Recontact: a survey of current practices and BRCA1/2 testing in Japan.

Genetic testing advances have enabled the provision of previously unavailable information on the pathogenicity of genetic variants, frequently necessitating the recontact of former patients by clinicians. In Japan, national health insurance coverage was extended to BRCA1/2 testing for the diagnosis of hereditary breast and ovarian cancer for patients who meet certain criteria in 2020, and conditions necessitating recontact were expected to increase. Studies and discussions regarding recontact have been conducted in the U.S. and Europe; however, in Japan, the national discussion around recontact remains undeveloped. We conducted a cross-sectional study by interviewing 73 facilities accredited by the Japanese Organization of Hereditary Breast and Ovarian Cancer regarding the practice of recontacting patients at these facilities. Sixty-six facilities responded that they recontact patients, but only 17 facilities had a protocol for this. The most common reason for recontact was that it could benefit the patient. Facilities that did not recontact stated that they lacked the necessary personnel or services. Most facilities indicated that a recontact system should be implemented in their practice. The increased burden on too few medical personnel, unestablished systems, patient confusion, and the right not to know were cited as barriers to implementing recontact. Although developing recommendations on recontact would be useful for providing equitable healthcare in Japan, there is an urgent need to deepen the discussion on recontacting, as negative opinions about recontacting patients were observed.

Semiautomated Segmentation and Measurement of Cytoplasmic Vacuoles in a Neutrophil With General-Purpose Image Analysis Software.

BACKGROUND: Morphological observation of blood or marrow film is still described nonquantitatively. We developed a semiautomatic method for segmenting vacuoles from the cytoplasm using Photoshop (PS) and Image-J (IJ), called PS-IJ, and measured the relative entire cell area (rECA) and relative areas of vacuoles (rAV) in the cytoplasm of neutrophil with PS-IJ. METHODS: Whole-blood samples were stored at 4°C with ethylenediaminetetraacetate and in two different preserving manners (P1 and P2). Color-tone intensity levels of neutrophil images were semiautomatically compensated using PS, and then vacuole portions were automatically segmented by IJ. The rAV and rECA were measured by counting pixels by IJ. For evaluating the accuracy in segmentations of vacuoles with PS-IJ, the rAV/rECA ratios calculated with results from PS-IJ were compared with those calculated with human eye and IJ (HE-IJ). RESULTS: The rECA and rAV/ in P1 significantly (P < 0.05, P < 0.05) were enlarged and increased, but did not significantly (P = 0.46, P = 0.21) change in P2. The rAV/rECA ratios by PS-IJ were significantly correlated (r = 0.90, P < 0.01) with those by HE-IJ. CONCLUSION: PS-IJ method can successfully segment vacuoles and measure the rAV and rECA, becoming a useful tool for quantitative description of morphological observation of blood and marrow film.

Secondary Mutations of the EGFR Gene That Confer Resistance to Mobocertinib in EGFR Exon 20 Insertion.

INTRODUCTION: Approximately 10% of mutations in the EGFR gene in NSCLC are in-frame insertions in exon 20 (X20ins). These tumors usually do not respond to conventional EGFR tyrosine kinase inhibitors (TKIs). Several novel EGFR TKIs active for X20ins are in clinical development, including mobocertinib, which was recently approved by the U.S. Food and Drug Administration. However, acquired resistance during treatment with these TKIs still occurs as in the case of EGFR TKIs of earlier generations. METHODS: We chronically exposed murine pro-B-cell line cells transduced with the five most common X20ins (A763_Y764insFQEA, V769_D770insASV, D770_N771insSVD, H773_V774insNPH and H773_V774insH) to mobocertinib in the presence of N-ethyl-N-nitrosourea and searched for secondary EGFR mutations. We evaluated the efficacies of several EGFR X20ins inhibitors, including zipalertinib and sunvozertinib, against cells with acquired resistant mutations. RESULTS: All secondary mutations resulting in acquired resistance to mobocertinib were exclusively C797S in insFQEA and insSVD. However, in the case of other X20ins (insASV, insNPH, and insH), T790M or C797S secondary mutations contributed to acquired resistance to mobocertinib. The emergence of T790M was more frequent in cells treated with lower drug concentrations. Sunvozertinib exhibited good activity against resistant cells with T790M. Cells with C797S were refractory to all EGFR TKIs, except for erlotinib, which was active for insFQEA with C797S. CONCLUSIONS: T790M or C797S, depending on the original X20ins mutations, conferred acquired resistance to mobocertinib. Sunvozertinib may be the treatment of choice for patients with tumors resistant to mobocertinib because of T790M.

Clinical Relevance of Patient-Derived Organoid of Surgically Resected Lung Cancer as an In Vitro Model for Biomarker and Drug Testing.

Introduction: Lung tumor organoids (LTOs) have attracted attention as in vitro preclinical models; however, their clinical and experimental applications have not been fully established. Methods: We attempted to establish LTOs from resected specimens of patients with lung cancer who underwent lung resection. Clinicopathologic characteristics related to the establishment of LTOs were evaluated. Histologic assessment and genetic analysis were conducted for both LTOs and their parental tumors. Organoid-derived xenografts were generated in immunocompetent mice. Drug sensitivity was assessed using cell proliferation assays. Results: We established 53 LTOs from 79 lung cancer samples, including 10 long-term culture models. The establishment rate was significantly lower in squamous cell carcinomas than in other histologic types (48% versus 75%, p = 0.034). Histologic similarities were confirmed among LTOs, the parental tumors, and organoid-derived xenografts. Seven mutations, including two EGFR L858R and one EGFR exon 20 H773delinsYNPY mutations, were detected in both LTO and parental tumors; the other four mutations were detected in either LTO or parental tumors. The extensive culture ability of LTO (passaged >10 times) correlated with poor patient prognosis. LTO9 cells harboring EGFR H773delinsYNPY were sensitive to osimertinib. The parental patient, who had new metastatic lesions, was treated with osimertinib and exhibited a remarkable response. Conclusions: The establishment and growth rates of LTOs were associated with the histologic subtype and tumor size. LTOs derived from resected specimens have become preclinical models that can be used to predict drug responses and accelerate the development of treatment strategies for patients with rare mutations.

Inter- and Intratumor Heterogeneity of EGFR Compound Mutations in Non-Small Cell Lung Cancers: Analysis of Five Cases.

BACKGROUND: Several clinical and preclinical studies suggest that non-small cell lung cancers (NSCLCs) with EGFR compound mutations were associated with lower efficacies of first-generation EGFR inhibitors than tumors with single EGFR mutation. Some researchers hypothesize that EGFR mutation status is heterogeneous in such tumors and that second-generation EGFR inhibitors may eliminate cancer cells with uncommon EGFR mutations from tumors with EGFR compound mutations. However, this hypothesis is currently unproven; therefore, we performed the current study to determine if tumor cells with EGFR compound mutations are present in heterogeneous or homogeneous manners. PATIENTS AND METHODS: Multiregion analysis was performed for surgically resected primary NSCLC tumors with EGFR compound mutations to examine the intratumor heterogeneity of EGFR compound mutations. In addition, we evaluated the intertumor heterogeneity of EGFR compound mutations using 2 pleural disseminations obtained from a patient with NSCLC at exploratory thoracotomy and 9 primary or metastatic lesions obtained from 2 autopsied NSCLC patients. Digital polymerase chain reaction, target sequencing, or direct sequencing were used to detect EGFR mutations. RESULTS: This study included 5 NSCLC cases; their compound mutations were L858R+S768I, G719X+S768I, G719A+R776H, L858R+E709G, and L858R+I759M. Noncancerous pulmonary tissues from each patient did not harbor EGFR mutations, which revealed that all mutations were somatic. We did not detect any intra- or intertumor heterogeneity in these EGFR compound mutations. CONCLUSION: No intra- or intertumor heterogeneity was observed for EGFR compound mutations. Our results indicate that both EGFR mutations were truncal and selective elimination of cancer cells with uncommon EGFR mutations is unrealistic.

Spatial heterogeneity of acquired resistance mechanisms to 1st/2nd generation EGFR tyrosine kinase inhibitors in lung cancer.

BACKGROUND: Overcoming acquired resistance against targeted therapies to improve outcomes of lung cancer patients harboring driver mutations is a critical issue. While drug therapy oriented to a resistance mechanism appears attractive, spatial heterogeneity of resistance mechanisms in each patient will diminish treatment efficacy. However, the frequency, clinical backgrounds, clinical implications, and patterns of spatial heterogeneity in resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs) are largely unknown. PATIENTS AND METHODS: This study included 128 specimens from 24 autopsied patients with lung adenocarcinoma harboring EGFR mutation. Acquired resistance mechanisms reported as relatively frequent in lung cancer, e.g., T790 M and other secondary EGFR mutations, MET and ERBB2 gene amplification, and histological transformation, were retrospectively examined. All patients had received 1st/2nd generation EGFR-TKI and showed acquired resistance to the drug before death. No patient received osimertinib. RESULTS: No resistance mechanism was identified in two patients. T790M mutation was detected in 20 patients (83 %); however, nine of these patients also had lesions without T790M mutation. Among 22 patients whose resistance mechanisms were identified, ten had spatial heterogeneity of resistance mechanisms (45 %), and these patients had significantly shorter time-to-treatment failure compared with those without heterogeneity (median 4.7 months vs. 14.7 months, p = 0.0004). CONCLUSION: We observed significant spatial heterogeneity of acquired resistance mechanisms to EGFR-TKIs in lung adenocarcinoma. Our results also indicate that the incidence of resistance mechanisms may vary based on the biopsied tumor locations.

Fusion protein of retinoic acid receptor alpha with promyelocytic leukemia protein or promyelocytic leukemia zinc finger protein recruits N-CoR-TBLR1 corepressor complex to repress transcription in vivo.

Fusion proteins of retinoic acid receptor alpha (RARalpha) with promyelocytic leukemia protein (PML-RARalpha) or with promyelocytic leukemia zinc finger protein (PLZF-RARalpha) are associated with and likely responsible for the development of acute promyelocytic leukemia. These oncoproteins retain the ability to bind DNA and retinoic acid through the RARalpha moiety. This enables them to repress RARalpha target genes in the absence of retinoic acid, but the underlying mechanisms remain to be investigated. Here we use the frog oocyte system to study transcriptional regulation by PML-RARalpha and PLZF-RARalpha in the context of chromatin. We first show that the endogenous corepressor N-CoR forms a complex with TBLR1 (transducin beta-like protein 1-related protein) and that both N-CoR and TBLR1 can interact with unliganded PML-RARalpha and PLZF-RARalpha in vivo. Using chromatin immunoprecipitation, we demonstrate that both oncoproteins recruit TBLR1, as well as N-CoR, to its target promoter, leading to histone deacetylation and transcriptional repression. Furthermore, expression of a dominant negative N-CoR that contains the TBLR1-interacting domain blocks transcription repression by unliganded PML-RARalpha and PLZF-RARalpha. Thus, our studies provide in vivo evidence for targeted recruitment of N-CoR-TBLR1 complexes by PML-RARalpha and PLZF-RARalpha in transcriptional repression in the context of chromatin.