Effects of a fertility-sparing re-treatment for recurrent atypical endometrial hyperplasia and endometrial cancer: a systematic literature review.

OBJECTIVE: To examine the effectiveness of progestin re-treatment for recurrent endometrial intraepithelial neoplasia (EIN), atypical endometrial hyperplasia (AH) and endometrial cancer (EC) following initial fertility-sparing treatment. METHODS: A comprehensive systematic review and meta-analysis were conducted by an Expert Panel of the Japan Society of Gynecologic Oncology Endometrial Cancer Committee. Multiple search engines, including PubMed/MEDLINE and the Cochrane Database, were searched in December 2021 using the keywords "Endometrial neoplasms," "Endometrial hyperplasia," "Endometrial intraepithelial neoplasia," "Fertility preservation," "Progestins," AND "Recurrence." Cases describing progestin re-treatment for recurrent EIN, AH and EC were compared with cases that underwent conventional hysterectomy. The primary outcomes were survival and disease recurrence, and the secondary outcome was pregnancy. RESULTS: After screening 238 studies, 32 with results for recurrent treatment were identified. These studies included 365 patients (270 received progestin re-treatment and 95 underwent hysterectomy). Most progestin re-treatment involved medroxyprogesterone acetate or megestrol acetate (94.5%). Complete remission (CR) following progestin re-treatment was achieved in 219 (81.1%) cases, with 3-, 6- and 9-month cumulative CR rates of 22.8%, 51.7% and 82.6%, respectively. Progestin re-treatment was associated with higher risk of disease recurrence than conventional hysterectomy was (odds ratio [OR]=6.78; 95% confidence interval [CI]=1.99-23.10), and one patient (0.4%) died of disease. Fifty-one (14.0%) women became pregnant after recurrence, and progestin re-treatment demonstrated a possibility of pregnancy (OR=2.48; 95% CI=0.94-6.58). CONCLUSION: This meta-analysis suggests that repeat progestin therapy is an effective option for women with recurrent EIN, AH and EC, who wish to retain their fertility.

Reduced expression of gangliosides with GM2-determinant in cervical carcinoma-derived cells after subcutaneous transplantation into nude mice.

Human cervical carcinoma-derived cell lines have been frequently found to contain gangliosides with GM2-determinant, i.e., GM2, GalNAc-GM1b and GalNAc-GD1a, but GM2 was only detected in 5 of 15 tissues, and GalNAc-GM1b and GalNAc-GD1a were not found in any tissues from patients with several histological types of cervical carcinomas. To further characterize the ganglioside expression in cervical carcinomas, cells were grown by subcutaneous transplantation into nude mice, and gangliosides were quantitated by TLC-immunostaining with the anti-GM2 (YHD-06) antibody and a newly developed anti-GM3 (5H6) antibody, which reacts with GM3 and GM1b, but not with GD1a. Gangliosides with GM2-determinant in cells disappeared in transplanted cells, and the amount of GM3, a precursor for GM2, in transplanted cells was greater than in cultured cells. Also, transplanted cells containing GalNAc-GM1b newly expressed GM1b, suggesting that the activity of GalNAc transferase for synthesis of GalNAc-GM1b is retarded on subcutaneous transplantation. The ganglioside composition, with GM3 as the major one, in the transplanted cells was similar to that in cervical carcinoma tissues, and thus, the expression of gangliosides with GM2-determinant seemed to be accelerated under cell-cultivation conditions.

Intra-tumor and Inter-tumor Heterogeneity in MET Exon 14 Skipping Mutations and Co-mutations in Pulmonary Pleomorphic Carcinomas.

BACKGROUND: MET exon 14 skipping mutation is a driver mutation in lung cancer and is highly enriched in pulmonary pleomorphic carcinomas (PPCs). Whether there is intratumor or intertumor heterogeneity in MET exon 14 skipping status or in co-occurring genetic alterations in lung cancers driven by MET exon 14 skipping is unknown. METHODS: We analyzed tumor specimens obtained from 23 PPC patients (10 autopsied and 13 surgically resected). MET exon 14 skipping was detected by RT-PCR. For patients with MET exon 14 skipping mutation, further analyses were performed. Genomic DNA (gDNA) was extracted from various histological components for each patient who underwent surgical resection (to assess intratumor heterogeneity). In autopsied patients, gDNA and total RNA were extracted from all metastatic lesions (to assess intertumor heterogeneity). RESULTS: MET exon 14 skipping mutation was detected in 4 patients (4/23, 17.4%): two surgically resected and two autopsied patients. We found no intratumor or intertumor heterogeneity in MET exon 14 skipping mutation status in these patients. We observed intratumor and intertumor heterogeneity in the copy number variations and/or mutational status of cancer-related genes; some of these differences may have an impact on MET tyrosine kinase inhibitor (TKI) efficacy. CONCLUSION: In our exploratory analysis of four cases, we observed that MET exon 14 skipping mutations are distributed homogeneously throughout histological components and between metastatic lesions. Our results also suggest that there is marked intertumor and intratumor heterogeneity in co-occurring genetic alterations, and therapeutic implications of such heterogeneity should be evaluated in future studies.

Nucleosome Positioning on Episomal Human Papillomavirus DNA in Cultured Cells.

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140-200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that changes in nucleosome positions on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

ECG Changes Through Immunosuppressive Therapy Indicate Cardiac Abnormality in Anti-MDA5 Antibody-Positive Clinically Amyopathic Dermatomyositis.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody, a dermatomyositis (DM)-specific antibody, is strongly associated with interstitial lung disease (ILD). Patients with idiopathic inflammatory myopathy (IIM) who are anti-MDA5 antibody positive [anti-MDA5 (+)] often experience chest symptoms during the active disease phase. These symptoms are primarily explained by respiratory failure; nevertheless, cardiac involvement can also be symptomatic. Thus, the aim of this study was to investigate cardiac involvement in anti-MDA5 (+) DM. A total of 63 patients with IIM who underwent electrocardiography (ECG) and ultrasound cardiography (UCG) during the active disease phase from 2016 to 2021 [anti-MDA5 (+) group, n = 21; anti-MDA5-negative (-) group, n = 42] were enrolled in the study, and their clinical charts were retrospectively reviewed. The ECG and UCG findings were compared between the anti-MDA5 (+) and anti-MDA5 (-) groups. All anti-MDA5 (+) patients had DM with ILD. The anti-MDA5 (+) group showed more frequent skin ulcerations and lower levels of leukocytes, muscle enzymes, and electrolytes (Na, K, Cl, and Ca) than the anti-MDA5 (-) group. According to the ECG findings obtained during the active disease phase, the T wave amplitudes were significantly lower for the anti-MDA5 (+) group than for the anti-MDA5 (-) group (I, II, and V4-6 lead; p < 0.01; aVF and V3, p < 0.05). However, the lower amplitudes were restored during the remission phase. Except for the E wave, A wave and Sep e', the UCG results showed no significant differences between the groups. Four patients with anti-MDA5 (+) DM had many leads with lower T wave and cardiac abnormalities (heart failure, diastolic dysfunction, myocarditis) on and after admission. Though anti-MDA5 (+) patients clinically improved after immunosuppressive therapy, some of their ECG findings did not fully recover in remission phase. In conclusion, anti-MDA5 (+) DM appears to show cardiac involvement (electrical activity and function) during the active phase. Further studies are necessary to clarify the actual cardiac condition and mechanism of these findings in patients with anti-MDA5 (+) DM.

Analysis of pathological and clinical characteristics of cervical conization according to age group in Japan.

OBJECTIVE: With increased screening, more patients with precancerous or early cervical cancer are now being identified. Age at pregnancy, and thus number of patients requiring fertility preservation, have also increased, resulting in more diagnostic and therapeutic cervical conization (conization) procedures. We here investigated the pathological and clinical characteristics of patients undergoing conization, with a focus on age. The objectives of our study were to identify the risk factors potentially involved in cervical intraepithelial neoplasia (CIN) recurrence or persistence, additional treatment after conization, the effects of conization on pregnancy, and the actual status of conization in Japan. STUDY DESIGN: A "Subcommittee for Investigation of Cervical Conization" within the Gynecologic Oncology Committee in the Japan Society of Obstetrics and Gynecology investigated pathological and clinical characteristics of conization at 205 institutions in Japan. We analyzed pathological and clinical characteristics according to age ≤50 and >50 years. RESULTS: Patients aged 20-40 years accounted for 12904 (87%) of the 14,832 study patients (median: 37 years, range: 16-88 years). However, 1838 (12.4%) were aged >50 years. The commonest post-operative diagnosis was CIN grade 3 in all age groups. Rates of invasive cancer, post-operative detection of more advanced lesions, positive surgical margins, additional treatment, and recurrence were significantly higher in patients aged ≥50 years than those aged <50 years (all p < 0.01), whereas rates of post-operative complications did not differ significantly between age groups. The relationship between cerclage and the incidence of amniorrhexis or premature birth did not differ significantly in any age group. CONCLUSION: Post-conization management of patients aged ≥50 years requires considering the high rates of detection of more advanced lesions post-operatively, positive surgical margins, and recurrence. Cerclage should not be performed post-conization without careful consideration.

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.

Treatment rationale and design of the PROLONG study: safety and efficacy of pembrolizumab as first-line therapy for elderly patients with non-small cell lung cancer.

BACKGROUND: Pembrolizumab is recommended as first-line therapy for patients with advanced non-small cell lung cancer (NSCLC) and a Programmed cell death ligand-1 (PD-L1) tumor proportion score (TPS) of ≥50% without driver mutations. However, the safety and efficacy were not investigated among patients who were ≥75 years old. METHODS: This open-label single-arm phase II study is designed to evaluate pembrolizumab as first-line therapy for patients who are ≥75 years old with advanced NSCLC and a PD-L1 TPS of ≥50% without driver mutations. The primary endpoint is progression-free survival, and the secondary endpoints are overall survival, objective response rate, safety, and quality of life. Recruitment started in October 2017 and is expected to continue for approximately 3 years. CONCLUSIONS: Given the currently poor prognosis of elderly patients with advanced NSCLC, we hope that the findings of this study will facilitate more effective treatment in this setting.

Intake frequency of vegetables or seafoods negatively correlates with disease activity of rheumatoid arthritis.

OBJECTIVE: To clarify the relationship between dietary habit and disease activity of rheumatoid arthritis (RA). METHODS: This study enrolled RA patients who met the ACR/EULAR 2010 classification criteria from Kyoto University Rheumatoid Arthritis Management Alliance (KURAMA) cohort in 2015. 22-item food frequency questionnaire (FFQ) was taken for the measurement of dietary habit in a single-institution cohort of RA (Kyoto University Rheumatoid Arthritis Management Alliance: KURAMA) in 2015. The disease activities of RA using the Disease Activity Score calculated based on the erythrocyte sedimentation rate (DAS28-ESR), Simplified Disease Activity Index (SDAI), Health Assessment Questionnaire (HAQ), and serum matrix metalloproteinase-3 (MMP-3) level, the use of disease-modifying anti-rheumatic drugs (DMARDs), disease duration, rheumatoid factor, anti-cyclic citrullinated antibody, and body mass index were also examined. All of them were combined and statistically analyzed. RESULTS: 441 RA patients (81% women; mean age 65 years; mean disease duration 15 years) were enrolled from the KURAMA cohort. Average Disease Activity Score-28 using the erythrocyte sedimentation rate (DAS28-ESR) was 2.7. Univariate analysis showed that intake frequency of vegetables had a statistically significant negative correlation with disease activity markers, such as DAS28-ESR (ρ = -0.11, p<0.01), Simplified Disease Activity Index (SDAI) (ρ = -0.16, p<0.001), matrix metalloproteinase-3 (MMP-3) (ρ = -0.21, p<0.0001), and Health Assessment Questionnaire (HAQ) (ρ = -0.13, p<0.01). Factor analysis with varimax rotation was done to simplify the relevance of disease activity to various food items. 22 foods were categorized into five dietary patterns: "seafoods", "vegetables/fruits", "meats/fried foods", "snacks", and "processed foods". The multivariate analysis adjusted for clinically significant confounders showed that "seafoods" had statistically significant negative correlations with DAS28-ESR (ß = -0.15, p<0.01), SDAI (ß = -0.18, p<0.001), MMP-3 (ß = -0.15, p<0.01), and HAQ (ß = -0.24, p<0.0001). "Vegetables/fruits" had statistically significant negative correlations with SDAI (ß = -0.11 p<0.05), MMP-3 (ß = -0.12, p<0.01), and HAQ (ß = -0.11, p<0.05). CONCLUSIONS: These results suggest that high intake frequency of vegetables/fruits and/or seafoods might correlate with low disease activity.

Roles for E1-independent replication and E6-mediated p53 degradation during low-risk and high-risk human papillomavirus genome maintenance.

Human papillomaviruses (HPV) have genotype-specific disease associations, with high-risk alpha types causing at least 5% of all human cancers. Despite these conspicuous differences, our data show that high- and low- risk HPV types use similar approaches for genome maintenance and persistence. During the maintenance phase, viral episomes and the host cell genome are replicated synchronously, and for both the high- and low-risk HPV types, the E1 viral helicase is non-essential. During virus genome amplification, replication switches from an E1-independent to an E1-dependent mode, which can uncouple viral DNA replication from that of the host cell. It appears that the viral E2 protein, but not E6 and E7, is required for the synchronous maintenance-replication of both the high and the low-risk HPV types. Interestingly, the ability of the high-risk E6 protein to mediate the proteosomal degradation of p53 and to inhibit keratinocyte differentiation, was also seen with low-risk HPV E6, but in this case was regulated by cell density and the level of viral gene expression. This allows low-risk E6 to support genome amplification, while limiting the extent of E6-mediated cell proliferation during synchronous genome maintenance. Both high and low-risk E7s could facilitate cell cycle re-entry in differentiating cells and support E1-dependent replication. Despite the well-established differences in the viral pathogenesis and cancer risk, it appears that low- and high-risk HPV types use fundamentally similar molecular strategies to maintain their genomes, albeit with important differences in their regulatory control. Our results provide new insights into the regulation of high and low-risk HPV genome replication and persistence in the epithelial basal and parabasal cells layers. Understanding the minimum requirement for viral genome persistence will facilitate the development of therapeutic strategies for clearance.

Effects of Hochuekkito combined with pulmonary rehabilitation in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) has significant systemic effects, such as weight loss, which affects exercise capacity, health-related quality of life (HRQOL) and survival. The traditional herbal medicine, Hochuekkito (TJ-41), improves the nutritional status and decreases systemic inflammation in patients with COPD. However, to date, the additive effect of TJ-41 on pulmonary rehabilitation (PR) in patients with COPD has not been researched comprehensively. The purpose of the present study was to investigate the efficacy and safety of adding TJ-41 to PR for patients with COPD. Thirty-three malnourished patients with COPD were randomly assigned to receive low-intensity exercise with (TJ-41 group) or without (control group) TJ-41 treatment for 12 weeks. The primary outcome was the change in the 6-min walk distance (6MWD). Secondary outcomes included changes in the body composition, peripheral muscle strength, modified Medical Research Council dyspnea score, visual analog scale (VAS) score for dyspnea, VAS score for fatigue and COPD assessment test (CAT) score. After the 12-week treatment, body weight and percent ideal body weight were significantly increased in the TJ-41 group (P<0.05), but not in the control group. After the 12-week treatment, the modified Medical Research Council dyspnea score, VAS score for dyspnea, VAS score for fatigue and total CAT score decreased significantly in the TJ-41 group (all P<0.05), but not in the control group. There were no significant differences in the 6MWD and peripheral muscle strength between baseline and after 12 weeks of treatment in either group. No adverse effects were noted with the use of TJ-41. It was concluded that the addition of TJ-41 to PR may benefit malnourished patients with COPD with respect to dyspnea and HRQOL.

Genetic profiling-based prognostic prediction of patients with advanced small-cell lung cancer in large scale analysis.

OBJECTIVES: Comprehensive genomic analysis of small-cell lung cancer (SCLC) revealed various genetic alterations. However, obtaining suitable samples for genetic analysis is difficult in advanced SCLC. Thus, the prognostic effect of genetic alterations on the outcome of SCLC patients has not been well investigated. Therefore, this study evaluated the effect of genetic alterations on the survival of SCLC patients. MATERIALS AND METHODS: We collected samples obtained from 220 patients with advanced SCLC before cancer treatment. Genomic DNA extracted from the samples was subjected to a 1.499 Mb-sized custom panel that captured all exons of 244 cancer-related genes, and the captured DNA was analyzed through next-generation sequencing. The associations between genetic alterations and overall survival were evaluated. RESULTS: Genetic analysis was successful in 204 samples (93%). Genetic alterations in the PI3K/AKT/mTOR pathway and inactivating mutations inTP53 and RB1 were detected in 14 (7%), 150 (74%), and 85 (42%) of the tumors. In extensive disease (ED, N = 126) patients, multivariate analysis revealed that the presence of genetic alterations in the PI3K/AKT/mTOR pathway was significantly associated with unfavorable survival [hazard ratio (HR), 2.14; 95% CI 1.02-4.06; P = 0.04]. In limited disease (LD, N = 78) patients, the presence of TP53 mutation and the absence of RB1 mutation were significantly associated with unfavorable survival (HR, 2.41; 95% CI 1.21-5.34; P = 0.01, and HR, 0.45; 95% CI 0.25-0.79; P < 0.01, respectively). CONCLUSIONS: Sequencing-based genetic profiling is feasible and useful to predict the prognosis in advanced SCLC. Genetic alterations in the PI3K/AKT/mTOR pathway, TP53 mutations and RB1 mutations were associated with prognosis in SCLC patients. The genetic alterations associated with the prognosis were different between ED-SCLC and LD-SCLC.

CD44 Facilitates Epithelial-to-Mesenchymal Transition Phenotypic Change at Acquisition of Resistance to EGFR Kinase Inhibitors in Lung Cancer.

Epithelial-to-mesenchymal transition (EMT) is one of the acquired resistance mechanisms to EGFR tyrosine kinase inhibitors (TKI) in lung cancers. Because EMT is related to tumor invasion, metastases, and resistance to various treatments, it is important to prevent the emergence of EMT. However, molecular mechanism(s) underlying EMT phenotypic changes, as well as biomarker(s) that predict the emergence of EMT in EGFR-mutated lung cancers, are unclear to date. Through the comparison of expression data between isogenic lung cancer cell lines that acquired resistance to EGFR-TKI(s), we identified that high CD44 expression is related to a mesenchymal phenotype and that shRNA-mediated knockdown of CD44 reversed the EMT change. High membranous CD44 expression was identified in lesions with mesenchymal phenotype that were obtained from lung cancer patients who developed acquired resistance to gefitinib or afatinib, whereas isogenic lesions without EMT change showed negative/weak staining for CD44. Immunohistochemistry for treatment-naïve lung cancer cell lines with EGFR mutations found those that acquire resistance to EGFR-TKIs via EMT (HCC4006 and H1975 cells) had strong membranous CD44 expression compared with non-EMT-transforming lines which demonstrated negative or weak staining (Fisher exact test P value = 0.036). shRNA-mediated CD44 knockdown in HCC4006 cells prevented the emergence of EMT after chronic exposure to osimertinib. These results suggest that upregulation of CD44 facilitates EMT-phenotypic change in lung cancers with EGFR mutations when treated with EGFR-TKIs. In addition, our results suggest that CD44 can be a useful biomarker to predict the emergence of EMT upon EGFR-TKI monotherapy. Mol Cancer Ther; 17(10); 2257-65. ©2018 AACR.

Innate Genetic Evolution of Lung Cancers and Spatial Heterogeneity: Analysis of Treatment-Naïve Lesions.

INTRODUCTION: Data regarding the pre-treatment intertumor heterogeneity of potential biomarkers in advanced-stage lung cancers is limited. A finding of such heterogeneity between primary and metastatic lesions would prove valuable to determine if a metastatic lesion can be a surrogate for the primary tumor, as more biomarkers will likely be used in the future to inform treatment decisions. METHODS: We performed RNA sequencing to analyze intertumor heterogeneity in 30 specimens (primary tumors, intrathoracic, and extrathoracic metastatic lesions) obtained from five treatment-naïve lung cancer patients. RESULTS: The global unsupervised clustering analysis showed that the lesions clustered at the individual patient level rather than on the metastatic sites, suggesting that the characteristics of specific tumor cells have a greater impact on the gene expression signature than the microenvironment in which the metastasis develops. The mutational and transcriptional data highlight the presence of intertumor heterogeneity showing that the primary tumors are usually distinct from metastatic lesions. Through a comparison between metastatic lesions and the primary tumors, we observed that pathways related to cell proliferation were upregulated, whereas immune-related pathways were downregulated in metastatic lesions. CONCLUSION: These data not only provide insight into the evolution of lung cancers, but also imply possibilities and limitations of biomarker-based treatment in lung cancers.

HPV16 and 18 genome amplification show different E4-dependence, with 16E4 enhancing E1 nuclear accumulation and replicative efficiency via its cell cycle arrest and kinase activation functions.

To clarify E1^E4's role during high-risk HPV infection, the E4 proteins of HPV16 and 18 were compared side by side using an isogenic keratinocyte differentiation model. While no effect on cell proliferation or viral genome copy number was observed during the early phase of either virus life cycle, time-course experiments showed that viral genome amplification and L1 expression were differently affected upon differentiation, with HPV16 showing a much clearer E4 dependency. Although E4 loss never completely abolished genome amplification, its more obvious contribution in HPV16 focused our efforts on 16E4. As previously suggested, in the context of the virus life cycle, 16E4s G2-arrest capability was found to contribute to both genome amplification success and L1 accumulation. Loss of 16E4 also lead to a reduced maintenance of ERK, JNK and p38MAPK activity throughout the genome amplifying cell layers, with 16E4 (but not 18E4) co-localizing precisely with activated cytoplasmic JNK in both wild type raft tissue, and HPV16-induced patient biopsy tissue. When 16E1 was co-expressed with E4, as occurs during genome amplification in vivo, the E1 replication helicase accumulated preferentially in the nucleus, and in transient replication assays, E4 stimulated viral genome amplification. Interestingly, a 16E1 mutant deficient in its regulatory phosphorylation sites no longer accumulated in the nucleus following E4 co-expression. E4-mediated stabilisation of 16E2 was also apparent, with E2 levels declining in organotypic raft culture when 16E4 was absent. These results suggest that 16E4-mediated enhancement of genome amplification involves its cell cycle inhibition and cellular kinase activation functions, with E4 modifying the activity and function of viral replication proteins including E1. These activities of 16E4, and the different kinase patterns seen here with HPV18, 31 and 45, may reflect natural differences in the biology and tropisms of these viruses, as well as differences in E4 function.

Heterogeneity in Immune Marker Expression after Acquisition of Resistance to EGFR Kinase Inhibitors: Analysis of a Case with Small Cell Lung Cancer Transformation.

INTRODUCTION: Expression of immune markers is of scientific interest because of their potential roles as predictive biomarkers for immunotherapy. Although the microenvironment of metastatic tumors and/or therapy-inducible histological transformation may affect the expression of these immune markers, there are few data regarding this context. METHODS: A 76-year-old never-smoking female with EGFR-mutated lung adenocarcinoma (AC) acquired resistance to gefitinib. After her death, an autopsy revealed SCLC transformation and EGFR T790M secondary mutation (T790M) as mutually exclusive resistance mechanisms occurring differently in different metastases; two liver metastases (SCLC versus AC with T790M) and two lymph node metastases (SCLC versus AC with T790M) were analyzed to compare the expression status of immune markers by immunohistochemistry and by an immune oncology gene expression panel. RESULTS: Programmed death ligand 1 (PD-L1) protein was partially expressed in tumor cells with AC lesions (T790M) but not in tumor cells with SCLC transformation. The liver metastasis with SCLC transformation showed no stromal PD-L1 expression and scant tumor-infiltrating lymphocytes, whereas the other lesions demonstrated stromal PD-L1 staining and infiltration of CD8-positive T cells. Data generated using an immuno-oncology gene expression panel indicated a higher level of T-cell costimulatory molecules and lower expression of type I interferon-regulated genes in lesions with SCLC transformation. CONCLUSION: These data highlight the heterogeneity of expression of immune markers depending on the metastatic sites and histological transformation and indicate that the biopsy specimen from one lesion may not be representative of immune marker status for all lesions.

Carboplatin plus Weekly Paclitaxel Combined with Bevacizumab as First-line Treatment for Non-small Cell Lung Cancer.

AIM: We aimed to evaluate the efficacy and safety of carboplatin plus weekly paclitaxel with bevacizumab in patients with advanced non-squamous non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with stage IIIB/IV or postoperative recurrent NSCLC (n=33) were treated with carboplatin (area under the curve of 6) on day 1; paclitaxel (80 mg/m2) on days 1, 8, and 15; and bevacizumab (15 mg/kg) on day 1 repeated every 4 weeks, for four to six cycles; followed by maintenance bevacizumab (15 mg/kg) every 3 weeks. RESULTS: The overall response rate was 76%. The median progression-free survival and overall survival were 8.4 months and 22.2 months, respectively. Grade 3-4 toxicities included neutropenia in 55% of patients, anemia in 18%, febrile neutropenia in 12%, and anorexia in 9%. No treatment-related deaths were observed. CONCLUSION: Carboplatin plus weekly paclitaxel with bevacizumab was effective and well tolerated by patients with advanced NSCLC.

Heterogeneity of EGFR Aberrations and Correlation with Histological Structures: Analyses of Therapy-Naive Isogenic Lung Cancer Lesions with EGFR Mutation.

INTRODUCTION: EGFR gene somatic mutation is reportedly homogeneous. However, there are few data regarding the heterogeneity of expression of mutant EGFR protein and EGFR gene copy number, especially in extrathoracic lesions. These types of data may enhance our understanding of the biology of EGFR-mutated lung cancer and our understanding of the heterogeneous response patterns to EGFR TKIs. METHODS: An 81-year-old never-smoking female with lung adenocarcinoma could not receive any systemic therapy because of her poor performance status. After her death, 15 tumor specimens from different sites were obtained by autopsy. Expression of mutant EGFR protein and EGFR gene copy numbers were assessed by immunohistochemical analysis and by silver in situ hybridization, respectively. Heterogeneity in these EGFR aberrations was compared between metastatic sites (distant versus lymph node) or histological structures (micropapillary versus nonmicropapillary). RESULTS: All lesions showed positive staining for mutant EGFR protein, except for 40% of the papillary component in one of the pulmonary metastases (weak staining below the 1+ threshold). Expression of mutant-specific EGFR protein, evaluated by H-score, was significantly higher in the micropapillary components than in the nonmicropapillary components (Mann-Whitney U test, p = 0.014). EGFR gene copy number was quite different between lesions but not correlated with histological structure or metastatic form. However, EGFR gene copy numbers were similar between histological structures in each lesion. CONCLUSION: These data indicate that expression of EGFR mutant protein and EGFR gene copy number do not change as a consequence of tumor progression. This also justifies using the biopsy specimens from metastases as a surrogate for primary tumors.

Oncogene swap as a novel mechanism of acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer.

Mutant selective epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as rociletinib and AZD9291, are effective for tumors with T790M secondary mutation that become refractory to first-generation EGFR-TKI. However, acquired resistance to these prospective drugs is anticipated considering the high adaptability of cancer cells and the mechanisms remain largely obscure. Here, CNX-2006 (tool compound of rociletinib) resistant sublines were established by chronic exposure of HCC827EPR cells harboring exon 19 deletion and T790M to CNX-2006. Through the analyses of these resistant subclones, we identified two resistant mechanisms accompanied by MET amplification. One was bypass signaling by MET amplification in addition to T790M, which was inhibited by the combination of CNX-2006 and MET-TKI. Another was loss of amplified EGFR mutant allele including T790M while acquiring MET amplification. Interestingly, MET-TKI alone was able to overcome this resistance, suggesting that oncogenic dependence completely shifted from EGFR to MET. We propose describing this phenomenon as an "oncogene swap." Furthermore, we analyzed multiple lesions from a patient who died of acquired resistance to gefitinib, then found a clinical example of an oncogene swap in which the EGFR mutation was lost and a MET gene copy was gained. In conclusion, an "oncogene swap" from EGFR to MET is a novel resistant mechanism to the EGFR-TKI. This novel mechanism should be considered in order to avoid futile inhibition of the original oncogene.

Heterogeneity in resistance mechanisms causes shorter duration of epidermal growth factor receptor kinase inhibitor treatment in lung cancer.

OBJECTIVES: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are used as a first line therapy for metastatic lung cancer harboring somatic EGFR mutation. However, acquisition of resistance to these drugs is almost inevitable. T790M (threonine to methionine substitution at codon 790 of the EGFR gene) and MET amplification are well-known resistance mechanisms, and we previously demonstrated that three of six autopsied patients showed inter-tumor heterogeneity in resistance mechanisms by analyzing T790M and MET gene copy number (Suda et al., 2010). To further elucidate the role of heterogeneity in acquired resistance, here we performed further analyses including additional five patients. MATERIALS AND METHODS: We analyzed somatic mutations in 50 cancer-related genes for 26 EGFR-TKI refractory lesions from four autopsied patients using target sequencing. MET and ERBB2 copy numbers were analyzed by real-time PCR. Data for additional one patient was obtained from our recent study (Suda et al., 2015). Relationship between heterogeneity in resistance mechanism(s) and time to treatment failure (TTF) of EGFR-TKI and post-progression survival (PPS) were analyzed. RESULTS AND CONCLUSION: We observed heterogeneity of resistance mechanisms in two of four patients analyzed (T790M+MET gene copy number gain, and mutant EGFR loss+unknown). We also identified quantitative heterogeneity in EGFR T790M mutation ratio among EGFR-TKI refractory lesions. In analyzing patient outcomes, we found that patients who developed multiple resistance mechanisms had shorter TTF compared with those who developed single resistance mechanism (p=0.022). PPS after EGFR-TKI treatment failure was compatible between these two groups (p=0.42). These findings further our understanding of acquired resistance mechanisms to EGFR-TKIs, and may lead to better treatment strategies after acquisition of resistance to first generation EGFR-TKIs in lung cancer patients with EGFR mutations.