EGFR and ERBB2 exon 20 insertion/duplication in advanced non–small cell lung cancer: genomic profiling and clinicopathologic features

BackgroundExon 20 (ex20) in-frame insertions or duplications (ins/dup) in epidermal growth factor receptor (EGFR) and its analog erb-b2 receptor tyrosine kinase 2 (ERBB2) are each detected in 1.5% of non–small cell lung cancer (NSCLC). Unlike EGFR p.L858R or ex19 deletions, ex20 ins/dup is associated with de novo resistance to classic EGFR inhibitors, lack of response to immune checkpoint inhibitors, and poor prognosis. US Food and Drug Administration has approved mobocertinib and amivantamab for targeting tumors with this aberration, but the number of comprehensive studies on ex20 ins/dup NSCLC is limited. We identified 18 cases of NSCLCs with EGFR/ERBB2 ex20 ins/dup and correlated the findings with clinical and morphologic information including programed death-ligand 1 (PD-L1) expression.MethodsA total of 536 NSCLC cases tested at our institution between 2014 and 2023 were reviewed. A custom-designed 214-gene next-generation sequencing panel was used for detecting DNA variants, and the FusionPlex CTL panel (ArcherDx) was used for the detection of fusion transcripts from formalin-fixed, paraffin-embedded tissue. Immunohistochemistry (IHC)for PD-L1 was performed using 22C3 or E1L3N clones.ResultsNine EGFR and nine ERBB2 ex20 ins/dup variants were identified from an equal number of men and women, 14 were non- or light smokers, and 15 had stage IV disease. All 18 cases were adenocarcinomas. Seven of the 11 cases with available primary tumors had acinar predominant pattern, two had lepidic predominant pattern, and the remainder had papillary (one case) and mucinous (one case) patterns. Ex20 ins/dup variants were heterogenous in-frame one to four amino acids spanning A767–V774 in EGFR and Y772–P780 in ERBB2 and were clustered in the loop following the C-helix and α C-helix. Twelve cases (67%) had co-existing TP53 variants. Copy number variation in CDK4 amplification was identified in one case. No fusion or microsatellite instability was identified in any case. PD-L1 was positive in two cases, low positive in four cases, and negative in 11 cases.ConclusionsNSCLCs harboring EGFR/ERBB2 ex20 ins/dup are rare and tend to be acinar predominant, negative for PD-L1, more frequent in non- or light smokers, and mutually exclusive with other driver mutations in NSCLC. The correlation of different EGFR/ERBB2 ex20 ins/dup variants and co-existing mutations with response to targeted therapy and the possibility of developing resistant mutations after mobocertinib treatment warrants further investigation

The Stem Cell Discovery Engine: an integrated repository and analysis system for cancer stem cell comparisons

Mounting evidence suggests that malignant tumors are initiated and maintained by a subpopulation of cancerous cells with biological properties similar to those of normal stem cells. However, descriptions of stem-like gene and pathway signatures in cancers are inconsistent across experimental systems. Driven by a need to improve our understanding of molecular processes that are common and unique across cancer stem cells (CSCs), we have developed the Stem Cell Discovery Engine (SCDE)—an online database of curated CSC experiments coupled to the Galaxy analytical framework. The SCDE allows users to consistently describe, share and compare CSC data at the gene and pathway level. Our initial focus has been on carefully curating tissue and cancer stem cell-related experiments from blood, intestine and brain to create a high quality resource containing 53 public studies and 1098 assays. The experimental information is captured and stored in the multi-omics Investigation/Study/Assay (ISA-Tab) format and can be queried in the data repository. A linked Galaxy framework provides a comprehensive, flexible environment populated with novel tools for gene list comparisons against molecular signatures in GeneSigDB and MSigDB, curated experiments in the SCDE and pathways in WikiPathways. The SCDE is available at http://discovery.hsci.harvard.edu

Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations

Background: The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. Methods: We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. Results: We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. Conclusion: We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma

Transcription Profiling of Epstein-Barr Virus Nuclear Antigen (EBNA)-1 Expressing Cells Suggests Targeting of Chromatin Remodeling Complexes

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA)-1 regulates virus replication and transcription, and participates in the remodeling of the cellular environment that accompanies EBV induced B-cell immortalization and malignant transformation. The putative cellular targets of these effects of EBNA-1 are largely unknown. To address this issue we have profiled the transcriptional changes induced by short- and long-term expression of EBNA-1 in the EBV negative B-cell lymphoma BJAB. Three hundred and nineteen cellular genes were regulated in a conditional transfectant shortly after EBNA-1 induction while a ten fold higher number of genes was regulated upon continuous EBNA-1 expression. Promoter analysis of the differentially regulated genes demonstrated a significant enrichment of putative EBNA-1 binding sites suggesting that EBNA-1 may directly influence the transcription of a subset of genes. Gene ontology analysis of forty seven genes that were consistently regulated independently on the time of EBNA-1 expression revealed an unexpected enrichment of genes involved in the maintenance of chromatin architecture. The interaction network of the affected gene products suggests that EBNA-1 may promote a broad rearrangement of the cellular transcription landscape by altering the expression of key components of chromatin remodeling complexes

In silico analysis of pathways targeted by EBV infection and malignant transformation

Epstein-Barr virus (EBV) is a ubiquitous γ-herpes virus with dual cell tropism for human B-lymphocytes and epithelial cells. EBV infection is linked to several malignancies such as Burkitt s lymphoma (BL) and nasopharyngeal carcinoma (NPC). In vitro EBV is a potent transforming virus that converts resting B-lymphocytes into indefinitely proliferating lymphoblastoid cells (LCLs). The overall aim of this study was to develop and utilize bioinformatics methods to dissect the molecular mechanisms by which EBV modulates the cellular environment. EBV is a large DNA virus that encodes about 100 open reading frames (ORFs) expressed at various times during infection. Using established sequence search methods such as patterns and hidden Markov models (HMM), we have identified catalytic domains of ubiquitin specific proteases (deubiquitinating enzymes, DUBs) in a limited number of EBV ORFs. The DUB activity of three high scoring candidates: BPLF1, BSLF1 and BXLF1, was confirmed by functional and mutational analysis (Paper-I). EBV establishes distinct programs of viral gene expression in latently infected and malignant cells. The EBV nuclear antigen (EBNA)-1 is expressed in all EBV carrying proliferating cells. EBNA-1 has been associated with the induction of cellular oxidative stress due to the production of reactive oxygen species (ROS) and DNA damage. To search for the cellular genes involved in these effects we used gene expression data sets from public databases. By analyzing the expression of genes involved in ROS metabolism in EBV positive and negative Burkitt's lymphoma (BL) cell lines and lymphoblastoid cell lines (LCL) we found that the NADPH oxidase (NOX)-2 is induced in cells expressing EBNA-1. Activation of the NOX2 gene by EBNA-1 is associated with the induction and/or maintenance of genomic instability, a critical step in malignant transformation (Paper-II). The expression of EBNA-1 is associated with a global rearrangement of cellular transcription. To investigate the primary and secondary targets of this transcriptional effect we have analyzed the gene expression profiles of stable and inducible EBNA-1 expressing cells. Functional analysis of the regulated transcripts revealed that EBNA-1 influences the expression of genes involved in the maintenance of chromatin architecture. Several subunits of chromatin remodeling complexes were down-regulated on EBNA-1 expression (Paper-III). Microarray analysis and systems biology approaches were implemented to investigate the cellular pathways modulated by tripeptidyl peptidase II (TPPII) a cellular protein that is highly expressed in BL cells and participates in the induction of the malignant phenotype. Comparison of the gene expression profiles of control and TPPII knockdown BL cells and systems level analysis of differentially regulated genes demonstrated that the MAPK signaling pathway is selectively inactivated by TPPII knockdown (Paper-IV). In summary, this work demonstrated the potential of bioinformatics tools and highthroughput genomic approaches in identifying the novel strategies of EBV

The ubiquitin specific protease-4 (USP4) interacts with the S9/Rpn6 subunit of the proteasome

The proteasome is the major non-lysosomal proteolytic machine in cells that, through degradation of ubiquitylated substrates, regulates virtually all cellular functions. Numerous accessory proteins influence the activity of the proteasome by recruiting or deubiquitylating proteasomal substrates, or by maintaining the integrity of the complex. Here we show that the ubiquitin specific protease (USP)-4, a deubiquitylating enzyme with specificity for both Lys48 and Lys63 ubiquitin chains, interacts with the S9/Rpn6 subunit of the proteasome via an internal ubiquitin-like (UBL) domain. S9/Rpn6 acts as a molecular clamp that holds together the proteasomal core and regulatory sub-complexes. Thus, the interaction with USP4 may regulate the structure and function of the proteasome or the turnover of specific proteasomal substrates. © 2012 Elsevier Inc

The ubiquitin specific protease-4 (USP4) interacts with the S9/Rpn6 subunit of the proteasome

The proteasome is the major non-lysosomal proteolytic machine in cells that, through degradation of ubiquitylated substrates, regulates virtually all cellular functions. Numerous accessory proteins influence the activity of the proteasome by recruiting or deubiquitylating proteasomal substrates, or by maintaining the integrity of the complex. Here we show that the ubiquitin specific protease (USP)-4, a deubiquitylating enzyme with specificity for both Lys48 and Lys63 ubiquitin chains, interacts with the S9/Rpn6 subunit of the proteasome via an internal ubiquitin-like (UBL) domain. S9/Rpn6 acts as a molecular clamp that holds together the proteasomal core and regulatory sub-complexes. Thus, the interaction with USP4 may regulate the structure and function of the proteasome or the turnover of specific proteasomal substrates. © 2012 Elsevier Inc

Epstein-Barr Virus Encodes Three Bona Fide Ubiquitin-Specific Proteases▿ ‡

Manipulation of the ubiquitin proteasome system (UPS) is emerging as a common theme in viral pathogenesis. Some viruses have been shown to encode functional homologs of UPS enzymes, suggesting that a systematic identification of these products may provide new insights into virus-host cell interactions. Ubiquitin-specific proteases, collectively known as deubiquitinating enzymes (DUBs), regulate the activity of the UPS by hydrolyzing ubiquitin peptide or isopeptide bonds. The prediction of viral DUBs based on sequence similarity with known enzymes is hampered by the diversity of viral genomes. In this study sequence alignments, pattern searches, and hidden Markov models were developed for the conserved C- and H-boxes of the known DUB families and used to search the open reading frames (ORFs) of Epstein-Barr virus (EBV), a large gammaherpesvirus that has been implicated in the pathogenesis of a broad spectrum of human malignancies of lymphoid and epithelial cell origin. The searches identified a limited number of EBV ORFs that contain putative DUB catalytic domains. DUB activity was confirmed by functional assays and mutation analysis for three high scoring candidates, supporting the usefulness of this bioinformatics approach in predicting distant homologues of cellular enzymes