Efficacy of transperineal minimally invasive surgery with laparoscopic abdominoperineal excision for lower rectal cancer.

PURPOSE: In abdominoperineal excision (APE), the advantages of the "down-to-up" approach are expected to be more obvious when performed as a two-team approach, including transperineal minimally invasive surgery (TpMIS). We investigated the efficacy of TpMIS with laparoscopic APE for lower rectal cancer. METHODS: Patients who underwent laparoscopic APE with (n = 20) or without (n = 30) TpMIS between December 2013 and April 2020 were retrospectively reviewed. Patient and tumor characteristics, intraoperative outcome, short-term outcome, and pathological findings were compared. Additional subgroup analyses were performed in technically challenging cases, including male patients, obese patients, and patients with tumors located at the anterior wall. RESULTS: There was no marked difference in the patient or tumor characteristics or short-term outcomes, including morbidity and mortality between the two groups. Pathological results were comparable, and the circumferential resection margin (CRM) positive rate was 10% in both groups. TpMIS achieved a significant reduction in operative time (p = 0.02). In a subgroup analysis, the amount of blood loss was also smaller in males (p = 0.02) and patients with a high BMI (> 25) (p = 0.005) than in others. CONCLUSION: Simultaneously performing TpMIS and laparoscopic APE is feasible owing to the favorable complication and CRM-positive rates. In terms of operative time and blood loss, TpMIS is expected to be advantageous in both easy and challenging cases.

Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.

Cancer treatment, especially that for breast and lung cancer, has entered a new era and continues to evolve, with the development of genome analysis technology and the advent of molecular targeted drugs including tyrosine kinase inhibitors. Nevertheless, acquired drug resistance to molecular targeted drugs is unavoidable, creating a clinically challenging problem. We recently reported the antitumor effect of a pan-HER inhibitor, afatinib, against human epidermal growth factor receptor 2 (HER2)-amplified gastric cancer cells. The purpose of the present study was to identify the mechanisms of acquired afatinib resistance and to investigate the treatment strategies for HER2-amplified gastric cancer cells. Two afatinib-resistant gastric cancer cell lines were established from 2 HER2-amplified cell lines, N87 and SNU216. Subsequently, we investigated the molecular profiles of resistant cells. The activation of the HER2 pathway was downregulated in N87-derived resistant cells, whereas it was upregulated in SNU216-derived resistant cells. In the N87-derived cell line, both MET and AXL were activated, and combination treatment with afatinib and cabozantinib, a multikinase inhibitor that inhibits MET and AXL, suppressed the cell growth of cells with acquired resistance both in vitro and in vivo. In the SNU216-derived cell line, YES1, which is a member of the Src family, was remarkably activated, and dasatinib, a Src inhibitor, exerted a strong antitumor effect in these cells. In conclusion, we identified MET and AXL activation in addition to YES1 activation as novel mechanisms of afatinib resistance in HER2-driven gastric cancer. Our results also indicated that treatment strategies targeting individual mechanisms of resistance are key to overcoming such resistance.

Ganetespib in Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor-resistant Non-small Cell Lung Cancer.

BACKGROUND: The 90-kDa heat-shock protein (HSP90) is a chaperone protein expressed at high levels in cancer cells and is involved in the folding or stabilization of several client proteins, including epidermal growth factor receptor (EGFR). Ganetespib is a second-generation HSP90 inhibitor with a potent antitumor effect against various cancer types. MATERIALS AND METHODS: This study examined the antitumor effect of ganetespib in EGFR-mutant non-small cell lung cancer (NSCLC) cells and experimentally established EGFR-tyrosine kinase inhibitor (TKI)-resistant cells harboring various resistance mechanisms, including EGFR T790M mutation, met proto-oncogene amplification, and epithelial-mesenchymal transition. RESULTS: Ganetespib showed a potent antitumor effect at low concentrations, suppressing EGFR-related downstream pathway molecules and inducing cleavage of poly ADP-ribose polymerase in all examined EGFR-TKI-resistant cell lines in vitro. Ganetespib also inhibited in vivo tumor growth in resistant cells harboring EGFR T790M. CONCLUSION: Ganetespib might be a promising therapeutic option for the treatment of patients with EGFR-TKI-resistant NSCLC.

Anti-tumor effect of neratinib against lung cancer cells harboring HER2 oncogene alterations.

Human epidermal growth factor receptor 2 (HER2) is a member of the ErbB family of receptor tyrosine kinases. Numerous studies have reported the amplification and overexpression of HER2 in several types of cancer, including non-small cell lung cancer (NSCLC). However, the benefits of HER2-targeted therapy have not been fully established. In the present study, the anti-tumor effect of neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI), against NSCLC cells harboring HER2 alterations was investigated. The sensitivity of normal bronchial epithelial cells (BEAS-2B) ectopically overexpressing wild-type or mutant HER2 to neratinib was assessed. Furthermore, the anti-tumor activity of neratinib in several NSCLC cell lines harboring HER2 alterations was determined in vitro and in vivo, and the association between their genetic alterations and sensitivity to neratinib treatment was investigated. BEAS-2B cells ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, G660D and S310F) exhibited constitutive autophosphorylation of HER2, as determined by western blotting. While these BEAS-2B cells were sensitive to neratinib, they were insensitive to erlotinib, a first-generation epidermal growth factor receptor-TKI. Neratinib also exerted anti-proliferative effects on HER2-altered (H2170, Calu-3 and H1781) NSCLC cell lines. Neratinib was also demonstrated to exert strong tumor growth inhibitory activity in mouse xenograft models using HER2-altered lung cancer cells. The results of the present study strongly suggest that neratinib has potential as a promising therapeutic option for the treatment of HER2-altered NSCLC.

Application of amplicon-based targeted sequencing with the molecular barcoding system to detect uncommon minor EGFR mutations in patients with treatment-naïve lung adenocarcinoma.

BACKGROUND: In lung cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitizing mutations co-existing with rare minor EGFR mutations are known as compound mutations. These minor EGFR mutations can lead to acquired resistance after EGFR tyrosine kinase inhibitor treatment, so determining the mutation status of patients is important. However, using amplicon-based targeted deep sequencing based on next-generation sequencing to characterize mutations is prone to sequencing error. We therefore assessed the benefit of incorporating molecular barcoding with high-throughput sequencing to investigate genomic heterogeneity in treatment-naïve patients who have undergone resection of their non-small cell lung cancer (NSCLC) EGFR mutations. METHODS: We performed amplicon-based targeted sequencing with the molecular barcoding system (MBS) to detect major common EGFR mutations and uncommon minor mutations at a 0.5% allele frequency in fresh-frozen lung cancer samples. RESULTS: Profiles of the common mutations of EGFR identified by MBS corresponded with the results of clinical testing in 63 (98.4%) out of 64 cases. Uncommon mutations of EGFR were detected in seven cases (10.9%). Among the three types of major EGFR mutations, patients with the G719X mutation had a significantly higher incidence of compound mutations than those with the L858R mutation or exon 19 deletion (p = 0.0052). This was validated in an independent cohort from the Cancer Genome Atlas dataset (p = 0.018). CONCLUSIONS: Our findings demonstrate the feasibility of using the MBS to establish an accurate NSCLC patient genotype. This work will help understand the molecular basis of EGFR compound mutations in NSCLC, and could aid the development of new treatment modalities.

Activation of AXL as a Preclinical Acquired Resistance Mechanism Against Osimertinib Treatment in EGFR-Mutant Non-Small Cell Lung Cancer Cells.

Osimertinib (AZD9291) has an efficacy superior to that of standard EGFR-tyrosine kinase inhibitors for the first-line treatment of patients with EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, patients treated with osimertinib eventually acquire drug resistance, and novel therapeutic strategies to overcome acquired resistance are needed. In clinical or preclinical models, several mechanisms of acquired resistance to osimertinib have been elucidated. However, the acquired resistance mechanisms when osimertinib is initially used for EGFR-mutant NSCLC remain unclear. In this study, we experimentally established acquired osimertinib-resistant cell lines from EGFR-mutant NSCLC cell lines and investigated the molecular profiles of resistant cells to uncover the mechanisms of acquired resistance. Various resistance mechanisms were identified, including the acquisition of MET amplification, EMT induction, and the upregulation of AXL. Using targeted next-generation sequencing with a multigene panel, no secondary mutations were detected in our resistant cell lines. Among three MET-amplified cell lines, one cell line was sensitive to a combination of osimertinib and crizotinib. Acquired resistance cell lines derived from H1975 harboring the T790M mutation showed AXL upregulation, and the cell growth of these cell lines was suppressed by a combination of osimertinib and cabozantinib, an inhibitor of multiple tyrosine kinases including AXL, both in vitro and in vivo. Our results suggest that AXL might be a therapeutic target for overcoming acquired resistance to osimertinib. IMPLICATIONS: Upregulation of AXL is one of the mechanisms of acquired resistance to osimertinib, and combination of osimertinib and cabozantinib might be a key treatment for overcoming osimertinib resistance.

Comparative mutational evaluation of multiple lung cancers by multiplex oncogene mutation analysis.

In patients presenting with synchronous or metachronous multiple lung cancer (MLC), it is important to distinguish between multiple primary lung cancer (MP) and intrapulmonary metastasis (IM). The present study was aimed at investigating the mutational profiles of synchronous/metachronous MLC and to compare the classification of paired tumors by multiplex gene mutation analysis with the histopathological evaluation. We carried out targeted sequencing of 20 lung cancer-related oncogenes using next-generation sequencing (NGS) in 82 tumors from 37 MLC patients who underwent surgical resection at our department. The patients were diagnosed as MP or IM cases based on the Martini and Melamed criteria, histopathological and gene mutational evaluations. Matching mutations between paired tumors was observed in 20 (54%) patients, who were diagnosed as IM cases by mutational evaluation. Patients who could not be clearly diagnosed by histopathological evaluation were classified as equivocal cases. Among the histopathological IM cases (n = 7), six (86%) were confirmed as IM cases also by mutational evaluation, and most of the paired tumors of these cases (n = 5) harbored multiple matching mutations. Among the histopathological MP cases (n = 17), mutational evaluation yielded a discordant diagnosis in eight (47%) cases. Of these, the paired tumors of four cases harbored multiple matching mutations, suggesting that the mutational diagnosis might be more suitable in these patients. Our findings suggest that multiplex mutational analysis could be a useful complementary tool for distinguishing between MP and IM in addition to histopathological evaluation.

Successful wound treatment using negative pressure wound therapy without primary closure in a patient undergoing highly contaminated abdominal surgery.

BACKGROUND: The indications for negative pressure wound therapy (NPWT) continue to expand, and NPWT has become a powerful tool for the treatment of interactive wounds. Recently, the use of NPWT over closed incisions has been shown to prevent surgical site infection (SSI) in patients undergoing contaminated or acute care surgery as prophylactic NPWT. In this article, we present our successful experience using NPWT without primary skin closure for wound treatment after a highly contaminated enterological surgery. The procedure we present in this case report is considerably different from the conventional prophylactic NPWT and a novel method in the field of gastrointestinal surgery. CASE PRESENTATION: A 33-year-old man with Crohn's disease underwent a dirty, infected enterological surgical procedure for the treatment of abdominal wall abscess and multiple fistulas around his colonic stoma. The stoma reconstruction and wound debridement resulted in a broad skin defect, and the incision was strategically left open. In addition to the infected wound condition (class IV), Crohn's disease itself is a risk factor for SSI; consequently, we induced NPWT immediately after the surgery and closed the incision from both ends in a stepwise manner using sutures each time we changed the dressing. This procedure was effective, enabling complete healing and closure at the surgical site on postoperative day 14 without infection or a skin defect. CONCLUSION: For highly contaminated enterological surgery, purposely leaving the incision open and starting NPWT immediately after the procedure is an effective strategy for early wound closure and the prevention of SSI.

Combined inhibition of MEK and PI3K pathways overcomes acquired resistance to EGFR-TKIs in non-small cell lung cancer.

Compensatory activation of the signal transduction pathways is one of the major obstacles for the targeted therapy of non-small cell lung cancer (NSCLC). Herein, we present the therapeutic strategy of combined targeted therapy against the MEK and phosphoinositide-3 kinase (PI3K) pathways for acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in NSCLC. We investigated the efficacy of combined trametinib plus taselisib therapy using experimentally established EGFR-TKI-resistant NSCLC cell lines. The results showed that the feedback loop between MEK/ERK and PI3K/AKT pathways had developed in several resistant cell lines, which caused the resistance to single-agent treatment with either inhibitor alone. Meanwhile, the combined therapy successfully regulated the compensatory activation of the key intracellular signals and synergistically inhibited the cell growth of those cells in vitro and in vivo. The resistance mechanisms for which the dual kinase inhibitor therapy proved effective included (MET) mesenchymal-epithelial transition factor amplification, induction of epithelial-to-mesenchymal transition (EMT) and EGFR T790M mutation. In further analysis, the combination therapy induced the phosphorylation of p38 MAPK signaling, leading to the activation of apoptosis cascade. Additionally, long-term treatment with the combination therapy induced the conversion from EMT to mesenchymal-to-epithelial transition in the resistant cell line harboring EMT features, restoring the sensitivity to EGFR-TKI. In conclusion, our results indicate that the combined therapy using MEK and PI3K inhibitors is a potent therapeutic strategy for NSCLC with the acquired resistance to EGFR-TKIs.

Prolonged Epigastric Pain Caused by a Leaf Stem Embedded in the Gastric Wall.

A 67-year-old woman underwent esophagogastroduodenoscopy to determine the cause of her prolonged epigastric pain. During the examination, a sharp-pointed foreign body was observed; the edge of the object had been embedded in the gastric wall. The object was removed via an endoscope, and the patients' symptoms improved immediately. Based on a dietary history and an electron microscope examination, we identified the object as a stem of mizuna, a potherb mustard. Our report indicates that commonly eaten leafy green vegetables can act as sharp-pointed foreign bodies with the potential to injure the upper gastrointestinal tract.

Therapeutic strategies for afatinib-resistant lung cancer harboring HER2 alterations.

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non-small-cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan-HER inhibitor afatinib could be a useful therapeutic agent as HER2-targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib-resistant cell lines from NSCLC cell lines harboring HER2 alterations, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib-resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial-to-mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)-like features. The afatinib-resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines which showed EMT or had acquired CSC-like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.

Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer.

Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-human epidermal growth factor receptor 2 (HER2) drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. In this study, we propose novel treatment options for patients with HER2-positive gastric cancer. First, we determined the molecular profiles of 12 gastric cancer cell lines, and examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines. Additionally, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. In the drug sensitivity analysis, both afatinib and neratinib produced an antitumor effect in most of the HER2-amplified cell lines. However, some cells were not sensitive to the drugs. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene that inhibits the activation of insulin-like growth factor-1 receptor (IGF-1R), were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, showed a notable synergistic effect. Among 123 clinical samples, we found 19 cases of HER2 amplification and three cases of oncogenic mutations. In conclusion, afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer.

Therapeutic potential of targeting S100A11 in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with an unfavorable prognosis. The standard therapeutic approaches are limited to surgery, chemotherapy, and radiotherapy. Because the consequent clinical outcome is often unsatisfactory, a different approach in MPM treatment is required. S100A11, a Ca2+-binding small protein with two EF-hands, is frequently upregulated in various human cancers. Interestingly, it has been found that intracellular and extracellular S100A11 have different functions in cell viability. In this study, we focused on the impact of extracellular S100A11 in MPM and explored the therapeutic potential of an S100A11-targeting strategy. We examined the secretion level of S100A11 in various kinds of cell lines by enzyme-linked immunosorbent assay. Among them, six out of seven MPM cell lines actively secreted S100A11, whereas normal mesothelial cell lines did not secrete it. To investigate the role of secreted S100A11 in MPM, we inhibited its function by neutralizing S100A11 with an anti-S100A11 antibody. Interestingly, the antibody significantly inhibited the proliferation of S100A11-secreting MPM cells in vitro and in vivo. Microarray analysis revealed that several pathways including genes involved in cell proliferation were negatively enriched in the antibody-treated cell lines. In addition, we examined the secretion level of S100A11 in various types of pleural effusions. We found that the secretion of S100A11 was significantly higher in MPM pleural effusions, compared to others, suggesting the possibility for the use of S100A11 as a biomarker. In conclusion, our results indicate that extracellular S100A11 plays important roles in MPM and may be a therapeutic target in S100A11-secreting MPM.

Yes1 signaling mediates the resistance to Trastuzumab/Lap atinib in breast cancer.

BACKGROUND: Overexpression of human epidermal growth factor receptor 2 (HER2) is observed in approximately 15-23% of breast cancers and these cancers are classified as HER2-positive breast cancer. Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer and has improved patient overall survival. However, acquired resistance to trastuzumab is still a critical issue in breast cancer treatment. We previously established a trastuzumab-resistant breast cancer cell line (named as BT-474-R) from a trastuzumab-sensitive HER2-amplified cell line BT-474. Lapatinib is also a molecular-targeted drug for HER2-positive breast cancer, which acquired the resistance to trastuzumab. Acquired resistance to lapatinib is also an issue to be conquered. METHODS: We established trastuzumab/lapatinib-dual resistant cell line (named as BT-474-RL2) by additionally treating BT-474-R with lapatinib. We analyzed the mechanisms of resistance to trastuzumab and lapatinib. Besides, we analyzed the effect of the detected resistance mechanism in HER2-positive breast cancer patients. RESULTS: Proto-oncogene tyrosine-protein kinase Yes1, which is one of the Src family members, was amplified, overexpressed and activated in BT-474-R and BT-474-RL2. Silencing of Yes1 by siRNA induced both BT-474-R and BT-474-RL2 to restore the sensitivity to trastuzumab and lapatinib. Pharmaceutical inhibition of Yes1 by the Src inhibitor dasatinib was also effective to restore the sensitivity to trastuzumab and lapatinib in the two resistant cell lines. Combination treatment with dasatinib and trastuzumab induced down-regulation of signaling molecules such as HER2 and Akt. Moreover, the combination treatments induced G1-phase cell-cycle arrest and apoptosis. Consistent with cell line data, high expression of Yes1 mRNA was correlated with worse prognosis in patients with HER2-positive breast cancer. CONCLUSION: Yes1 plays an important role in acquired resistance to trastuzumab and lapatinib in HER2-positive breast cancer. Our data suggest that pharmacological inhibition of Yes1 may be an effective strategy to overcome resistance to trastuzumab and lapatinib.

Ultrastructural analysis of an enterolith composed of deoxycholic acid.

A 67-year-old Japanese man underwent enterotomy because of enterolith ileus. Component analysis by infrared spectroscopy revealed that the enterolith was composed of a high concentration of deoxycholic acid. We further analyzed and compared the ultrastructure of the enterolith and a commercially available powdered form of deoxycholic acid by means of scanning electron microscopy and energy dispersive X-ray spectroscopy. Energy dispersive X-ray spectroscopy analysis revealed that the ratios of carbon and oxygen in the enterolith were equal to those in the deoxycholic acid powder. Scanning electron microscopy analysis showed rectangular prism-shaped particles on the surface of the enterolith. This structure was similar to that of the deoxycholic acid powder. The surgically removed enterolith had a twisted and coiled appearance. Possible mechanisms underlying the formation of this unique form are discussed.

Temperature- and time-dependent changes in TLR2-activated microglial NF-κB activity and concentrations of inflammatory and anti-inflammatory factors.

PURPOSE: Therapeutic hypothermia protects neurons following injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in pathological processes in sterile CNS injury. We have examined the effects of culture temperature on the TLR2-activated microglial production of cytokines and nitric oxide (NO), which are known to be associated with CNS damage, and the possible involvement of nuclear factor-κB (NF-κB) activation underlying such effects. METHODS: Rat microglia were cultured with a selective TLR2 agonist, Pam(3)CSK(4), under hypothermic, normothermic, and hyperthermic conditions, and with Pam(3)CSK(4) in the presence of a NF-κB activation inhibitor at 37 °C. Cytokine and NO levels and NF-κB p65 activation were measured. RESULTS: The production of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), and NO and the activation of NF-κB p65 were reduced by hypothermia, but augmented by hyperthermia at 3-6, 24-48, 48, and 0.5 h, post-treatment initiation, respectively. Pharmacological inhibition of NF-κB activation impaired the Pam(3)CSK(4)-induced TNF-α, IL-10, and NO production. CONCLUSIONS: In TLR2-activated microglia, hypothermia reduced, while hyperthermia increased, the early activation of NF-κB and the subsequent NF-κB-mediated production of TNF-α, IL-10, and NO in a time-dependent manner, suggesting that attenuation of these factors via suppression of NF-κB in microglia is one possible neuroprotective mechanism of therapeutic hypothermia. Moreover, temperature-dependent changes in microglial TNF-α production during the early phase and IL-10 and NO production during the late phase indicate that these factors might be useful as clinical markers to monitor hypothermia-related neuronal protection and hyperthermia-related neuronal injury.