State of the art and upcoming trends in HER2-directed therapies in gastrointestinal malignancies.

PURPOSE OF REVIEW: This review critically evaluates the evolution and current status of human epidermal growth factor receptor 2 (HER2)-directed therapies in upper gastrointestinal (GI) malignancies, a timely and relevant inquiry given the dynamic shifts in therapeutic strategies over the past decade. Initial enthusiasm following the Trastuzumab for Gastric Cancer (ToGA) study's demonstration of trastuzumab's efficacy, however, encountered hurdles due to subsequent trials showing limited progress, underscoring the necessity for a reevaluation of therapeutic approaches and the exploration of novel agents. RECENT FINDINGS: The review highlights significant breakthroughs in the form of immune checkpoint inhibitors and innovative therapeutic technologies, which have redefined treatment paradigms and shown promising efficacy in HER2-positive cases. Emerging treatments such as trastuzumab deruxtecan (T-DXd), zanidatamab and evorpacept further illustrate the ongoing efforts to leverage unique mechanisms of action for improved HER2-positive antitumor activity. SUMMARY: The advancements in HER2-directed therapies underscore a pivotal era in the management of upper GI malignancies. These developments not only reflect the profound impact of integrating novel therapeutic combinations but also highlight the critical role of ongoing research in overcoming resistance mechanisms and tailoring treatment to individual disease profiles.

Advances in Human Epidermal Growth Factor Receptor 2-Targeted Therapy in Upper Gastrointestinal Cancers.

The Trastuzumab for Gastric Cancer (ToGA) trial marked a pivotal moment in the adoption of trastuzumab for treating advanced human epidermal growth factor receptor 2 (HER2)-positive esophagogastric (EG) cancer. The KEYNOTE-811 trial brought to light the synergistic effect of immune modulation and HER2 targeting. Additionally, the emergence of trastuzumab deruxtecan (T-DXd) highlighted the potential of new pharmaceutical technologies to extend response, particularly for patients who have advanced beyond initial HER2-targeted therapies. This review aims to navigate through both the successes and challenges encountered historically, as well as promising current trials on innovative and transformative therapeutic strategies, including promising first-in-class and novel first-in-human agents.

Clonal Analysis of Human Dendritic Cell Progenitors Using a Stromal Cell Culture.

Dendritic cells (DCs) are a heterogenous population of professional antigen-presenting cells that play an "educator" role in immunity. Multiple DC subsets collaboratively initiate and orchestrate innate and adaptive immune responses. Recent advances in our ability to investigate cellular transcription, signaling, and function at the single-cell level have opened opportunities to examine heterogeneous populations at unprecedented resolutions. Culturing of mouse DC subsets from single bone marrow hematopoietic progenitor cells, that is, clonal analysis, has enabled identification of multiple progenitors with distinct potentials and furthered understanding of mouse DC development. However, studies of human DC development have been hampered by the lack of a corresponding system to generate multiple human DC subsets. Here, we describe a protocol to functionally profile the differentiation potentials of single human hematopoietic stem and progenitor cells (HSPCs) to multiple DC subsets, myeloid and lymphoid cells that will facilitate investigation of human DC lineage specification and reveal its molecular bases.

Reduced calcium levels and accumulation of abnormal insulin granules in stem cell models of HNF1A deficiency.

Mutations in HNF1A cause Maturity Onset Diabetes of the Young (HNF1A-MODY). To understand mechanisms of ß-cell dysfunction, we generated stem cell-derived pancreatic endocrine cells with hypomorphic mutations in HNF1A. HNF1A-deficient ß-cells display impaired basal and glucose stimulated-insulin secretion, reduced intracellular calcium levels in association with a reduction in CACNA1A expression, and accumulation of abnormal insulin granules in association with SYT13 down-regulation. Knockout of CACNA1A and SYT13 reproduce the relevant phenotypes. In HNF1A deficient ß-cells, glibenclamide, a sulfonylurea drug used in the treatment of HNF1A-MODY patients, increases intracellular calcium, and restores insulin secretion. While insulin secretion defects are constitutive in ß-cells null for HNF1A, ß-cells heterozygous for hypomorphic HNF1A (R200Q) mutations lose the ability to secrete insulin gradually; this phenotype is prevented by correction of the mutation. Our studies illuminate the molecular basis for the efficacy of treatment of HNF1A-MODY with sulfonylureas, and suggest promise for the use of cell therapies.

Defects in Long-Term APC Repopulation Ability of Adult Human Bone Marrow Hematopoietic Stem Cells (HSCs) Compared with Fetal Liver HSCs.

Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.

Single cell RNA-Seq reveals pre-cDCs fate determined by transcription factor combinatorial dose.

BACKGROUND: Classic dendritic cells (cDCs) play a central role in the immune system by processing and presenting antigens to activate T cells, and consist of two major subsets: CD141+ cDC (cDC1) and CD1c+ cDC (cDC2). A population of migratory precursor cells, the pre-cDCs, is the immediate precursors to both cDC subsets. Previous studies showed that there were two pre-committed pre-cDC subpopulations. However, the key molecular drivers of pre-commitment in human pre-cDCs were not investigated. RESULTS: To identify the key molecular drivers for pre-commitment in human pre-cDCs, we performed single cell RNA sequencing (RNA-Seq) of two cDC subsets and pre-cDCs, and bulk RNA-Seq of pre-cDCs and cDCs from human peripheral blood. We found that pre-DC subpopulations cannot be separated by either variable genes within pre-cDCs or differentially expressed genes between cDC1 and cDC2. In contrast, they were separated by 16 transcription factors that are themselves differentially expressed or have regulated targets enriched in the differentially expressed genes between bulk cDC1 and cDC2, with one subpopulation close to cDC1 and the other close to cDC2. More importantly, these two pre-cDC sub-populations are correlated with ratio of IRF8 to IRF4 expression level more than their individual expression level. We also verified these findings using three recently published datasets. CONCLUSIONS: In this study, we demonstrate that single cell transcriptome profiling can reveal pre-cDCs differentiation map, and our results suggest the concept that combinatorial dose of transcription factors determines cell differentiation fate.

High Dimensional Functionomic Analysis of Human Hematopoietic Stem and Progenitor Cells at a Single Cell Level.

The ability to conduct investigation of cellular transcription, signaling, and function at the single-cell level has opened opportunities to examine heterogeneous populations at unprecedented resolutions. Although methods have been developed to evaluate high-dimensional transcriptomic and proteomic data (relating to cellular mRNA and protein), there has not been a method to evaluate corresponding high-dimensional functionomic data (relating to cellular functions) from single cells. Here, we present a protocol to quantitatively measure the differentiation potentials of single human hematopoietic stem and progenitor cells, and then cluster the cells according to these measurements. High dimensional functionomic analysis of cell potential allows cell function to be linked to molecular mechanisms within the same progenitor population.

Lineage specification of human dendritic cells is marked by IRF8 expression in hematopoietic stem cells and multipotent progenitors.

The origin and specification of human dendritic cells (DCs) have not been investigated at the clonal level. Through the use of clonal assays, combined with statistical computation, to quantify the yield of granulocytes, monocytes, lymphocytes and three subsets of DCs from single human CD34+ progenitor cells, we found that specification to the DC lineage occurred in parallel with specification of hematopoietic stem cells (HSCs) to the myeloid and lymphoid lineages. This started as a lineage bias defined by specific transcriptional programs that correlated with the combinatorial 'dose' of the transcription factors IRF8 and PU.1, which was transmitted to most progeny cells and was reinforced by upregulation of IRF8 expression driven by the hematopoietic cytokine FLT3L during cell division. We propose a model in which specification to the DC lineage is driven by parallel and inheritable transcriptional programs in HSCs and is reinforced over cell division by recursive interactions between transcriptional programs and extrinsic signals.

Defining human dendritic cell progenitors by multiparametric flow cytometry.

Human dendritic cells (DCs) develop from progressively restricted bone marrow (BM) progenitors: these progenitor cells include granulocyte, monocyte and DC progenitor (GMDP) cells; monocyte and DC progenitor (MDP) cells; and common DC progenitor (CDP) and DC precursor (pre-DC) cells. These four DC progenitors can be defined on the basis of the expression of surface markers such as CD34 and hematopoietin receptors. In this protocol, we describe five multiparametric flow cytometry panels that can be used as a tool (i) to simultaneously detect or phenotype the four DC progenitors, (ii) to isolate DC progenitors to enable in vitro differentiation or (iii) to assess the in vitro differentiation and proliferation of DC progenitors. The entire procedure from isolation of cells to flow cytometry can be completed in 3-7 h. This protocol provides optimized antibody panels, as well as gating strategies, for immunostaining of BM and cord blood specimens to study human DC hematopoiesis in health, disease and vaccine settings.

Clonal analysis of human dendritic cell progenitor using a stromal cell culture.

Different dendritic cell (DC) subsets co-exist in humans and coordinate the immune response. Having a short life, DCs must be constantly replenished from their progenitors in the bone marrow through hematopoiesis. Identification of a DC-restricted progenitor in mouse has improved our understanding of how DC lineage diverges from myeloid and lymphoid lineages. However, identification of the DC-restricted progenitor in humans has not been possible because a system that simultaneously nurtures differentiation of human DCs, myeloid and lymphoid cells, is lacking. Here we report a cytokine and stromal cell culture that allows evaluation of CD34(+) progenitor potential to all three DC subsets as well as other myeloid and lymphoid cells, at a single cell level. Using this system, we show that human granulocyte-macrophage progenitors are heterogeneous and contain restricted progenitors to DCs.

Circulating precursors of human CD1c+ and CD141+ dendritic cells.

Two subsets of conventional dendritic cells (cDCs) with distinct cell surface markers and functions exist in mouse and human. The two subsets of cDCs are specialized antigen-presenting cells that initiate T cell immunity and tolerance. In the mouse, a migratory cDC precursor (pre-CDC) originates from defined progenitors in the bone marrow (BM). Small numbers of short-lived pre-CDCs travel through the blood and replace cDCs in the peripheral organs, maintaining homeostasis of the highly dynamic cDC pool. However, the identity and distribution of the immediate precursor to human cDCs has not been defined. Using a tissue culture system that supports the development of human DCs, we identify a migratory precursor (hpre-CDC) that exists in human cord blood, BM, blood, and peripheral lymphoid organs. hpre-CDCs differ from premonocytes that are restricted to the BM. In contrast to earlier progenitors with greater developmental potential, the hpre-CDC is restricted to producing CD1c(+) and CD141(+) Clec9a(+) cDCs. Studies in human volunteers demonstrate that hpre-CDCs are a dynamic population that increases in response to levels of circulating Flt3L.

Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow.

In mice, two restricted dendritic cell (DC) progenitors, macrophage/dendritic progenitors (MDPs) and common dendritic progenitors (CDPs), demonstrate increasing commitment to the DC lineage, as they sequentially lose granulocyte and monocyte potential, respectively. Identifying these progenitors has enabled us to understand the role of DCs and monocytes in immunity and tolerance in mice. In humans, however, restricted monocyte and DC progenitors remain unknown. Progress in studying human DC development has been hampered by lack of an in vitro culture system that recapitulates in vivo DC hematopoiesis. Here we report a culture system that supports development of CD34(+) hematopoietic stem cell progenitors into the three major human DC subsets, monocytes, granulocytes, and NK and B cells. Using this culture system, we defined the pathway for human DC development and revealed the sequential origin of human DCs from increasingly restricted progenitors: a human granulocyte-monocyte-DC progenitor (hGMDP) that develops into a human monocyte-dendritic progenitor (hMDP), which in turn develops into monocytes, and a human CDP (hCDP) that is restricted to produce the three major DC subsets. The phenotype of the DC progenitors partially overlaps with granulocyte-macrophage progenitors (GMPs). These progenitors reside in human cord blood and bone marrow but not in the blood or lymphoid tissues.

Dendritic cell development-History, advances, and open questions.

Dendritic cells (DCs) are uniquely potent in orchestrating T cell immune response, thus they are indispensable immune sentinels. They originate from progenitors in the bone marrow through hematopoiesis, a highly regulated developmental process involving multiple cellular and molecular events. This review highlights studies of DC development-from the discovery of DCs as glass-adherent antigen presenting cells to the debate and resolution of their origin and lineage map. In particular, we summarize the roles of lineage-specific cytokines, the placement of distinct hematopoietic progenitors within the DC lineage and transcriptional programs governing DC development, which together have allowed us to diagram the current view of DC hematopoiesis. Important open questions and debates on the DC development and relevant models are also discussed.