Trastuzumab deruxtecan in HER2-positive advanced gastric cancer: exploratory biomarker analysis of the randomized, phase 2 DESTINY-Gastric01 trial.

Trastuzumab deruxtecan (T-DXd) showed statistically significant clinical improvement in patients with human epidermal growth factor receptor 2-positive (HER2+) gastric cancer in the DESTINY-Gastric01 trial. Exploratory results from DESTINY-Gastric01 suggested a potential benefit in patients with HER2-low gastric cancer. Spatial and temporal heterogeneity in HER2 expression or gene alteration, an inherent characteristic of gastric cancer tumors, presents a challenge in identifying patients who may respond to T-DXd. Specific biomarkers related to therapeutic response have not been explored extensively. Exploratory analyses were conducted to assess baseline HER2-associated biomarkers in circulating tumor DNA and tissue samples, and to investigate mechanisms of resistance to T-DXd. Baseline HER2-associated biomarkers were correlated with objective response rate (ORR) in the primary cohort of patients with HER2+ gastric cancer. The primary cohort had 64% concordance between HER2 positivity and HER2 (ERBB2) plasma gene amplification. Other key driver gene amplifications, specifically MET, EGFR and FGFR2, in circulating tumor DNA were associated with numerically lower ORR. Among 12 patients with HER2 gain-of-function mutations, ORR was 58.3% (7 of 12). ORR was consistent regardless of timing of immunohistochemistry sample collection. Further investigations are required in larger studies.

Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals.

CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we discovered a novel rat monoclonal antibody #147D. This humanized IgG4-formatted antibody, h4#147D, showed potent antitumor efficacy in xenograft mouse models harboring the human PDAC cell line MIA PaCa-2, HCC cell line Hep G2, and CML cell line KU812, which featured low sensitivity to the corresponding standard-of-care drugs (gemcitabine, sorafenib, and imatinib, respectively). An analysis of tumor cells derived from MIA PaCa-2 xenograft mice treated with h4#147D revealed that cell surface expression of CD147 and its binding partners, including CD44 and integrin α3ß1/α6ß1, was significantly reduced by h4#147D. Inhibition of focal adhesion kinase (FAK), activation of multiple stress responsible signal proteins such as c-JunN-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38MAPK), and expression of SMAD4, as well as activation of caspase-3 were obviously observed in the tumor cells, suggesting that h4#147D induced tumor shrinkage by inducing multiple stress responsible signals. These results suggest that the anti-CD147 antibody h4#147D offers promise as a new antibody drug candidate.

Pan-cancer gene expression analysis of tissue microarray using EdgeSeq oncology biomarker panel and a cross-comparison with HER2 and HER3 immunohistochemical analysis.

Molecular and protein biomarker profiling are key to oncology drug development. Antibody-drug conjugates (ADCs) directly deliver chemotherapeutic agents into tumor cells based on unique cancer cell biomarkers. A pan-cancer tissue microarray (TMA) data set and gene panel were validated and gene signature analyses were conducted on normal and cancer tissues to refine selection of ADC targets. Correlation of mRNA and protein levels, and human epidermal growth factor receptor (HER) expression patterns were assessed. An EdgeSeq biomarker panel (2862 genes) was used across 8531 samples (23 solid cancer types/subtypes; 16 normal tissues) with an established TMA data set, and immune cell and cell cycle gene signatures were analyzed. Discriminating gene expression signatures were defined based on pathological classification of cancer subtypes. Correlative analyses of HER2 and HER3 mRNA (EdgeSeq) and protein expression (immunohistochemistry [IHC]) were performed and compared with publicly available data (The Cancer Genome Atlas [TCGA]; Cancer Cell Line Encyclopedia [CCLE]). Gene expression patterns among cancer types in the TMA (EdgeSeq) and TCGA (RNA-seq) were similar. EdgeSeq gene signature analyses aligned with the majority of pathological cancer types/subtypes and identified cancer-specific gene expression patterns. TMA IHC H-scores for HER3 varied across cancer types/subtypes. In a few cancer types, HER3 mRNA and protein expression did not align, including lower liver hepatocellular carcinoma IHC H-score, compared with mRNA. Although all TNBC and ovarian cancer subtypes expressed mRNA, some had lower protein expression. This was seen in TMA and TCGA data sets, but not in CCLE. The EdgeSeq TMA data set can expand upon current biomarker data by including cancers not currently in TCGA. The primary analysis of EdgeSeq and IHC comparison suggested a unique protein-level regulation of HER3 in some tumor subtypes and highlights the importance of investigating protein levels of ADC targets in both tumor and normal tissues.

CDKN1C-mediated growth inhibition by an EZH1/2 dual inhibitor overcomes resistance of mantle cell lymphoma to ibrutinib.

Mantle cell lymphoma (MCL) is a rare subtype of non-Hodgkin's lymphoma, which is characterized by overexpression of cyclin D1. Although novel drugs, such as ibrutinib, show promising clinical outcomes, relapsed MCL often acquires drug resistance. Therefore, alternative approaches for refractory and relapsed MCL are needed. Here, we examined whether a novel inhibitor of enhancer of zeste homologs 1 and 2 (EZH1/2), OR-S1 (a close analog of the clinical-stage compound valemetostat), had an antitumor effect on MCL cells. In an ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model, OR-S1 treatment by oral administration significantly inhibited MCL tumor growth, whereas ibrutinib did not. In vitro growth assays showed that compared with an established EZH2-specific inhibitor GSK126, OR-S1 had a marked antitumor effect on MCL cell lines. Furthermore, comprehensive gene expression analysis was performed using OR-S1-sensitive or insensitive MCL cell lines and showed that OR-S1 treatment modulated B-cell activation, differentiation, and cell cycle. In addition, we identified Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, also known as p57, KIP2), which contributes to cell cycle arrest, as a direct target of EZH1/2 and showed that its expression influenced MCL cell proliferation. These results suggest that EZH1/2 may be a potential novel target for the treatment of aggressive ibrutinib-resistant MCL via CDKN1C-mediated cell cycle arrest.

Identification and Therapeutic Targeting of GPR20, Selectively Expressed in Gastrointestinal Stromal Tumors, with DS-6157a, a First-in-Class Antibody-Drug Conjugate.

Currently, the only approved treatments for gastrointestinal stromal tumor (GIST) are tyrosine kinase inhibitors (TKI), which eventually lead to the development of secondary resistance mutations in KIT or PDGFRA and disease progression. Herein, we identified G protein-coupled receptor 20 (GPR20) as a novel non-tyrosine kinase target in GIST, developed new GPR20 IHC, and assessed GPR20 expression in cell lines, patient-derived xenografts, and clinical samples from two institutes (United States and Japan). We studied GPR20 expression stratified by treatment line, KIT expression, GIST molecular subtype, and primary tumor location. We produced DS-6157a, an anti-GPR20 antibody-drug conjugate with a novel tetrapeptide-based linker and DNA topoisomerase I inhibitor exatecan derivative (DXd). DS-6157a exhibited GPR20 expression-dependent antitumor activity in GIST xenograft models including a GIST model resistant to imatinib, sunitinib, and regorafenib. Preclinical pharmacokinetics and safety profile of DS-6157a support its clinical development as a potential novel GIST therapy in patients who are refractory or have resistance or intolerance to approved TKIs. SIGNIFICANCE: GPR20 is selectively expressed in GIST across all treatment lines, regardless of KIT/PDGFRA genotypes. We generated DS-6157a, a DXd-based antibody-drug conjugate that exhibited antitumor activity in GIST models by a different mode of action than currently approved TKIs, showing favorable pharmacokinetics and safety profiles.This article is highlighted in the In This Issue feature, p. 1307.

Potentiality of multiple modalities for single-cell analyses to evaluate the tumor microenvironment in clinical specimens.

Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.

Dual inhibition of enhancer of zeste homolog 1/2 overactivates WNT signaling to deplete cancer stem cells in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy caused by accumulation of abnormal clonal plasma cells. Despite the recent development of novel therapies, relapse of MM eventually occurs as a result of a remaining population of drug-resistant myeloma stem cells. Side population (SP) cells show cancer stem cell-like characteristics in MM; thus, targeting these cells is a promising strategy to completely cure this malignancy. Herein, we showed that SP cells expressed higher levels of enhancer of zeste homolog (EZH) 1 and EZH2, which encode the catalytic subunits of Polycomb repressive complex 2 (PRC2), than non-SP cells, suggesting that EZH1 as well as EZH2 contributes to the stemness maintenance of the MM cells and that targeting both EZH1/2 is potentially a significant therapeutic approach for eradicating myeloma stem cells. A novel orally bioavailable EZH1/2 dual inhibitor, OR-S1, effectively eradicated SP cells and had a greater antitumor effect than a selective EZH2 inhibitor in vitro and in vivo, including a unique patient-derived xenograft model. Moreover, long-term continuous dosing of OR-S1 completely cured mice bearing orthotopic xenografts. Additionally, PRC2 directly regulated WNT signaling in MM, and overactivation of this signaling induced by dual inhibition of EZH1/2 eradicated myeloma stem cells and negatively affected tumorigenesis, suggesting that repression of WNT signaling by PRC2 plays an important role in stemness maintenance of MM cells. Our results show the role of EZH1/2 in the maintenance of myeloma stem cells and provide a preclinical rationale for therapeutic application of OR-S1, leading to significant advances in the treatment of MM.

The tandem duplicator phenotype as a distinct genomic configuration in cancer.

Next-generation sequencing studies have revealed genome-wide structural variation patterns in cancer, such as chromothripsis and chromoplexy, that do not engage a single discernable driver mutation, and whose clinical relevance is unclear. We devised a robust genomic metric able to identify cancers with a chromotype called tandem duplicator phenotype (TDP) characterized by frequent and distributed tandem duplications (TDs). Enriched only in triple-negative breast cancer (TNBC) and in ovarian, endometrial, and liver cancers, TDP tumors conjointly exhibit tumor protein p53 (TP53) mutations, disruption of breast cancer 1 (BRCA1), and increased expression of DNA replication genes pointing at rereplication in a defective checkpoint environment as a plausible causal mechanism. The resultant TDs in TDP augment global oncogene expression and disrupt tumor suppressor genes. Importantly, the TDP strongly correlates with cisplatin sensitivity in both TNBC cell lines and primary patient-derived xenografts. We conclude that the TDP is a common cancer chromotype that coordinately alters oncogene/tumor suppressor expression with potential as a marker for chemotherapeutic response.

Characteristics of depression in community-dwelling elderly people as indicated by the tree-drawing test.

BACKGROUND: The tree-drawing test (TDT) is a typical projective method, but previous studies have paid little attention to it for elderly people. We investigated the characteristics of depression in community-dwelling elderly people as indicated by the TDT. METHODS: This study was a complete enumeration survey of elderly people conducted through home visits. The contents of the survey included gender, age, presence or absence of housemates, frequency of going out, the 15-item Geriatric Depression Scale, and TDT. The subjects were divided into three groups (normal, depressed tendency, and depressed) according to the total 15-item Geriatric Depression Scale score. RESULTS: In TDT, no significant difference was observed in drooping crown, shadow of the whole tree, or shadow near the base, which have been regarded as indices of depression in younger people. However, the values concerning the size of the tree, such as the height and width of the whole tree, height and width of the crown, and number of occupied areas (of the paper), were significantly lower in the depressed group than in the other groups. In addition, the width of the trunk was significantly smaller in the depressed group than in the normal group. Subjects were classified as being in a 'depressed state' if they used 40 or fewer areas for drawing (i.e. occupied areas) and a 'non-depressed state' if they used 41 or more areas. This enabled depression to be detected (sensitivity: 71.4%; specificity: 79.9%). CONCLUSIONS: The size of the tree in TDT is suggested to reflect characteristics of depression in elderly people, such as introversion, reserve, antisocial attitude, a feeling of inferiority, weakness of ego, and lack of vigour. Furthermore, the numbers of occupied areas were found to be relatively useful in detecting depression in elderly people.

Functional chromatin features are associated with structural mutations in cancer.

BACKGROUND: Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers. RESULTS: We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound. CONCLUSIONS: Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers.

Systems consequences of amplicon formation in human breast cancer.

Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer.

Structural mutations in cancer: mechanistic and functional insights.

Next-generation sequencing (NGS) has enabled the comprehensive and precise identification of many somatic structural mutations in cancer. Analyses integrating point mutation information with data on rearrangements and copy number variation have revealed a higher-order organization of the seemingly random genetic events that lead to cancer. These meta-analyses provide a more refined view of the mutational mechanisms, genomic evolution, and combinations of mutations that contribute to tumorigenesis. Structural mutations, or genome-scale rearrangements of segments of DNA, may play a hitherto unappreciated role in cancer through their ability to move blocks of adjacent genes simultaneously, leading to concurrent oncogenic events. Moreover, whole-genome sequencing (WGS) data from tumors have revealed global rearrangements, such as those seen in the tandem duplicator phenotype and in chromothripsis, suggesting that massive rearrangements are a specific cancer phenotype. Taken together, the emerging data suggest that the chromosome structure itself functions as a systems oncogenic organizer.

Transcriptional consequences of genomic structural aberrations in breast cancer.

Using a long-span, paired-end deep sequencing strategy, we have comprehensively identified cancer genome rearrangements in eight breast cancer genomes. Herein, we show that 40%-54% of these structural genomic rearrangements result in different forms of fusion transcripts and that 44% are potentially translated. We find that single segmental tandem duplication spanning several genes is a major source of the fusion gene transcripts in both cell lines and primary tumors involving adjacent genes placed in the reverse-order position by the duplication event. Certain other structural mutations, however, tend to attenuate gene expression. From these candidate gene fusions, we have found a fusion transcript (RPS6KB1-VMP1) recurrently expressed in ∼30% of breast cancers associated with potential clinical consequences. This gene fusion is caused by tandem duplication on 17q23 and appears to be an indicator of local genomic instability altering the expression of oncogenic components such as MIR21 and RPS6KB1.

Comprehensive long-span paired-end-tag mapping reveals characteristic patterns of structural variations in epithelial cancer genomes.

Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long-span paired-end-tag (PET) sequencing approach using 10-Kb genomic DNA inserts to study human genome structural variations (SVs). The use of a 10-Kb insert size allows the identification of breakpoints within repetitive or homology-containing regions of a few kilobases in size and results in a higher physical coverage compared with small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and two noncancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germ-line SVs, whereas tandem duplications, unpaired inversions, interchromosomal translocations, and complex rearrangements are over-represented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA-PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures that are compatible with breakage-fusion-bridge cycles, and we discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers.

Laminar organization of the developing lateral olfactory tract revealed by differential expression of cell recognition molecules.

The projection neurons in the olfactory bulb (mitral and tufted cells) send axons through the lateral olfactory tract (LOT) onto several structures of the olfactory cortex. However, little is known of the molecular and cellular mechanisms underlying establishment of functional connectivity from the bulb to the cortex. Here, we investigated the developmental process of LOT formation by observing expression patterns of cell recognition molecules in embryonic mice. We immunohistochemically identified a dozen molecules expressed in the developing LOT and some of them were localized to subsets of mitral cell axons. Combinatorial immunostaining for these molecules revealed that the developing LOT consists of three laminas: superficial, middle, and deep. Detailed immunohistochemical, in situ hybridization, and 5-bromodeoxyuridine labeling analyses suggested that the laminar organization reflects: 1) the segregated pathways from the accessory and main olfactory bulbs, and 2) the different maturity of mitral cell axons. Mitral cell axons of the accessory olfactory bulb were localized to the deep lamina, segregated from those of the main olfactory bulb. In the main olfactory pathway, axons of mature mitral cells, whose somata is located in the apical sublayer of the mitral cell layer, were localized to the middle lamina within LOT, while those of immature mitral cells that located in the basal sublayer were complementarily localized to the superficial lamina. These results suggest that newly generated immature axons are added to the most superficial lamina of LOT successively, leading to the formation of piled laminas with different maturational stages of the mitral cell axons.

[Effects of emotion work on burnout and stress among human service professionals].

This study examined the relationship between emotion work (Zapf, 2002) and burnout. One hundred and eighty-two human service professionals (nurses and caregivers) completed questionnaires. A factor analysis revealed that the concept of emotion work had four main factors: "Negative emotions display", "Positive emotions display", "Emotional dissonance", and "Sensitivity requirements". In addition, the hierarchical regression analyses revealed only the main effect of "Emotional dissonance" on psychological stress reaction, whereas the main effects of all emotion work variables and two interaction effects of those on burnout. These results suggested that burnout was distinguished from psychological stress reaction by the differences in its relationship to emotion work.

Molecular-feature domains with posterodorsal-anteroventral polarity in the symmetrical sensory maps of the mouse olfactory bulb: mapping of odourant-induced Zif268 expression.

Individual glomeruli in the mammalian olfactory bulb presumably represent a single type of odourant receptor. Thus, the glomerular sheet provides odourant receptor maps at the surface of the olfactory bulb. To understand the basic spatial organization of the olfactory sensory maps, we first compared the spatial distribution of odourant-induced responses measured by the optical imaging of intrinsic signals with that detected immunohistochemically by expressions of Zif268, one of the immediate early gene products in juxtaglomerular cells. In the dorsal surface of the bulb, we detected a clear correlation in the spatial pattern between these responses. In addition, the molecular-feature domains and their polarities (spatial shifts of responses with an increase in carbon chain length) that were defined by the optical imaging method could be also detected by the Zif268 mapping method. We then mapped the Zif268 signals over the entire olfactory bulb using a homologous series of fatty acids and aliphatic alcohols as stimulus odourants. We superimposed the Zif268 signals onto the standard unrolled map with the help of cell adhesion molecule compartments. Each odourant typically elicited two pairs of clusters of dense Zif268 signals. The results showed that molecular-feature domains and their polarities were arranged symmetrically at stereotypical positions in a mirror-image fashion between the lateral and the medial sensory maps. The polarity of each domain was roughly in parallel with the posterodorsal-anteroventral axis that was defined by the cell adhesion molecule compartments. These results suggest that the molecular-feature domain with its fixed polarity is one of the basic structural units in the spatial organization of the odourant receptor maps in the olfactory bulb.