Small-cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers, and is characterized as extremely aggressive, often displaying rapid tumor growth and multiple organ metastases. In addition, the clinical outcome of SCLC patients is poor due to early relapse and acquired resistance to standard chemotherapy treatments. Hence, novel therapeutic strategies for the treatment of SCLC are urgently required. Accordingly, several molecular targeted therapies were evaluated in SCLC; however, they failed to improve the clinical outcome. The receptor tyrosine kinase MET is a receptor for hepatocyte growth factor (HGF), and aberrant activation of HGF/MET signaling is known as one of the crucial mechanisms enabling cancer progression and invasion. Here, we found that the HGF/MET signaling was aberrantly activated in chemoresistant or chemorelapsed SCLC cell lines (SBC-5, DMS273, and DMS273-G3H) by the secretion of HGF and/or MET copy number gain. A cell-based in vitro assay revealed that HGF/MET inhibition, induced either by MET inhibitors (crizotinib and golvatinib), or by siRNA-mediated knockdown of HGF or MET, constrained growth of chemoresistant SCLC cells through the inhibition of ERK and AKT signals. Furthermore, treatment with either crizotinib or golvatinib suppressed the systemic metastasis of SBC-5 cell tumors in natural killer cell-depleted SCID mice, predominantly through cell cycle arrest. These findings reveal the therapeutic potential of targeting the HGF/MET pathway for inhibition, to constrain tumor progression of SCLC cells showing aberrant activation of HGF/MET signaling. We suggest that it would be clinically valuable to further investigate HGF/MET-mediated signaling in SCLC cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. Although improvement in both surgical techniques and neoadjuvant chemotherapy has been achieved, the 5-year survival rate of locally advanced tumors was, at best, still 55%. Therefore, elucidation of mechanisms of the malignancy is eagerly awaited. Epithelial-mesenchymal transition (EMT) by transforming growth factor-β (TGF-β) has been reported to have critical biological roles for cancer cell stemness, whereas little is known about it in ESCC. In the current study, a transcriptional factor SIX1 was found to be aberrantly expressed in ESCCs. SIX1 cDNA transfection induced overexpression of transforming growth factors (TGFB1 and TGFB2) and its receptor (TGFBR2). Cell invasion was reduced by SIX1 knockdown and was increased in stable SIX1-transfectants. Furthermore, the SIX1-transfectants highly expressed tumor basal cell markers such as NGFR, SOX2, ALDH1A1, and PDPN. Although mock-transfectants had only a 20% PDPN-high population, SIX1-transfectants had 60-70%. In two sets of 42 and 85 ESCC patients receiving surgery alone or neoadjuvant chemoradiotherapy followed by surgery, the cases with high SIX1 mRNA and protein expression level significantly showed a poor prognosis compared with those with low levels. These SIX1 high cases also expressed the above basal cell markers, but suppressed the differentiation markers. Finally, TGF-β signaling blockade suppressed ESCC cell growth in association with the reduction of PDPN-positive tumor basal cell population. The present results suggest that SIX1 accelerates self-renewal of tumor basal cells, resulting in a poor prognosis for ESCC patients.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in patients with non-small-cell lung cancer (NSCLC) harboring EGFR mutations. However, due to acquired resistance to EGFR-TKIs, even patients on third-generation osimertinib have a poor prognosis. Resistance mechanisms are still not fully understood. Here, we demonstrate that the increased expression of MUSASHI-2 (MSI2), an RNA-binding protein, is a novel mechanism for resistance to EGFR-TKIs. We found that after a long-term exposure to gefitinib, the first-generation EGFR-TKI lung cancer cells harboring the EGFR-TKI-sensitive mutations became resistant to both gefitinib and osimertinib. Although other mutations in EGFR were not found, expression levels of Nanog, a stemness core protein, and activities of aldehyde dehydrogenase (ALDH) were increased, suggesting that cancer stem-like properties were increased. Transcriptome analysis revealed that MSI2 was among the stemness-related genes highly upregulated in EGFR-TKI-resistant cells. Knockdown of MSI2 reduced cancer stem-like properties, including the expression levels of Nanog, a core stemness factor. We demonstrated that knockdown of MSI2 restored sensitivity to osimertinib or gefitinib in EGFR-TKI-resistant cells to levels similar to those of parental cells in vitro. An RNA immunoprecipitation (RIP) assay revealed that antibodies against MSI2 were bound to Nanog mRNA, suggesting that MSI2 increases Nanog expression by binding to Nanog mRNA. Moreover, overexpression of MSI2 or Nanog conferred resistance to osimertinib or gefitinib in parental cells. Finally, MSI2 knockdown greatly increased the sensitivity to osimertinib in vivo. Collectively, our findings provide proof of principle that targeting the MSI2-Nanog axis in combination with EGFR-TKIs would effectively prevent the emergence of acquired resistance.

Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) with a broad coverage against ALK mutations, has demonstrated dramatic effects in patients with ALK-rearranged lung cancer. The mechanisms of acquired resistance to lorlatinib by secondary ALK compound mutations have recently been reported; however, resistance mechanisms other than secondary mutations remain unclear. Here, we investigated the molecular mechanisms of the acquired resistance in ALK-rearranged lung cancer cells in vitro. We established two different lorlatinib-resistant ALK-rearranged lung cancer cell lines (H3122LR and A925LLR) via long-term administration of lorlatinib. These resistant cells did not harbor the secondary ALK mutations and showed cross-resistance to the other kinds of ALK-TKIs (crizotinib or alectinib) compared with the parental cells; however, these resistant cells overexpressed the phosphorylated human epidermal growth factor receptor 3 (HER3) protein and the ligand of HER3 (neuregulin 1; NRG1). Pharmacological inhibition of HER3 with pan-HER inhibitors or genetic knockdown of HER3 with siRNA resensitized H3122LR and A925LLR cells to lorlatinib in vitro, indicating that H3122LR and A925LLR acquired resistance by NRG1/HER3 activation. These findings demonstrated that targeting NRG1/HER3 is a potential novel therapeutic option for lorlatinib-resistant ALK-rearranged lung cancer.

To identify prognostic factors in patients with grade 3 (high-grade) endometrial endometrioid carcinoma, we evaluated the spectrum of genomic alterations and examined whether previously reported molecular subtypes of endometrial carcinoma were adapted to clinical outcome prediction. Seventy-five Japanese patients with grade 3 endometrial endometrioid carcinoma, who underwent a potentially curative resection procedure between 1997 and 2018 at the National Cancer Center Hospital, were included. We classified the patients into four risk groups of the disease based on the Proactive Molecular Risk Classifier for Endometrial Cancer. Genomic alterations in PTEN, ARID1A, TP53, and PIK3CA were detected in more than 30% of the patients. Overall survival and recurrence-free survival of patients with genomic alterations in CTNNB1 were poorer than those of patients with wild-type CTNNB1 (p = 0.006 and p = 0.004, respectively). Compared with that of alterations prevalent in Caucasians, the frequency of genomic alterations in POLE and TP53 was higher in our study than in The Cancer Genome Atlas dataset (p = 0.01 and p = 0.01, respectively). The tendency for recurrence-free survival in the POLE exonuclease domain mutation group was better than that in the TP53 mutation and mismatch repair-deficient groups (p = 0.08 and p = 0.07, respectively), consistent with the Proactive Molecular Risk Classifier for Endometrial Cancer risk classifier definition. The CTNNB1 mutation is a potential novel biomarker for the prognosis of patients with grade 3 endometrial endometrioid carcinoma, and prognosis classification using Proactive Molecular Risk Classifier for Endometrial Cancer may help screen Japanese patients with the disease.

Identification of genetic alterations through next-generation sequencing (NGS) can guide treatment decision-making by providing information on diagnosis, therapy selection, and prognostic stratification in patients with hematological malignancies. Although the utility of NGS-based genomic profiling assays was investigated in hematological malignancies, no assays sufficiently cover driver mutations, including recently discovered ones, as well as fusions and/or pathogenic germline variants. To address these issues, here we have devised an integrated DNA/RNA profiling assay to detect various types of somatic alterations and germline variants at once. Particularly, our assay can successfully identify copy number alterations and structural variations, including immunoglobulin heavy chain translocations, IKZF1 intragenic deletions, and rare fusions. Using this assay, we conducted a prospective study to investigate the feasibility and clinical usefulness of comprehensive genomic profiling for 452 recurrently altered genes in hematological malignancies. In total, 176 patients (with 188 specimens) were analyzed, in which at least one alteration was detected in 171 (97%) patients, with a median number of total alterations of 7 (0-55). Among them, 145 (82%), 86 (49%), and 102 (58%) patients harbored at least one clinically relevant alteration for diagnosis, treatment, and prognosis, respectively. The proportion of patients with clinically relevant alterations was the highest in acute myeloid leukemia, whereas this assay was less informative in T/natural killer-cell lymphoma. These results suggest the clinical utility of NGS-based genomic profiling, particularly for their diagnosis and prognostic prediction, thereby highlighting the promise of precision medicine in hematological malignancies.

Advances in cancer genome care over the past few years have included the development of gene panel testing for various biomarkers. This article summarizes issues and provides recommendations related to analytical performance evaluations for new oncology gene panels. The scope of these recommendations includes comprehensive genomic profiling assays related to gene panel testing that uses histological or serum specimens to detect gene mutations. As a research project of the Japan Agency for Medical Research and Development Research on Regulatory Science of Pharmaceuticals and Medical Devices, we convened the working group committee that consisted of more than 30 experts from academia, industry, and government. We have discussed the points that should be considered to allow maximal simplification of assessments using clinical specimens in evaluating accuracy and limit of detection in equivalence and analytical performance for 3 years. We provide recommendations specific to each type of gene mutation as well as to reference standards or specimens used for evaluations. In addition, in order to facilitate the discussion on the analytical performance of gene panel tests by multidisciplinary tumor boards of hospitals, the present recommendations also describe the items that companies are expected to provide information on in their packaging inserts and reports, and the items that are expected to be discussed by multidisciplinary tumor boards. Our working group document will be important for participants in multidisciplinary tumor boards, including medical oncologists and genome scientists, and developers of gene panels not only in Japan but also in other countries.

Next-generation sequencing (NGS) of tumor tissue (ie, clinical sequencing) can guide clinical management by providing information about actionable gene aberrations that have diagnostic and therapeutic significance. Here, we undertook a hospital-based prospective study (TOP-GEAR project, 2nd stage) to investigate the feasibility and utility of NGS-based analysis of 114 cancer-associated genes (the NCC Oncopanel test). We examined 230 cases (comprising more than 30 tumor types) of advanced solid tumors, all of which were matched with nontumor samples. Gene profiling data were obtained for 187 cases (81.3%), 111 (59.4%) of which harbored actionable gene aberrations according to the Clinical Practice Guidelines for Next Generation Sequencing in Cancer Diagnosis and Treatment (Edition 1.0) issued by 3 major Japanese cancer-related societies. Twenty-five (13.3%) cases have since received molecular-targeted therapy according to their gene aberrations. These results indicate the utility of tumor-profiling multiplex gene panel testing in a clinical setting in Japan. This study is registered with UMIN Clinical Trials Registry (UMIN 000011141).