V9302. Experiments to perform PET with 4-[ 18F]FGln in TCL1 mice to demonstrate an in vivo proof of concept are ongoing. Overall, these data support a crucial role for ASCT2 and glutamine in leukemia development and VEN resistance in CLL. This provides a basis for targeting the transporter for therapeutic options and/or taking advantage of its function for diagnostic approaches using 4-[ 18F]FGln as PET tracer. 1. Chen, Z. et al. Characterization of metabolic alterations of chronic lymphocytic leukemia in the lymph node microenvironment. Blood140, 630-643, doi:10.1182/blood.2021013990 (2022). 2. Qu, W. et al. Synthesis of optically pure 4-fluoro-glutamines as potential metabolic imaging agents for tumors. J Am Chem Soc133, 1122-1133, doi:10.1021/ja109203d (2011). 3. Nardi, F. et al. Assessing gene function in human B cells: CRISPR/Cas9-based gene editing and mRNA-based gene expression in healthy and tumor cells. Eur J Immunol52, 1362-1365, doi:10.1002/eji.202149784 (2022). 4. Rhodes, J. M. & Mato, A. R. PET/Computed Tomography in Chronic Lymphocytic Leukemia and Richter Transformation. PET Clin14, 405-410, doi:10.1016/j.cpet.2019.03.007 (2019).

Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy

BackgroundThe wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons...

The thin red line between the immune system and cancer evolution

The cancer immunoediting theory describes the dual ability of endogenous antitumor immunity to inhibit or promote progressing cancers. Tumor-specific neoantigens arising from somatic mutations serve as targets for the endogenous T-cell-mediated antitumor immunity and therefore possess a crucial role for tumor development. Additionally, targeting these molecules is conceptually appealing because neoantigens are not expressed in healthy tissue and therefore confer less toxicity and greater specificity when used in therapeutic interventions. Moreover, intratumor neo-antigenic heterogeneity is believed to play a pivotal role in the activation of adaptive immunity and in the efficacy of immunotherapies that are based on immune checkpoint inhibition. In this respect, mutual interactions between tumor cells and immune lymphocytes regulate the levels of antitumor immunity, but also shape tumor heterogeneity through the selective outgrowth of tumor subclones. Therefore, the exploration of the mechanistic pathways and the identification of the genomic aberrations underlying the clonal evolution of tumors is considered mandatory for improving the clinical outcomes of therapies, as it will assist in the selection of the appropriate therapeutic decisions so as to delay, avoid, or overcome resistance through the identification of the most effective therapeutic strategies.

Unravelling Tumour Microenvironment in Melanoma at Single-Cell Level and Challenges to Checkpoint Immunotherapy.

Melanoma is known as one of the most immunogenic tumours and is often characterised by high mutation burden, neoantigen load and immune infiltrate. The application of immunotherapies has led to impressive improvements in the clinical outcomes of advanced stage melanoma patients. The standard of care immunotherapies leverage the host immunological influence on tumour cells, which entail complex interactions among the tumour, stroma, and immune cells at the tumour microenvironmental level. However, not all cancer patients can achieve a long-term durable response to immunotherapy, and a significant proportion of patients develops resistance and still die from their disease. Owing to the multi-faceted problems of tumour and microenvironmental heterogeneity, identifying the key factors underlying tumour progression and immunotherapy resistance poses a great challenge. In this review, we outline the main challenges to current cancer immunotherapy research posed by tumour heterogeneity and microenvironment complexities including genomic and transcriptomic variability, selective outgrowth of tumour subpopulations, spatial and temporal tumour heterogeneity and the dynamic state of host immunity and microenvironment orchestration. We also highlight the opportunities to dissect tumour heterogeneity using single-cell sequencing and spatial platforms. Integrative analyses of large-scale datasets will enable in-depth exploration of biological questions, which facilitates the clinical application of translational research.

CRISPR/Cas9-Mediated Insertion of HIV Long Terminal Repeat within BACH2 Promotes Expansion of T Regulatory-like Cells.

One key barrier to curative therapies for HIV is the limited understanding of HIV persistence. HIV provirus integration sites (ISs) within BACH2 are common, and almost all sites mapped to date are located upstream of the start codon in the same transcriptional orientation as the gene. These unique features suggest the possibility of insertional mutagenesis at this location. Using CRISPR/Cas9-based homology-directed repair in primary human CD4+ T cells, we directly modeled the effects of HIV integration within BACH2 Integration of the HIV long terminal repeat (LTR) and major splice donor increased BACH2 mRNA and protein levels, altered gene expression, and promoted selective outgrowth of an activated, proliferative, and T regulatory-like cell population. In contrast, introduction of the HIV-LTR alone or an HIV-LTR-major splice donor construct into STAT5B, a second common HIV IS, had no functional impact. Thus, HIV LTR-driven BACH2 expression modulates T cell programming and leads to cellular outgrowth and unique phenotypic changes, findings that support a direct role for IS-dependent HIV-1 persistence.

Potentiation of Efficacy of Chimeric Antigen Receptor (CAR) T-cell Therapy in Hematological Malignancies using FDA-Approved Small Molecule Sensitizing Agents

Chimeric antigen receptor CD19 CAR T-cell therapy has received FDA-approval for treatment of B cell malignancies. CD19 is an ideal target for B cell malignancies due to its limited expression by B lineage cells. Non-Hodgkin’s Lymphoma of B-cell origin (NHL B-cell) accounts for about 4% of all cancers in the United States. Traditionally, combination chemotherapy consisting of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) was considered as standard treatment option for NHL patients. However, a subset of individuals was inherently resistant to CHOP or developed resistance upon continued exposure to chemotherapy. The development of drug-resistance, plus the undesired toxic side effects of this regimen, led to the inclusion of anti-CD20 mAb, Rituximab, to chemotherapy protocols of NHL patients (R-CHOP). Superior improvement was observed in patients undergoing R-CHOP compared to CHOP. More recently, chimeric antigen receptor (CAR) T-Cells redirected against CD19 (CD19 CAR T-cell) has proven to be an effective immunotherapy against various cancers including NHL. Despite initial success, and like other approaches, NHL patients become unresponsive to CD19 CAR T-Cells due to selective outgrowth of NHLs with deregulated expression of apoptotic proteins. Histone deacetylase inhibitors (HDACis) and celecoxib have gene regulatory effects and skew the tumor intracellular environment into a proapoptotic milieu. Thus, resistant NHL cells will become sensitized to apoptotic death signals delivered by CD19 CAR T-Cells. We propose the inclusion of FDA-approved small molecules as sensitizing agents to reduce the apoptosis threshold of resistant NHL and boost CD19 CAR T-cell efficacy

IR-Surviving NSCLC Cells Exhibit Different Patterns of Molecular and Cellular Reactions Relating to the Multifraction Irradiation Regimen and p53-Family Proteins Expression.

Simple SummaryFor the first time, we demonstrated that the significant decrease in p63/p73 expression together with the absence of functional p53 could underlie an increase in the fraction of polyploid cells, transformation rates, and the glycolytic NAD(P)H production in multifraction X-ray radiation exposure (MFR)-surviving cancer cells, providing conditions for radioresistance associated with epithelial–mesenchymal transition (EMT)-like process activation. During radiation therapy (RT), the treatment dose, fractionation, and dose limits for organs at risk (OARs) do not change between patients and are still prescribed mainly based on the Tumor, Node, Metastasis (TNM) stage, performance status, and comorbidities, taking no account of the tumor biology. Our data once again emphasize that non-small cell lung cancer (NSCLC) therapy approaches should become more personalized according to RT regimen, tumor histology, and molecular status of critical proteins.Radiotherapy is a primary treatment modality for patients with unresectable non-small cell lung cancer (NSCLC). Tumor heterogeneity still poses the central question of cancer radioresistance, whether the presence of a particular cell population inside a tumor undergoing a selective outgrowth during radio- and chemotherapy give rise to metastasis and tumor recurrence. In this study, we examined the impact of two different multifraction X-ray radiation exposure (MFR) regimens, fraction dose escalation (FDE) in the split course and the conventional hypofractionation (HF), on the phenotypic and molecular signatures of four MFR-surviving NSCLC cell sublines derived from parental A549 (p53 wild-type) and H1299 (p53-null) cells, namely A549FR/A549HR, H1299FR/H1299HR cells. We demonstrate that sublines surviving different MFR regimens in a total dose of 60 Gy significantly diverge in their molecular traits related to irradiation regimen and p53 status. The observed changes regarding radiosensitivity, transformation, proliferation, metabolic activity, partial epithelial-to-mesenchymal transition (EMT) program activation and 1D confined migratory behavior (wound healing). For the first time, we demonstrated that MFR exposure led to the significant decrease in the expression of p63 and p73, the p53-family members, in p53null cells, which correlated with the increase in cell polyploidy. We could not find significant differences in FRA1 expression between parental cells and their sublines that survived after any MFR regimen regardless of p53 status. In our study, the FDE regimen probably causes partial EMT program activation in MFR-survived NSCLC cells through either Vimentin upregulation in p53null or an aberrant N-cadherin upregulation in p53wt cells. The HF regimen likely less influences the EMT activation irrespectively of the p53 status of MFR-survived NSCLC cells. Our data highlight that both MFR regimens caused overall higher cell transformation of p53null H1299FR and H1299HR cells than their parental H1299 cells. Moreover, our results indicate that the FDE regimen raised the radioresistance and transformation of MFR-surviving NSCLC cells irrespectively of their p53 status, though the HF regimen demonstrated a similar effect on p53null NSCLC cells only. Our data once again emphasize that NSCLC therapy approaches should become more personalized according to radiation therapy (RT) regimen, tumor histology, and molecular status of critical proteins.

070 Comprehensive single-cell analysis of Sézary syndrome reveals novel expression and therapeutic biomarkers

Cutaneous T cell lymphomas (CTCL) are a group of malignancies thought to derive from a clonal proliferation of skin-tropic T cells. Diagnosis is often delayed several years due to variations in presentation and lack of definitive biomarkers, which result in missed early intervention opportunities that may prevent disease progression. In the setting of more aggressive forms of CTCL, most treatments can initially decrease the tumor burden, but often ultimately fail to prevent the selective outgrowth of a resistant population. The lack of effective therapies is due in part to an incomplete understanding of tumor biology. Using single-cell mRNA and adaptive immune receptor sequencing of peripheral blood immune cells in SS, transcriptomic variations were quantified of almost 50,000 malignant and nonmalignant T cells across six SS patients. Within the population of single cells, we identified both quiescent and proliferative populations across multiple patients, and were able to newly identify a large number of genes associated with malignancy. Furthermore, in a single patient, we characterized differences in cell populations comparing malignant T cells after disease progression in the setting of treatment with histone deacetylase inhibition (HDACi) and photopheresis. HDACi therapy led to new transcriptional profiles in a subset of SS cells, characterized by the increased expression of the transcriptional factor FOXP3. These data are critical in defining a compendium of gene expression profiles and intratumoral heterogeneity in SS and CTCL, which are pivotal in our understanding of CTCL biology and in the development of targeted treatments.

Polymicrobial communities in periodontal disease: Their quasi-organismal nature and dialogue with the host.

In health, indigenous polymicrobial communities at mucosal surfaces maintain an ecological balance via both inter-microbial and host-microbial interactions that promote their own and the host's fitness, while preventing invasion by exogenous pathogens. However, genetic and acquired destabilizing factors (including immune deficiencies, immunoregulatory defects, smoking, diet, obesity, diabetes and other systemic diseases, and aging) may disrupt this homeostatic balance, leading to selective outgrowth of species with the potential for destructive inflammation. This process, known as dysbiosis, underlies the development of periodontitis in susceptible hosts. The pathogenic process is not linear but involves a positive-feedback loop between dysbiosis and the host inflammatory response. The dysbiotic community is essentially a quasi-organismal entity, where constituent organisms communicate via sophisticated physical and chemical signals and display functional specialization (eg, accessory pathogens, keystone pathogens, pathobionts), which enables polymicrobial synergy and dictates the community's pathogenic potential or nososymbiocity. In this review, we discuss early and recent studies in support of the polymicrobial synergy and dysbiosis model of periodontal disease pathogenesis. According to this concept, disease is not caused by individual "causative pathogens" but rather by reciprocally reinforced interactions between physically and metabolically integrated polymicrobial communities and a dysregulated host inflammatory response.

Damaging effects of BMAA on retina neurons and Müller glial cells

B–N-methylamino-L-alanine (BMAA), a cyanotoxin produced by most cyanobacteria, has been proposed to cause long term damages leading to neurodegenerative diseases, including Amyotrophic Lateral Sclerosis/Parkinsonism Dementia complex (ALS/PDC) and retinal pathologies. Previous work has shown diverse mechanisms leading to BMAA-induced degeneration; however, the underlying mechanisms of toxicity affecting retina cells are not fully elucidated. We here show that BMAA treatment of rat retina neurons in vitro induced nuclear fragmentation and cell death in both photoreceptors (PHRs) and amacrine neurons, provoking mitochondrial membrane depolarization. Pretreatment with the N-Methyl-D-aspartate (NMDA) receptor antagonist MK-801 prevented BMAA-induced death of amacrine neurons, but not that of PHRs, implying activation of NMDA receptors participated only in amacrine cell death. Noteworthy, BMAA stimulated a selective axonal outgrowth in amacrine neurons, simultaneously promoting growth cone destabilization. BMAA partially decreased the viability of Müller glial cells (MGC), the main glial cell type in the retina, induced marked alterations in their actin cytoskeleton and impaired their capacity to protect retinal neurons. BMAA also induced cell death and promoted axonal outgrowth in differentiated rat pheochromocytoma (PC12) cells, implying these effects were not limited to amacrine neurons. These results suggest that BMAA is toxic for retina neurons and MGC and point to the involvement of NMDA receptors in amacrine cell death, providing new insight into the mechanisms involved in BMAA neurotoxic effects in the retina.

Abstract PO-106: Genomic heterogeneity and clonal dynamics of resistance evolution and metastatic progression in rectal cancer

Abstract The standard treatment for locally advanced rectal cancer is chemoradiotherapy followed by surgery. However, patient response to pre-operative chemoradiotherapy is highly heterogeneous, ranging from complete tumor regression to no response. Over the course of disease, up to 40% of patients suffer from local relapse or the development of metachronous metastatic disease, compromising survival. It is not yet known how treatment resistance and disease progression in these patients evolve – whether from the selective outgrowth of pre-existing resistant clones during treatment or through the acquisition of additional, resistance conferring genomic alterations. We aim to delineate the genomic evolution and clonal architecture underlying treatment resistance and metastatic progression in rectal cancer based on a cohort of 42 patients, from which 98 tumor samples were collected longitudinally over the course of treatment and metastatic progression. Tumor diagnosis and treatment response assessment are routinely based on the histopathologic evaluation of formalin-fixed paraffin-embedded (FFPE) patient tissues. While formalin fixation and paraffin embedding optimally preserve histomorphology and allow long-term tissue storage at ambient temperature, they, on the other hand, lead to DNA crosslinks posing a challenge for sequencing analyses. To overcome this limitation, we have successfully developed a protocol for dissociation of FFPE tissues into single cells that are suitable for immunolabeling, flow sorting and genomic analysis by whole exome sequencing of pure tumor cell populations, single cell copy number variation (CNV) sequencing and multiplex interphase fluorescence in situ hybridization (miFISH). Our genomic analyses at the population as well as individual cell level revealed divergent aberration patterns and distinct levels of intra-tumor heterogeneity. Major tumor clones were characterized by gains of EGFR (7p11.2), MYC (8q24.21), CDX2 (13q12.2) and ZNF217 (20q13.2) along with losses of SMAD4 (18q21.2) and TP53 (17p13.1). While in some patients the clonal composition remained largely stable throughout treatment, in others major clonal shifts occurred favoring clones with TP53 loss. Our preliminary results indicate different propensities of genetically distinct tumor cell clones in therapy response and metastatic progression. Furthermore, our data show the feasibility of high-resolution clonal reconstruction from whole exome sequencing and single cell CNV sequencing data of FFPE cells. Citation Format: Daniela Hirsch, Judith Lieberich, Kerstin Heselmeyer-Haddad, Yonca Ceribas, Michael Kelly, Keyur Talsania, Yongmei Zhao, Timo Gaiser, Thomas Ried. Genomic heterogeneity and clonal dynamics of resistance evolution and metastatic progression in rectal cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-106.

3094 – ROLE OF BONE MARROW INJURY IN PROMOTING TET2-MEDIATED CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL (CHIP)

CHIP is characterized by selective outgrowth of hematopoietic stem/progenitor cell (HSPC) in the bone marrow (BM) which frequently harbors oncogenic mutations in genes such as TET2. This leads to aberrant expansion of hyperinflammatory immune cells that can contribute to cardiovascular disease and other comorbidities. Prior genotoxic treatments such as chemotherapy and radiation reportedly correlate with the expansion of CHIP mutant HSPC. However, the mechanism(s) remain poorly understood. To address this question, we modeled human CHIP via adoptively transferring Tet2-/- mouse BM into wild-type (WT) recipient mice. In this setting, Tet2-/- HSPC and their progeny preferentially expanded in the BM and blood. Interestingly, Tet2-/- HSPC also colonized non-hematopoietic organs including lung, liver and gonadal adipose tissue. To test whether genotoxic stress caused by radiation accelerated this process, we subjected Tet2-/- CHIP mice to 2.5Gy radiation. Indeed, radiation accelerated Tet2-/- HSPC expansion in nearly all tissues. To assess the impact of microenvironmental factors induced by radiation on CHIP progression, we modeled radiation-induced inflammation and tissue oxygenation using in vitro HSPC culture. Surprisingly, inflammatory cytokines were not sufficient to drive selective expansion of Tet2-/- HSPC. On the other hand, Tet2-/- HSPC displayed a significant proliferative advantage over WT when cultured at high oxygen tension (20%O2) but not in hypoxic conditions representative of healthy BM (1%O2), suggesting high O2 tension caused by BM injury may play a vital role in the selective expansion of Tet2-/- HSPC. Ongoing studies are aimed at understanding the impact of increased oxygen levels in the BM using non-genotoxic approaches and identifying mechanism(s) by which Tet2-/- HSPC harness high O2 tension to proliferate. Such investigations could provide a basis for preventing CHIP and its associated comorbidities.

Artificial feeder cells expressing ligands for killer cell immunoglobulin-like receptors and CD94/NKG2A for expansion of functional primary natural killer cells with tolerance to self

Background aimsNatural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells. MethodsPC3PSCA-derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells. ResultsOnly combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen. ConclusionsCollectively, the results demonstrate the feasibility of PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.

Intestinal microbiota domination under extreme selective pressures characterized by metagenomic read cloud sequencing and assembly

BackgroundLow diversity of the gut microbiome, often progressing to the point of intestinal domination by a single species, has been linked to poor outcomes in patients undergoing hematopoietic cell transplantation (HCT). Our ability to understand how certain organisms attain intestinal domination over others has been restricted in part by current metagenomic sequencing technologies that are typically unable to reconstruct complete genomes for individual organisms present within a sequenced microbial community. We recently developed a metagenomic read cloud sequencing and assembly approach that generates improved draft genomes for individual organisms compared to conventional short-read sequencing and assembly methods. Herein, we applied metagenomic read cloud sequencing to four stool samples collected longitudinally from an HCT patient preceding treatment and over the course of heavy antibiotic exposure.ResultsCharacterization of microbiome composition by taxonomic classification of reads reveals that that upon antibiotic exposure, the subject’s gut microbiome experienced a marked decrease in diversity and became dominated by Escherichia coli. While diversity is restored at the final time point, this occurs without recovery of the original species and strain-level composition. Draft genomes for individual organisms within each sample were generated using both read cloud and conventional assembly. Read clouds were found to improve the completeness and contiguity of genome assemblies compared to conventional assembly. Moreover, read clouds enabled the placement of antibiotic resistance genes present in multiple copies both within a single draft genome and across multiple organisms. The occurrence of resistance genes associates with the timing of antibiotics administered to the patient, and comparative genomic analysis of the various intestinal E. coli strains across time points as well as the bloodstream isolate showed that the subject’s E. coli bloodstream infection likely originated from the intestine. The E. coli genome from the initial pre-transplant stool sample harbors 46 known antimicrobial resistance genes, while all other species from the pre-transplant sample each contain at most 5 genes, consistent with a model of heavy antibiotic exposure resulting in selective outgrowth of the highly antibiotic-resistant E. coli.ConclusionThis study demonstrates the application and utility of metagenomic read cloud sequencing and assembly to study the underlying strain-level genomic factors influencing gut microbiome dynamics under extreme selective pressures in the clinical context of HCT.

Abstract 751: Ductal carcinoma in situ of the breast: Cancer precursor or not

Abstract Background. Ductal carcinoma in situ (DCIS) is a potential precursor of invasive breast cancer (IBC). However, the natural course of a particular DCIS lesion is unknown, because almost all women with DCIS are treated. Furthermore, most studies are biased because these comprise DCIS adjacent to IBC, also known as synchronous DCIS and IBC, indicating that such DCIS lesions already have the capacity to progress to IBC. It is still unknown which proportion and type of subsequent ipsilateral IBCs (iIBC) are related to the initial primary “pure” DCIS lesion. Therefore, we performed an extensive molecular characterization of DCIS and matched subsequent iIBC, to better understand the natural course of DCIS. Patients and methods. We used a unique series of 78 women diagnosed with DCIS and treated by breast conserving surgery (BCS) alone, which subsequently developed iIBC. Mean time to iIBC event was 6.3 years (range 0.5-17.0). These 78 women are a representative sample of a case-control series, nested in a nation-wide, population-based cohort including all patients diagnosed with DCIS between 1989 and 2005 in the Netherlands (Visser, et al Clin Can Res 2018). Data on tumor location (ICD-10) was available for all lesions. DNA and RNA was simultaneously extracted for 78 DCIS lesions and 78 matched subsequent iIBC (DNA >20ng; RNA >100ng), and RNA sequencing (RNAseq) and low coverage whole genome sequencing (CNVseq) was performed. Panel sequencing (PanelSeq), using a custom panel of 53 breast cancer driver genes, was performed with the remaining DNA of 42 DCIS and iIBC matched pairs. We determined if the iIBC lesion and DCIS lesion were related, by comparing tumor location and genomic features. Results. Based on tumor location and histological grade, >95% of the subsequent iIBC reflected outgrowth of residual disease. Based on RNAseq data, 77% of all DCIS and IBC lesions classified into the same PAM50 subtypes. The CNVseq data showed that the DCIS lesions contained copy number aberrations on typical breast cancer-associated loci, such as 1q gain, 8q gain, 16q loss, 20q gain. However, when we compared DCIS with their matched iIBC, we observed in 41% of the cases very distinct copy number profiles, indicating either outgrow of a different tumor (minority) subclone or a second primary tumor. Analysis of PanelSeq mutation data supported this clonal or independent origin of the subsequent iIBC. Conclusion. To our knowledge, our study is the first to investigate if subsequent iIBC is likely to originate from the initial primary “pure” DCIS in a large series with long-term follow-up. Surprisingly, our CNVseq and PanelSeq results indicate that more than one third of the subsequent iIBCs after BCS alone treated primary DCIS are likely to be second primary tumors or represent selective outgrowth of a minority DCIS subclone. Citation Format: Lindy Visser, Marlous Hoogstraat, Tycho Bismeijer, Lotte Elshof, Koen van de Vijver, Emilie Groen, Mathilde Almekinders, Joyce Sanders, Carolien Bierman, Dennis Peters, Ingrid Hofland, Frank Nieboer, Michiel de Maaker, Petra Kristel, Lennart Mulder, Annegien Broeks, Michael Schaapveld, Marjanka Schmidt, Lodewyk Wessels, Esther Lips, Jelle Wesseling, On behalf of the PRECISION team. Ductal carcinoma in situ of the breast: Cancer precursor or not [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 751.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Quantification and localization of oncogenic receptor tyrosine kinase variant transcripts using molecular inversion probes

Oncogenic membrane receptor tyrosine kinases such as MET and EGFR, or auto-active variants thereof, are important targets for cancer precision therapy. Targeted inhibition of these oncogenic receptors however invariably leads to resistance, resulting from acquisition of resistance-inducing mutations or from selective outgrowth of a priori resistant tumour cells. Most applied molecular protocols cannot distinguish between intracellular and intercellular heterogeneity of oncogene (variant) expression, which may lead to misinterpretation of the molecular make-up of a cancer and suboptimal application of targeted therapies. We here combined two related techniques to allow semiquantitative and localized in situ detection of specific transcript splice variants using single molecule molecular inversion probe (smMIP)-based next generation sequencing and padlock probe-based rolling circle amplification, respectively. We show highly specific padlock probe-based multiplex detection of MET, METΔ7-8 and METΔ14 transcripts, lacking exons 7–8 and exon 14 respectively, and of EGFR and the auto-active EGFRvIII, lacking exons 2–7. The combination of quantitative transcript variant detection with smMIPs and transcript localization using padlock probes can be used for detection of oncogenic transcripts on the single-cell level, allowing study of tumour heterogeneity. Visualization of tumour heterogeneity can shed light on the biology underlying drug resistance and potentially improve targeted therapeutics.

Analysis and dissociation of anti-HIV effects of shRNA to CCR5 and the fusion inhibitor C46.

The gene therapeutic Cal-1 comprises the anti-HIV agents: (i) sh5, a short hairpin RNA to CCR5 that down-regulates CCR5 expression and (ii) maC46 (C46), a peptide that inhibits viral fusion with the cell membrane. These constructs were assessed for inhibition of viral replication and selective cell expansion in a number of settings. HIV replication, selective outgrowth and cell surface viral binding were analysed with a single cycle infection assay of six pseudotyped HIV strains and a static and longitudinal passaging of MOLT4/CCR5 cells with HIV. Pronase digestion of surface virus and fluorescence microscopy assessed interactions between HIV virions and transduced cells. Cal-1 reduced CCR5 expression in peripheral blood mononuclear cells to CCR5Δ32 heterozygote levels. Even low level transduction resulted in significant preferential expansion in MOLT4/CCR5 gene-containing cells over a 3-week HIV challenge regardless of viral suppression [12.5% to 47.0% (C46), 46.7% (sh5), 62.2% (Dual), respectively]. The sh5 and Dual constructs at > 95% transduction also significantly suppressed virus to day 12 in the passage assay and all constructs, at varying percentage transduction inhibited virus in static culture. No escape mutations were present through 9weeks of challenge. The Dual construct significantly suppressed infection by a panel of CCR5-using viruses, with its efficacy being independently determined from the single constructs. Dual and sh5 inhibited virion internalisation, as determined via pronase digestion of surface bound virus, by 70% compared to 13% for C46. The use of two anti-HIV genes allows optimal preferential survival and inhibition of HIV replication, with the impact on viral load being dependent on the percentage of gene marked cells.

Biodegradable neural cell culture sheet made of poly(lactic-co-glycolic acid) thin film with micropatterns of Dulbecco’s phosphate-buffered saline (−) containing laminin layers

In the regenerative medicine field of nervous systems, techniques used to fabricate microstructures of neurons on flexible and biodegradable substrates have attracted attention. In this research, biodegradable and flexible neuron culture thin films that enable the selective axonal outgrowth of neurons were fabricated using poly(lactic-co-glycolic acid) (PLGA) thin films with micropatterns of Dulbecco’s phosphate-buffered saline (D-PBS) (−) containing laminin layers. The 100-µm-thick PLGA thin films were fabricated by diluting PLGA in acetone (5% w/w) and the solution was distributed onto a poly(dimethylsiloxane) (PDMS) mold. D-PBS (−) micropatterns containing laminin layers with widths of 10–150 µm were fabricated by micromolding in capillaries (MIMIC) and the microstencil method. Rat neurons were selectively cultured for 3 d on the laminin micropatterns; using the MIMIC method, the cells properly adhered to a pattern wider than 30 µm, while with the microstencil method, the necessary pattern width for proper adhesion was more than 50 µm.

ERRα Maintains Mitochondrial Oxidative Metabolism and Constitutes an Actionable Target in PGC1α-Elevated Melanomas.

The uncontrolled growth of tumors provides metabolic dependencies that can be harnessed for therapeutic benefit. Although tumor cells exhibit these increased metabolic demands due to their rapid proliferation, these metabolic processes are general to all cells, and furthermore, targeted therapeutic intervention can provoke compensatory adaptation that alters tumors' characteristics. As an example, a subset of melanomas depends on the transcriptional coactivator PGC1α function to sustain their mitochondrial energy-dependent survival. However, selective outgrowth of resistant PGC1α-independent tumor cells becomes endowed with an augmented metastatic phenotype. To find PGC1α-dependent components that would not affect metastasis in melanomas, an unbiased proteomic analyses was performed and uncovered the orphan nuclear receptor ERRα, which supports PGC1α's control of mitochondrial energetic metabolism, but does not affect the antioxidant nor antimetastatic regulatory roles. Specifically, genetic or pharmacologic inhibition of ERRα reduces the inherent bioenergetic capacity and decreases melanoma cell growth, but without altering the invasive characteristics. Thus, within this particularly aggressive subset of melanomas, which is characterized by heighted expression of PGC1α, ERRα specifically mediates prosurvival functions and represents a tangible therapeutic target.Implications: ERRα, a druggable protein, mediates the bioenergetic effects in melanomas defined by high PGC1α expression, suggesting a rational means for therapeutic targeting of this particularly aggressive melanoma subtype. Mol Cancer Res; 15(10); 1366-75. ©2017 AACR.