Ancestry-, Sex-, and Age-Based Differences of Gene Alterations in NSCLC: From the Real-World Data of Cancer Genomic Profiling Tests.

BACKGROUND: Some genomic alterations in non-small cell lung cancer (NSCLC) are known to differ according to race, sex, or age. These studies have been limited in sample size and thus they cannot detect the differences precisely and comprehensively. METHODS: Tissue-based comprehensive genomic profiling was performed on 75,362 patients with NSCLC from the United States during routine clinical care. Additionally, we examined data of a Japanese NSCLC cohort with 1,019 patients. In the US cohort, 296 genes were examined for pathogenic alterations. Predominant genetic ancestry was inferred using a SNP-based approach, and patients were categorized into European (EUR), African (AFR), East Asian (EAS), Admixed American (AMR), and South Asian (SAS) ancestry groups. Patients were additionally stratified by histologic type, age (<40/≥40 years, <75/≥75 years), and sex. The prevalence of high tumor mutational burden (TMB-High) and microsatellite instability status was also calculated. RESULTS: Stratified by ancestry, EGFR alterations were significantly enriched in EAS versus other ancestry groups. The prevalence of ALK was significantly higher in the AMR, EAS, and SAS patients than in AFR and EUR patients. KRAS and STK11 were enriched in EUR and AFR patients versus other groups. TMB-High was significantly enriched in AFR patients versus all other groups. An analysis based on sex revealed differences in prevalence of alterations in 80 genes and TMB-High status. For example, EGFR, ALK, BRAF, and KRAS alterations were significantly enriched in females, whereas TP53, STK11, KEAP1, and TMB-High were significantly enriched in males. With respect to age, the prevalence of alterations in 41 genes, including ALK, RET, MET, EGFR, STK11, KEAP1, BRAF, and KRAS, as well as TMB-High, were significantly different between patients aged <40 years and those aged ≥40 years. CONCLUSIONS: Comprehensive analysis from a large real-world dataset revealed ancestry-associated differences in genomic alterations in NSCLC. Age- and sex-related differences in prevalence of genomic alterations and TMB-High status were also observed.

Characterization of colorectal cancer by hierarchical clustering analyses of five immune cell markers.

The present study analyzed the expression of five independent immunohistochemical markers, CD4, CD8, CD66b, CD68, and CD163, on immune cells within the colorectal cancer (CRC) tumor microenvironment (TME). Using hierarchical clustering, patients were successfully classified according to significant associations with clinicopathological features and/or survival. Patients with mismatch repair-proficient (pMMR) CRC were categorized into four groups with survival differences (p = 0.0084): CD4Low , CD4High , MΦHigh , and CD8Low . MΦHigh tumors showed significantly higher expression of CD47 (p < 0.0001), a phagocytosis checkpoint molecule. These tumors contained significantly greater numbers of PD-1+ (p < 0.0001), TIM-3+ (p < 0.0001), and SIRPA+ (p < 0.0001) immune cells. Notably, 10% of the patients with pMMR CRC expressed PD-L1 (CD274) on tumor cells with significantly worse survival (p = 0.00064). The Cox proportional hazards model identified MΦ High (hazard ratio [HR] = 2.02, 95%, p = 0.032), CD8Low (HR = 2.45, p = 0.011), and tumor PD-L1 expression (HR = 2.74, p = 0.0061) as potential risk factors. PD-L1-PD-1 and/or CD47-SIRPA axes targeting immune checkpoint therapies might be considered for patients with pMMR CRC according to their tumor cells and tumor immune microenvironment characteristics.

Recurrent nocturnal hypoglycemic hemiplegia: a case report and review of the literature.

A 67-year-old man with type 1 diabetes, Cronkhite-Canada syndrome, and membranous nephropathy who received insulin therapy was admitted to our hospital with right hemiplegia and dysarthria. Brain magnetic resonance imaging revealed a lesion with a high diffusion-weighted imaging signal and low apparent diffusion coefficient signal in the posterior limb of the left internal capsule. He was hypoglycemic with a blood glucose level of 56 mg/dL (3.1 mmol/L). Following glucose administration, the patient's symptoms resolved within several hours. The patient experienced similar transient hypoglycemic hemiplegia at midnight, three times within 10 days. In a literature review of 170 cases of hypoglycemic hemiplegia, 26 cases of recurrent hemiplegia were investigated. Recurrent hypoglycemic hemiplegia occurs more frequently on the right side than on the left side, and most recurrences occur within approximately a week, almost exclusively at midnight and in the early morning. We speculate that hypoglycemia-associated autonomic failure may be involved in the nocturnal recurrence of episodes. In our patient, depleted endogenous insulin secretion and lipodystrophy at the injection site, may have acted as additional factors, leading to severe hypoglycemia despite the absence of apparent autonomic neuropathy. Clinically, it is important to recognize hypoglycemia as a cause of hemiplegia to avoid unnecessary intervention and to maintain an appropriate blood glucose level at midnight and early in the morning to prevent recurrent hypoglycemic hemiplegia.

Detection of Novel Tyrosine Kinase Fusion Genes as Potential Therapeutic Targets in Bone and Soft Tissue Sarcomas Using DNA/RNA-based Clinical Sequencing.

BACKGROUND: Approximately 1% of clinically treatable tyrosine kinase fusions, including anaplastic lymphoma kinase, neurotrophic tyrosine receptor kinase, RET proto-oncogene, and ROS proto-oncogene 1, have been identified in soft tissue sarcomas via comprehensive genome profiling based on DNA sequencing. Histologic tumor-specific fusion genes have been reported in approximately 20% of soft tissue sarcomas; however, unlike tyrosine kinase fusion genes, these fusions cannot be directly targeted in therapy. Approximately 80% of tumor-specific fusion-negative sarcomas, including myxofibrosarcoma and leiomyosarcoma, that are defined in complex karyotype sarcomas remain genetically uncharacterized; this mutually exclusive pattern of mutations suggests that other mutually exclusive driver oncogenes are yet to be discovered. Tumor-specific, fusion-negative sarcomas may be associated with unique translocations, and oncogenic fusion genes, including tyrosine kinase fusions, may have been overlooked in these sarcomas. QUESTIONS/PURPOSES: (1) Can DNA- or RNA-based analysis reveal any characteristic gene alterations in bone and soft tissue sarcomas? (2) Can useful and potential tyrosine kinase fusions in tumors from tumor-specific, fusion-negative sarcomas be detected using an RNA-based screening system? (3) Do the identified potential fusion tumors, especially in neurotrophic tyrosine receptor kinase gene fusions in bone sarcoma, transform cells and respond to targeted drug treatment in in vitro assays? (4) Can the identified tyrosine kinase fusion genes in sarcomas be useful therapeutic targets? METHODS: Between 2017 and 2020, we treated 100 patients for bone and soft tissue sarcomas at five institutions. Any biopsy or surgery from which a specimen could be obtained was included as potentially eligible. Ninety percent (90 patients) of patients were eligible; a further 8% (8 patients) were excluded because they were either lost to follow-up or their diagnosis was changed, leaving 82% (82 patients) for analysis here. To answer our first and second questions regarding gene alterations and potential tyrosine kinase fusions in eight bone and 74 soft tissue sarcomas, we used the TruSight Tumor 170 assay to detect mutations, copy number variations, and gene fusions in the samples. To answer our third question, we performed functional analyses involving in vitro assays to determine whether the identified tyrosine kinase fusions were associated with oncogenic abilities and drug responses. Finally, to determine usefulness as therapeutic targets, two pediatric patients harboring an NTRK fusion and an ALK fusion were treated with tyrosine kinase inhibitors in clinical trials. RESULTS: DNA/RNA-based analysis demonstrated characteristic alterations in bone and soft tissue sarcomas; DNA-based analyses detected TP53 and copy number alterations of MDM2 and CDK4 . These single-nucleotide variants and copy number variations were enriched in specific fusion-negative sarcomas. RNA-based screening detected fusion genes in 24% (20 of 82) of patients. Useful potential fusions were detected in 19% (11 of 58) of tumor-specific fusion-negative sarcomas, with nine of these patients harboring tyrosine kinase fusion genes; five of these patients had in-frame tyrosine kinase fusion genes ( STRN3-NTRK3, VWC2-EGFR, ICK-KDR, FOXP2-MET , and CEP290-MET ) with unknown pathologic significance. The functional analysis revealed that STRN3-NTRK3 rearrangement that was identified in bone had a strong transforming potential in 3T3 cells, and that STRN3-NTRK3 -positive cells were sensitive to larotrectinib in vitro. To confirm the usefulness of identified tyrosine kinase fusion genes as therapeutic targets, patients with well-characterized LMNA-NTRK1 and CLTC-ALK fusions were treated with tyrosine kinase inhibitors in clinical trials, and a complete response was achieved. CONCLUSION: We identified useful potential therapeutic targets for tyrosine kinase fusions in bone and soft tissue sarcomas using RNA-based analysis. We successfully identified STRN3-NTRK3 fusion in a patient with leiomyosarcoma of bone and determined the malignant potential of this fusion gene via functional analyses and drug effects. In light of these discoveries, comprehensive genome profiling should be considered even if the sarcoma is a bone sarcoma. There seem to be some limitations regarding current DNA-based comprehensive genome profiling tests, and it is important to use RNA testing for proper diagnosis and accurate identification of fusion genes. Studies on more patients, validation of results, and further functional analysis of unknown tyrosine kinase fusion genes are required to establish future treatments. CLINICAL RELEVANCE: DNA- and RNA-based screening systems may be useful for detecting tyrosine kinase fusion genes in specific fusion-negative sarcomas and identifying key therapeutic targets, leading to possible breakthroughs in the treatment of bone and soft tissue sarcomas. Given that current DNA sequencing misses fusion genes, RNA-based screening systems should be widely considered as a worldwide test for sarcoma. If standard treatments such as chemotherapy are not effective, or even if the sarcoma is of bone, RNA sequencing should be considered to identify as many therapeutic targets as possible.

Simultaneous Expression of CD70 and POSTN in Cancer-Associated Fibroblasts Predicts Worse Survival of Colorectal Cancer Patients.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide, with high morbidity and mortality rates. The evidence for the tumor-supporting capacities of cancer-associated fibroblasts (CAFs) that modulate cancer cell proliferation, invasion, metastasis, and tumor immunity, including in CRC, has been attracting attention. The present study examined the expression status of CD70 and POSTN in CRC and analyzed their association with clinicopathological features and clinical outcomes. In the present study, in total 15% (40/269) and 44% (119/269) of cases exhibited CD70 and POSTN expression on CAFs, respectively. Co-expression of CD70 and POSTN was detected in 8% (21/269) of patients. Fluorescent immunohistochemistry identified the co-expression of CD70 and POSTN with FAP and PDPN, respectively. ACTA2 was not co-expressed with CD70 or POSTN in CRC CAFs. CRC with CD70+/POSTN+ status in CAFs was significantly associated with distant organ metastasis (p = 0.0020) or incomplete resection status (p = 0.0011). CD70+/POSTN+ status tended to associate with advanced pT stage (p = 0.032) or peritoneal metastasis (p = 0.0059). Multivariate Cox hazards regression analysis identified CD70+/POSTN+ status in CAFs [hazard ratio (HR) = 3.78] as a potential independent risk factor. In vitro experiments revealed the activated phenotypes of colonic fibroblasts induced by CD70 and POSTN, while migration and invasion assays identified enhanced migration and invasion of CRC cells co-cultured with CD70- and POSTN-expressing colonic fibroblasts. On the basis of our observations, CD70 and POSTN immunohistochemistry can be used in the prognostication of CRC patients. CRC CAFs may be a promising target in the treatment of CRC patients.

Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric ß-Barrel Protein Scaffold.

Evolutionary engineering of our previously reported Cp*Rh(III)-linked artificial metalloenzyme was performed based on a DNA recombination strategy to improve its catalytic activity toward C(sp2)-H bond functionalization. Improved scaffold design was achieved with α-helical cap domains of fatty acid binding protein (FABP) embedded within the ß-barrel structure of nitrobindin (NB) as a chimeric protein scaffold for the artificial metalloenzyme. After optimization of the amino acid sequence by directed evolution methodology, an engineered variant, designated NBHLH1(Y119A/G149P) with enhanced performance and enhanced stability was obtained. Additional rounds of metalloenzyme evolution provided a Cp*Rh(III)-linked NBHLH1(Y119A/G149P) variant with a >35-fold increase in catalytic efficiency (kcat/KM) for cycloaddition of oxime and alkyne. Kinetic studies and MD simulations revealed that aromatic amino acid residues in the confined active-site form a hydrophobic core which binds to aromatic substrates adjacent to the Cp*Rh(III) complex. The metalloenzyme engineering process based on this DNA recombination strategy will serve as a powerful method for extensive optimization of the active-sites of artificial metalloenzymes.

Long-term effect of letrozole on metastatic uterine smooth muscle tumors of uncertain malignant potential: A case report.

A 48-year-old woman underwent total abdominal hysterectomy and right salpingo-oophorectomy and was initially diagnosed with a uterine leiomyoma and right ovarian cystadenoma. After 4 years, multiple pulmonary metastases were identified, and treatment with gonadotropin-releasing hormone agonists was started, but stopped later due to disease progression. The patient developed dyspnea and underwent right upper lobectomy. The histopathological findings were consistent with those of pulmonary metastases secondary to a uterine smooth muscle tumor of uncertain malignant potential. Slow disease progression after a poor response to adriamycin and hormone receptor positivity led to the start of letrozole. Letrozole induced spontaneous regression of the pulmonary metastases, and about 2 years into the treatment, sustained response was achieved with minimal side effects. This may be the first case supporting the long-term efficacy and safety of letrozole in the management of adriamycin-resistant lung metastases of uterine smooth muscle tumors of uncertain malignant potential.

Chitin- and Streptavidin-Mediated Affinity Purification Systems: A Screening Platform for Enzyme Discovery.

Affinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost-intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin- and streptavidin-mediated affinity purification) for low-cost purification of Strep-tag II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost-efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96-well format as a demonstration. Through the screening of 96 types of purified hemoproteins, several proteins capable of the catalytic diastereodivergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction.

Characteristics of Li-Fraumeni Syndrome in Japan; A Review Study by the Special Committee of JSHT.

Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome, and the majority of patients with LFS have been identified with germline variants in the p53 tumor suppressor (TP53) gene. In the past three decades, considerable case reports of TP53 germline variants have been published in Japan. To the best of our knowledge, there have been no large-scale studies of Japanese patients with LFS. In this study, we aimed to identify Japanese patients with TP53 germline variants and to reveal the characteristics of LFS in Japan. We collected reported cases by reviewing the medical literature and cases diagnosed at the institutions of the authors. We identified 68 individuals from 48 families with TP53 germline pathogenic or likely pathogenic variants. Of the 48 families, 35 (72.9%) had missense variants, most of which were located within the DNA-binding loop. A total of 128 tumors were identified in the 68 affected individuals. The 128 tumor sites were as follows: breast, 25; bones, 16; brain, 12; hematological, 11; soft tissues, 10; stomach, 10; lung, 10; colorectum, 10; adrenal gland, 9; liver, 4; and others, 11. Unique phenotype patterns of LFS were shown in Japan in comparison to those in a large national LFS cohort study in France. Above all, a higher frequency of patients with stomach cancer was observed in Japanese TP53 germline variant carriers. These results may provide useful information for the clinical management of LFS in Japan.

Directed Evolution of a Cp*RhIII -Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification.

Directed evolution of Cp*RhIII -linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII -linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.

Reduction of Superoxide Dismutase 1 Delays Regeneration of Cardiotoxin-Injured Skeletal Muscle in KK/Ta-Ins2Akita Mice with Progressive Diabetic Nephropathy.

Superoxide dismutase (SOD) is a major antioxidant enzyme for superoxide removal, and cytoplasmic SOD (SOD1) is expressed as a predominant isoform in all cells. We previously reported that renal SOD1 deficiency accelerates the progression of diabetic nephropathy (DN) via increasing renal oxidative stress. To evaluate whether the degree of SOD1 expression determines regeneration capacity and sarcopenic phenotypes of skeletal muscles under incipient and advanced DN conditions, we investigated the alterations of SOD1 expression, oxidative stress marker, inflammation, fibrosis, and regeneration capacity in cardiotoxin (CTX)-injured tibialis anterior (TA) muscles of two Akita diabetic mouse models with different susceptibility to DN, DN-resistant C57BL/6-Ins2Akita and DN-prone KK/Ta-Ins2Akita mice. Here, we report that KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, exhibit delayed muscle regeneration after CTX injection, as demonstrated by the finding indicating significantly smaller average cross-sectional areas of regenerating TA muscle myofibers relative to KK/Ta-wild-type mice. Furthermore, we observed markedly reduced SOD1 expression in CTX-injected TA muscles of KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, along with increased inflammatory cell infiltration, prominent fibrosis and superoxide overproduction. Our study provides the first evidence that SOD1 reduction and the following superoxide overproduction delay skeletal muscle regeneration through induction of overt inflammation and fibrosis in a mouse model of progressive DN.

Incorporation of a Cp*Rh(III)-dithiophosphate Cofactor with Latent Activity into a Protein Scaffold Generates a Biohybrid Catalyst Promoting C(sp2)-H Bond Functionalization.

A Cp*Rh(III)-dithiophosphate cofactor with "latent" catalytic activity was developed to construct an artificial metalloenzyme representing a new type of biohybrid catalyst which is capable of promoting C(sp2)-H bond functionalization within the ß-barrel structure of nitrobindin (NB). To covalently conjugate the Cp*Rh(III) cofactor into a specific position of the hydrophobic cavity of NB via a maleimide-Cys linkage, strong chelation of the dithiophosphate ligand is employed to protect the rhodium metal center against attack by nucleophilic amino acid residues in the protein. It is found that subsequent addition of the Ag+ ion induces dissociation of the dithiophosphate ligands, thereby activating the catalytic activity of the Cp*Rh(III) cofactor. The resulting "active" biohybrid catalyst promotes cycloaddition of acetophenone oxime with diphenylacetylene via C(sp2)-H bond activation. This catalytic activity is enhanced 2.3-fold with the introduction of two glutamate residues (A100E/L125E) adjacent to the Cp*Rh(III) cofactor. The Cp*Rh(III) cofactor with switchable activity from a "latent" form to an "active" form provides a new strategy for generating biohybrid catalysts incorporating a variety of highly reactive transition metal complexes specifically within its protein scaffolds.

Japan society of clinical oncology/Japanese society of medical oncology-led clinical recommendations on the diagnosis and use of tropomyosin receptor kinase inhibitors in adult and pediatric patients with neurotrophic receptor tyrosine kinase fusion-positive advanced solid tumors, cooperated by the Japanese society of pediatric hematology/oncology.

BACKGROUND: The development of novel antitumor agents and accompanying biomarkers has improved survival across several tumor types. Previously, we published provisional clinical opinion for the diagnosis and use of immunotherapy in patients with deficient DNA mismatch repair tumors. Recently, efficacy of tropomyosin receptor kinase inhibitors against neurotrophic receptor tyrosine kinase (NTRK) fusion gene-positive advanced solid tumors have been established as the second tumor-agnostic treatment, making it necessary to develop the guideline prioritized for these patients. METHODS: Clinical questions regarding medical care were formulated for patients with NTRK-positive advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by Japan Society of Clinical Oncology (JSCO) and Japanese Society of Medical Oncology (JSMO) voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and Japanese Society of Pediatric Hematology/Oncology, and the public comments among all Societies' members was done. RESULTS: The current guideline describes 3 clinical questions and 15 recommendations for whom, when, and how NTRK fusion should be tested, and what is recommended for patients with NTRK fusion-positive advanced solid tumors. CONCLUSION: In the NTRK guideline, the committee proposed 15 recommendations for performing NTRK testing properly to select patients who are likely to benefit from tropomyosin receptor kinase inhibitors.

Assessment of Predictive Biomarkers of the Response to Pazopanib Based on an Integrative Analysis of High-grade Soft-tissue Sarcomas: Analysis of a Tumor Sample from a Responder and Patients with Other Soft-tissue Sarcomas.

BACKGROUND: Soft-tissue sarcomas are a rare group of malignant tumors that usually are treated with surgical excision and radiation therapy, but recently, pazopanib, an oral tyrosine kinase inhibitor, has been used in patients with metastases who do not respond to standard chemotherapy regimens. Based on patients with advanced soft-tissue sarcomas who had received prior chemotherapy, several clinical studies have reported the survival and sensitivity (approximately 5% to 10% sensitive) of patients with soft-tissue sarcomas treated with pazopanib. Recently, next-generation sequencing (NGS) technologies have been used to provide a wide genetic information and to develop personalized medicine in cancer treatment. However, there are few reports and no genetic analyses of patients with soft-tissue sarcomas who had a complete response (CR) to pazopanib. QUESTIONS/PURPOSES: We described the clinicopathologic features of a patient with a rare, advanced soft-tissue sarcoma who achieved a CR to pazopanib treatment. Furthermore, integrative analyses using NGS and arrays were performed to elucidate characteristic alterations, including gene mutations, copy number changes, and protein expression that were associated with response to pazopanib. Additionally, functional analyses consisting of in vitro and in vivo assays were also performed to elucidate whether the identified alterations were associated with oncogenic abilities and drug responses. METHODS: In a sample from a 70-year-old woman with an advanced soft-tissue sarcoma treated for 1 month with 800 mg of oral pazopanib daily, CT scans demonstrated a CR to treatment. To our knowledge, there have been no patients with soft-tissue sarcomas among several clinical trials of pazopanib that have achieved a CR and therefore, our patient is considered to be extremely rare. We performed an integrative analysis including whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of receptor tyrosine kinases (RTK) using tumor samples from a patient with a CR matched to normal samples. From here on we will refer to this patient as having a CR, although a short term high-grade partial response may be more accurate. These analyses were performed using NGS and the phosphoreceptor tyrosine kinase (phospho-RTK) array. As a validation study, we also performed target sequencing using three samples from patients with long-term stable disease and two samples from patients with progressive disease who responded to pazopanib treatment. In addition, characteristic gene alterations that were identified according to the response to pazopanib in one patient with a CR, in three patients with long-term stable disease, and in 27 patients with high-grade soft-tissue sarcomas with different histologic subtypes and different responses to pazopanib were verified by quantitative real-time polymerase chain reaction. We conducted a focus formation assay to evaluate the transforming activities of these genomic alterations. RESULTS: In the patient with a CR to pazopanib, we identified several somatic mutations including Fms related receptor tyrosine kinase 1 (FLT1) p.G38S, platelet-derived growth factor receptor alpha (PDGFRA) p.T83S, and platelet-derived growth factor receptor beta (PDGFRB) exon 13 skipping. Amplification at chromosome 12q13-14 encompassing GLI family zinc finger 1 (GLI1) and cyclin-dependent kinase-4 (CDK4) was also detected. Furthermore, an elevated PDGFRB phosphorylation level was observed in the tumor. In target sequencing analyses in five patients, one of three patients with long-term stable disease had 12q13-14 amplification. The mRNA expression of GLI1, CDK4, and pazopanib targets including PDGFRA, PDGFRB, vascular endothelial growth factor receptor (VEGFR)1-3, and stem cell factor receptor (KIT) in samples from the patient with a CR, and 27 patients with high-grade soft-tissue sarcomas was verified. The expression of GLI1 was characteristically increased in the patient with a CR and in those with long-term stable disease relative to other patients with soft-tissue sarcomas. Overexpression of GLI1 showed strong transforming potential in 3T3 cells. Moreover, the overexpression of GLI1 upregulated the expression of the PDGFRB protein and promoted phosphorylation, which was dose-dependently inhibited by pazopanib. However, inhibition of GLI1-induced transformation by pazopanib was limited in the focus formation assay; therefore, mechanisms other than PDGFRB activation may contribute to transformation. CONCLUSIONS: We identified several gene alterations that might be associated with a CR and long-term stable disease in patients who received pazopanib for advanced soft-tissue sarcomas. We therefore believe that this distinct molecular profile warrants further investigation to identify predictive biomarkers of the response to pazopanib. CLINICAL RELEVANCE: Our findings identify molecular mechanisms that possibly explain the high sensitivity of soft-tissue sarcomas to pazopanib and may lead to the development of predictive biomarkers and novel therapies in patients with this and other types of soft-tissue sarcomas.

Inhibition of GIP signaling extends lifespan without caloric restriction.

AIMS/INTRODUCTION: Caloric restriction (CR) promotes longevity and exerts anti-aging effects by increasing Sirtuin production and activation. Gastric inhibitory polypeptide (GIP), a gastrointestinal peptide hormone, exerts various effects on pancreatic ß-cells and extra-pancreatic tissues. GIP promotes glucose-dependent augmentation of insulin secretion and uptake of nutrients into the adipose tissue. MATERIALS AND METHODS: Gipr-/- and Gipr+/+ mice were used for lifespan analysis, behavior experiments and gene expression of adipose tissue and muscles. 3T3-L1 differentiated adipocytes were used for Sirt1 and Nampt expression followed by treatment with GIP and α-lipoic acid. RESULTS: We observed that GIP receptor-knockout (Gipr-/-) mice fed normal diet showed an extended lifespan, increased exploratory and decreased anxiety-based behaviors, which are characteristic behavioral changes under CR. Moreover, Gipr-/- mice showed increased Sirt1 and Nampt expression in the adipose tissue. GIP suppressed α-lipoic acid-induced Sirt1 expression and activity in differentiated adipocytes. CONCLUSIONS: Although maintenance of CR is difficult, food intake and muscle endurance of Gipr-/- mice were similar to those of wild-type mice. Inhibition of GIP signaling may be a novel strategy to extend the lifespan of diabetic patients.

[An Effective Treatment of Relapsed Leiomyosarcoma with Fourth-Line Trabectedin].

A 56-year-old women underwent retroperitoneal leiomyosarcoma resection in December 2011. In July 2015, CT showed multiple liver metastases, and she received first-line chemotherapy treatment with doxorubicin. After 2 courses of doxorubicin, her performance status deteriorated; therefore, she was treated with pazopanib as second-line chemotherapy. PFS with pazopanib was 5 months and that with eribulin was 2.5 months. She was then treated with trabectedin as fourth-line chemotherapy from July 2016. The liver metastases reduced, and the disease was controlled for 1 year and 6 months following administration of trabectedin. We continue to treat this patient with trabectedin, and no serious side effects have been observed.

[A Case of Pediatric Soft Tissue Sarcoma with LMNA-NTRK1 Gene Fusion Treated with Larotrectinib under Single Patient Expanded Access System].

Tropomyosin-related kinase(TRK)fusion proteins are oncogenic drivers in multiple tumors in adults and children.Larotrectinib, an orally administered selective TRK inhibitor approved in the US, exhibits inhibitory activity against tumors harboring TRK fusions and is well tolerated.Here, we report the case of an 8-year-old female child with recurrence of an NTRK fusion low-grade sarcoma treated with larotrectinib monotherapy.The patient previously underwent resection of low-grade sarcoma in the right brachialis at 6 years of age, but local recurrence occurred after 16 months.As re-operation likely required amputation, larotrectinib was commenced at a dose of 100 mg BID.Complete radiographic remission was achieved after 3 months.There were no adverse events attributed to larotrectinib treatment.After dosing for 6 months, we performed local resection, confirming pathological complete remission.The drug was stopped, and the patient showed no evidence of relapse at 4 months after resection.In this case, larotrectinib was obtained using Single Patient Expanded Access under the FDA.In this paper, we also discuss the issues faced while accessing unapproved drugs in the precision medicine era in Japan.

Intermittent Withdrawal of Oxaliplatin for Alleviating Neurotoxicity during Oxaliplatin-Based Chemotherapy for Japanese Patients with Inoperable or Metastatic Colorectal Cancer: A Phase 2 Multicenter Study.

Oxaliplatin-based chemotherapy is a well-established regimen for patients with inoperable and metastatic colorectal cancer. However, one of the major limitations of oxaliplatin-based chemotherapy is sensory neuropathy. It was previously reported that introduction of intermittent oxaliplatin treatment to an oxaliplatin-based regimen has a significant benefit on efficacy or safety. Here, we prospectively assessed whether efficacy and safety of first-line chemotherapy for advanced colorectal cancer are achieved by introduction of withdrawal of oxaliplatin treatment for a certain period (intermittent oxaliplatin treatment). The primary endpoint of the present study is to assess the progression free survival time on patients treated with chemotherapy (mFOLFOX6 (levofolinate, 5-fluorouracil and oxaliplatin combination therapy) plus bevacizumab or CapeOX (oxaliplatin and capecitabine combination therapy) plus bevacizumab) with intermittent oxaliplatin treatment. Bevacizumab is a humanized anti-vascular endothelial growth factor antibody. Median progression-free survival by the mFOLFOX6 plus bevacizumab with intermittent oxaliplatin treatment or the CapeOX plus bevacizumab with intermittent oxaliplatin treatment were 10.6 months (95% confidential interval [CI], 8.3-13.4 months) or 8.0 months (95% CI, 4.2-16.8 months), respectively. Overall response rate by the mFOLFOX6 plus bevacizumab with intermittent oxaliplatin treatment or CapeOX plus bevacizumab with intermittent oxaliplatin treatment was 55.1% or 42.1%, respectively. Grade 3 or 4 neuropathy was observed in 4.1% or 10.5% of patients treated with mFOLFOX6 plus bevacizumab with intermittent oxaliplatin treatment or CapeOX plus bevacizumab with intermittent oxaliplatin treatment, respectively. Introduction of intermittent oxaliplatin treatment has improved severe neuropathy in mFOLFOX6 plus bevacizumab regimen without reducing treatment efficacy.

A Pyrene-Linked Cavity within a ß-Barrel Protein Promotes an Asymmetric Diels-Alder Reaction.

A unique π-expanded reaction cavity tethering a polycyclic moiety which provides a platform for substrate binding was constructed within the robust ß-barrel structure of nitrobindin (NB). NB variants with cavities of different sizes and shapes are coupled with N-(1-pyrenyl)maleimide (Pyr) to prepare a series of NB-Pyr conjugates. The orientation of the pyrene moiety is fixed within the cavity by the coupling reaction. The fluorescent quenching analysis of NB-Pyr indicates that azachalcone (aza), which is a dienophile for a Diels-Alder (DA) reaction, is efficiently incorporated within the pyrene-linked reaction cavity by the aromatic interaction. The DA reaction between aza and cyclopentadiene proceeds within the reaction cavity of NB-Pyr in the presence of CuII ion in high yield and high enantio- and regioselectivity.