Keita Kudo

Detection of epidermal growth factor receptor T790M mutation in plasma DNA from patients refractory to epidermal growth factor receptor tyrosine kinase inhibitor

A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR‐tyrosine kinase inhibitors (EGFR‐TKIs). A non‐invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non‐small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR‐TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression‐free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M‐positive patients (n = 11) was significantly shorter than that of the T790M‐negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR‐TKI therapy.

Activin A inhibits vascular endothelial cell growth and suppresses tumour angiogenesis in gastric cancer.

Background:Activin A is a multi-functional cytokine belonging to the transforming growth factor-β (TGF-β) superfamily; however, the effect of activin A on angiogenesis remains largely unclear. We found that inhibin β A subunit (INHBA) mRNA is overexpressed in gastric cancer (GC) specimens and investigated the effect of activin A, a homodimer of INHBA, on angiogenesis in GC.Methods:Anti-angiogenic effects of activin A via p21 induction were evaluated using human umbilical vein endothelial cells (HUVECs) in vitro and a stable INHBA-introduced GC cell line in vivo.Results:Compared with TGF-β, activin A potently inhibited the cellular proliferation and tube formation of HUVECs with induction of p21. A promoter assay and a chromatin immunoprecipitation assay revealed that activin A directly regulates p21 transcriptional activity through Smads. Stable p21-knockdown significantly enhanced the cellular proliferation of HUVECs. Notably, stable p21-knockdown exhibited a resistance to activin-mediated growth inhibition in HUVECs, indicating that p21 induction has a key role on activin A-mediated growth inhibition in vascular endothelial cells. Finally, a stable INHBA-introduced GC cell line exhibited a decrease in tumour growth and angiogenesis in vivo.Conclusion:Our findings highlight the suppressive role of activin A, unlike TGF-β, on tumour growth and angiogenesis in GC.

Association of Immune-Related Adverse Events With Nivolumab Efficacy in Non–Small-Cell Lung Cancer

Importance Immune-related adverse events (irAEs) have been associated with the efficacy of PD-1 (programmed cell death protein 1) inhibitors in patients with melanoma, but whether such an association exists for non–small-cell lung cancer (NSCLC) has remained unknown. Objective To evaluate the relation of irAEs to nivolumab efficacy in NSCLC. Design, Setting, and Participants In this study based on landmark and multivariable analyses, a total of 134 patients with advanced or recurrent NSCLC who were treated with nivolumab in the second-line setting or later between December 2015 and August 2016 were identified from a review of medical records from multiple institutions, including a university hospital and community hospitals. Data were updated as of December 31, 2016. Exposures The absence or presence of any irAE before the landmark date. Main Outcomes and Measures Kaplan-Meier curves of progression-free survival (PFS) according to the development of irAEs in 6-week landmark analysis were evaluated with the log-rank test as a preplanned primary objective. Overall survival (OS) was similarly evaluated. Multivariable analysis of both PFS and OS was performed with Cox proportional hazard regression models. Results In a cohort of 134 patients (median [range] age, 68 [33-85] years; 90 men [67%], 44 women [33%]), irAEs were observed in 69 of the 134 study patients (51%), including 12 patients (9%) with such events of grade 3 or 4, and 24 patients (18%) requiring systemic corticosteroid therapy. In 6-week landmark analysis, median PFS was 9.2 months (95% CI, 4.4 to not reached [NR]) and 4.8 months (95% CI, 3.0 to 7.5) (P = .04) whereas median OS was NR (95% CI, 12.3 to NR) and 11.1 months (95% CI, 9.6 to NR) (P = .01) for patients with or without irAEs, respectively. Multivariable analysis also revealed that irAEs were positively associated with survival outcome, with hazard ratios of 0.525 (95% CI, 0.287 to 0.937; P = .03) for PFS and 0.282 (95% CI, 0.101 to 0.667; P = .003) for OS. Conclusions and Relevance Development of irAEs was associated with survival outcome of nivolumab treatment in patients with advanced or recurrent NSCLC. Further studies are needed to confirm our findings.

Clinical application of amplicon-based next-generation sequencing to therapeutic decision making in lung cancer

BACKGROUND The clinical implementation of genomic profiling for lung cancer with high-throughput, multiplex tests is warranted to allow prioritization of appropriate therapies for individual patients. We have now applied such testing to detect actionable mutations that may inform treatment recommendations in lung cancer. PATIENTS AND METHODS We prospectively applied amplicon sequencing panels that cover both mutational hotspots in 22 genes related to lung and colon tumorigenesis as well as 72 major variants of ALK, RET, ROS1, and NTRK1 fusion transcripts. We then determined the proportion of patients who received genotype-directed therapy and their overall survival (OS). RESULTS Tumor specimens from 110 patients with lung cancer recruited between July 2013 and March 2015 were analyzed. The most common genetic alterations were TP53 mutations in 42 patients, followed by EGFR mutations in 25, STK11 mutations in 12, and KRAS mutations in 10. Potentially actionable mutations were identified in 44 patients including 50% of those with adenocarcinoma and 14% of those with squamous cell carcinoma. The OS of patients with advanced or recurrent cancer who had an actionable mutation and received targeted therapy (median OS not achieved) was significantly longer than that of those with no mutation (18.1 months, P = 0.041) or of those with a mutation not so treated (6.1 months, P = 0.0027). CONCLUSIONS Multiplex genomic testing was performed on formalin-fixed, paraffin-embedded tumor specimens with a success rate of ≥95%. Such testing can assist physicians in matching patients with approved or experimental targeted treatments. CLINICAL TRIAL REGISTRATION The University Medical Hospital Information Network (UMIN) Clinical Trials Registry under the identifier UMIN000014782.

The pan-HER family tyrosine kinase inhibitor afatinib overcomes HER3 ligand heregulin-mediated resistance to EGFR inhibitors in non-small cell lung cancer

Afatinib is a second generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) characterized as an irreversible pan-human EGFR (HER) family inhibitor. Afatinib remains effective for a subpopulation of patients with non-small cell lung cancer (NSCLC) with acquired resistance to first generation EGFF-TKIs such as erlotinib. Heregulin activates HER3 in an autocrine fashion and causes erlotinib resistance in NSCLC. Here we examine whether afatinib is effective against heregulin-overexpressing NSCLCs harboring EGFR activating mutations. Afatinib but not erlotinib decreased EGFR mutant NSCLC PC9HRG cell proliferation in vitro and in mouse xenografts. Afatinib inhibited phosphorylation of the cell signaling pathway proteins HER3, EGFR, HER2, and HER4, likely by prevention of trans-phosphorylation as HER3 kinase activity is inadequate for auto-phosphorylation. Afatinib, unlike erlotinib, inhibited AKT activation, resulting in elevated apoptosis in PC9HRG cells. Clinically, a subpopulation of 33 patients with EGFR mutations and NSCLC who had received first generation EGFR-TKIs exhibited elevated plasma heregulin levels compared to healthy volunteers; one of these achieved a response with afatinib therapy despite having previously developed erlotinib resistance. Afatinib can overcome heregulin-mediated resistance to erlotinib in EGFR mutant NSCLC. Further studies are necessary to determine whether heregulin can predict afatinib efficacy after development offirst generation EGFR-TKI resistance.

Characterization of tumour cell aggregation promoting factor from rat ascites hepatoma cells: Separation of two factors with different antigenic property.

The previously described glycoprotein that promotes tumour cell aggregation, derived from rat ascites hepatoma cells and capable of partial purification by chromatography, was found to be a mixture of 2 factors with different antigenic property. One was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. The action of the unabsorbed factor was clearly more potent than that of the absorbed factor. Both the factors were found in the serum of tumour bearing rats and the action of the unabsorbed factor was also more potent than that of the absorbed factor; its amount increased with time after i.p. inoculation of the cells. The serum of healthy rats contained the absorbed factor but not the unabsorbed factor. It was thus assumed that the unabsorbed factor was associated with the hepatoma cell surface itself and released into the serum, while the absorbed factor was associated with serum protein coating the cell.

Early onset recall pneumonitis during targeted therapy with sunitinib

BackgroundSunitinib interacts with radiation therapy, leading to synergism of the toxicities of these treatments. Radiation recall pneumonitis is a rare but serious complication of targeted therapy with tyrosine kinase inhibitors.Case presentationThe case of a patient with metastatic renal cell cancer (RCC) who developed recall pneumonitis on the first cycle of systemic sunitinib treatment is reported here. A 65-year-old man with RCC and bone metastasis underwent radiation therapy on his thoracic vertebrae (Th5-8) with a total dose of 24 Gy. Sunitinib (37.5 mg) was started 14 days after completing the radiation therapy. On the 14th day of sunitinib treatment, the patient developed progressive fever with worsening of dyspnea and general weakness. Treatment with pulse administration of prednisolone 1,000 mg for 3 days was initiated. Thereafter, the symptoms and the radiological findings regarding the interstitial filtration gradually improved over 7 days.ConclusionTo our knowledge, this is the first report of early onset recall pneumonitis during sunitinib therapy. At present, how sunitinib interacts with radiation therapy remains unclear. The possibility that tyrosine kinase inhibitor therapy, including with sunitinib, after radiation therapy may lead to adverse effects should be kept in mind.

Phase I study of irinotecan and gefitinib in patients with gefitinib treatment failure for non-small cell lung cancer.

Background:Currently, no effective treatments exist for non-small cell lung cancer (NSCLC) after failure of gefitinib therapy. Pre-clinical studies have demonstrated that gefitinib-resistant NSCLC cells are more sensitive to irinotecan than parental cells, and that combined administration of irinotecan and gefitinib has a synergistic additive effect. We conducted a phase I study to evaluate the combination of irinotecan and gefitinib as a therapeutic option for NSCLC patients with progressive disease (PD) after initial gefitinib treatment.Methods:Eligibility criteria included histologically confirmed NSCLC, age range of 20–74 years, refractory to or relapsed after gefitinib treatment, one or more previous chemotherapy regimens, Eastern Cooperative Oncology Group performance status 0–2, adequate organ function, and informed consent. Patients were treated with irinotecan on days 1 and 15, and treated daily with gefitinib from day 2 every 4 weeks. The treatment was continued until disease progression. The gefitinib dose was fixed at 250 mg. Irinotecan dosing started at 50 mg m−2 and was escalated in patients by 25 mg m−2 increments up to a maximum dose of 150 mg m−2.Results:Twenty-seven patients were enrolled: male/female=14/13; median age=60 (45–75); histology, adenocarcinoma/non-adenocarcinoma=25/2; performance status 0–1/2=19/8; previous response to gefitinib, partial response/stable disease/PD=21/2/4. Dose-limiting toxicities were observed in 2 patients at level 3. Maximum tolerated dose was not determined, and the full dose of irinotecan could be combined with the full dose of gefitinib. The disease control rate (DCR) and response rate (RR) were 69.2 and 26.9%, respectively. For 12 patients at level 5 (the recommended phase II dose), the DCR and RR were 75.0% and 41.7%, respectively. The median treatment cycles were 4; median time to treatment failure, 57 days (95% confidence interval (CI), 32–82 days); median overall survival, 244 days (95% CI, 185–303 days); and 1-year survival rate, 32.6%.Conclusion:The combination of irinotecan and gefitinib was well tolerated and potentially beneficial for NSCLC patients failing initial gefitinib monotherapy.

The induction of tumour cell adhesiveness and intercellular junctions by a glycoprotein of rat ascites hepatoma cell surface.

Rat ascites hepatoma AH109A cells (present as a free form in vivo) can aggregate and then develop well-defined tripartite junctional complexes, including intermediate junctions, desmosomes and focal tight junctions, on incubation with a glycoprotein separated from rat ascites hepatoma AH136B cells (forming cell islnds in vivo). The development of binding structures was strongly inhibited by actinomycin D. AH109A cells or rat ascites hepatoma YS cells (present as a free form in vivo) previously treated with the glycoprotein for 24 h, when inoculated i.p., proliferated as free cells in the ascitic fluid, like the untreated cells. AH109A cells actively proliferating in the skin do not form any junctional complexes. The reason for the failure of island formation by AH109A cells or YS cells in vivo is discussed.

Sorafenib treatment for patients with RET fusion-positive non-small cell lung cancer

BACKGROUND RET fusions were recently identified in non-small cell lung cancer (NSCLC) and are considered as a potential therapeutic target of NSCLC. Sorafenib, a multi-kinase inhibitor, has potent anti-RET activity. We conducted a study to evaluate the efficacy of sorafenib in a small number of patients with RET fusion-positive NSCLC. MATERIALS AND METHODS Eligible patients had advanced or recurrent NSCLC, were more than 20 years old, had undergone treatment with one or more previous chemotherapy regimens, had an Eastern Cooperative Oncology Group performance status 0-2, had adequate organ function, and provided informed consent. The presence of the RET fusion gene was confirmed by a split FISH assay. The patients were treated twice daily with 400mg of sorafenib taken orally. The treatment was continued until either disease progression or unacceptable toxicity. RESULTS From March 2012 to April 2013, three patients were enrolled. The responses to sorafenib included one patient with stable disease (SD) and two patients with progressive disease (PD). One patient took sorafenib for twelve months. The most common toxicities were palmar-plantar erythrodysesthesia syndrome, hypertension, and diarrhea. CONCLUSION Since sorafenib did not show dramatic responses, we suggest testing other RET inhibitors for the treatment of RET fusion-positive NSCLC. This study was registered at UMIN as trial number 000007515.