Phase II study of the efficacy and safety of lenvatinib for anaplastic thyroid cancer (HOPE).

PURPOSE: Anaplastic thyroid cancer (ATC) is a rare and highly aggressive cancer for which effective systemic therapy has long been sought. Here, we assessed the efficacy and safety of lenvatinib in patients with unresectable ATC. PATIENTS AND METHODS: The study was investigator-initiated and conducted under a multicenter, open-label, nonrandomized, phase II design. Eligibility criteria included pathologically proven ATC; unresectable measurable lesion as defined by RECIST 1.1; age 20 years or older; ECOG PS 0-2; and adequate organ function. The primary end-point was overall survival. Secondary end-points were progression-free survival, objective response rate, disease control rate, clinical benefit rate, and safety. RESULTS: Of 52 patients enrolled from 17 institutions, 42 patients who were confirmed to have ATC were included for efficacy analysis, and 50 patients were included for safety analysis. The estimated 1-year overall survival rate was 11.9% (95% CI, 4.4%-23.6%). One patient (2.4%) achieved complete response, four patients (9.5%) partial response, and 26 patients (61.9%) stable disease, including nine patients (21.4%) who demonstrated durable stable disease, giving an objective response rate of 11.9%, disease control rate of 73.8%, and clinical benefit rate of 33.3%. Adverse events of any grade were observed in 45 patients (90.0%), the most common of which of any grade included loss of appetite (48.0%), fatigue (48.0%), hypertension (44.0%), and palmar-plantar erythrodysesthesia syndrome (26.0%). CONCLUSION: Lenvatinib treatment resulted in disappointing survival for unresectable ATC patients. Although the number of responders was small, responses were durable, indicating that lenvatinib may be beneficial for selected patients. Further investigation to identify suitable candidates for lenvatinib monotherapy is needed.

Role of debulking surgery in combination with immune therapy: A successfully treated case of locally advanced mucosal melanoma.

Immune checkpoint inhibitors (ICIs) have markedly changed the treatment landscape for melanoma; however, their efficacy and applications are currently limited and medical requirements remain unmet. The present case study reports on a 85-year-old female patient who visited our outpatient clinic with a 1-month history of a buccal mucosa mass and was diagnosed with locally advanced mucosal melanoma of the head and neck. The patient's tumor progressed right after the administration of nivolumab, compromising oral intake. Palliative debulking surgery was performed. Subsequently, the other part of the melanoma on the hard palate slightly decreased in size without forming new lesions for more than one year after surgery. The present case exemplifies that tumor volume reduction surgery may increase the response to ICI and may prolong the duration of response. This combination therapy may be more effective in patients whose tumors increase in size after administration of ICIs or whose tumor is already large at the beginning of treatment. The combination of ICIs and debulking surgery may become an important treatment option in the future for locally advanced mucosal melanoma.

Factors predictive of the development of hypothyroidism after intensity-modulated radiation therapy for pharyngeal cancer.

BACKGROUND: Hypothyroidism is a common adverse event after radiotherapy for head and neck tumors and the incidence need to be re-evaluated because of using intensity-modulated radiotherapy (IMRT). AIMS/OBJECTIVES: Confirm the dose-volume effect of IMRT for pharyngeal cancer on hypothyroidism. MATERIALS AND METHODS: This was a retrospective analysis of patients underwent IMRT for pharyngeal cancer from June 2011 to May 2018. Patients were classified into group A (thyroid stimulating hormone (TSH) <5µU/ml), group B (5< =TSH < 10), and group C (10< =TSH) based on TSH over 36 months post-radiation. Radiation dose, thyroid volume, and the proportion of the thyroid that received X Gy or greater (Vx) were measured. RESULTS: Fifty-two patients were included in this work. Hypothyroidism developed in 33/52 (63%) patients, 13 in group B and 20 in group C. The mean radiation dose to the thyroid was 49.4 Gy and the median time until hypothyroidism was 39 months after irradiation. Hypothyroidism was significantly related to neck dissection (ND) and radiation dose to the thyroid. Patients whose thyroid received 45 Gy or more (V45) >67% had a significantly higher incidence of hypothyroidism. CONCLUSIONS AND SIGNIFICANCE: Patients with pharyngeal cancer who had ND and V45 to the thyroid >67% are at risk of hypothyroidism.

Docetaxel plus cisplatin in recurrent and/or metastatic non-squamous-cell head and neck cancer: a multicenter phase II trial.

The clinical utility of systemic therapy and genomic profiling in non-squamous-cell head and neck cancer (NSCHNC) has not been fully elucidated. This phase II trial evaluated the efficacy and safety of docetaxel and cisplatin combination in the first-line setting. Eligibility criteria were recurrent and/or metastatic NSCHNC; progressive disease within the last 6 months; no prior systemic therapy; and ECOG performance status of 0-1. Patients received docetaxel (75 mg/m2 on day 1) and cisplatin (75 mg/m2 on day 1), repeated every 21 days for 6 cycles. The primary endpoint was confirmed objective response rate (ORR). The secondary endpoints included progression-free survival (PFS), overall survival (OS), and adverse events. Next-generation sequencing (NGS) was performed using the Ion AmpliSeq Cancer Hotspot Panel v2. Twenty-three patients were enrolled from November 2012 to October 2016, of whom 8 were male. Median age was 57 years. Ninety-six percent of cases were metastatic. Among 22 evaluable patients, confirmed ORR was 45% (95% confidential interval 24-68%). With a median follow-up period of 18.8 months, median PFS and OS were 6.7 and 20.1 months, respectively. Grade 3/4 adverse events included febrile neutropenia (39%) and anemia (22%). No treatment-related deaths were observed. NGS analysis revealed potential treatment targets, including ERBB2, KIT, and ALK. The docetaxel and cisplatin combination regimen can be considered a new treatment option in recurrent and/or metastatic NSCHNC, although primary prophylaxis for febrile neutropenia should be considered. Diverse genomic alterations may lead novel treatment options.This trial was registered with the UMIN Clinical Trials Registry as UMIN000008333 on [September 1st, 2012].

A study of 24 cases of salivary gland carcinoma with distant metastasis.

Salivary gland carcinoma is a relatively rare disease of the head and neck. Although it frequently presents with distant metastases, few reports have been published on this subject. The present study investigated the prognosis of patients with distant metastases from salivary gland cancer. A total of 24 cases of salivary gland carcinoma with distant metastasis who were initially treated at the Department of Otolaryngology-Head and Neck Surgery of Nara Medical University during a 16-year period from August 2004 to July 2020 were included. The histopathological types included salivary duct carcinoma (8 cases), adenoid cystic carcinoma (6 cases), myoepithelial carcinoma (3 cases), Squamous cell carcinoma (2 cases), adenocarcinoma (2 cases), acinic cell carcinoma (2 cases) and mucoepidermoid carcinoma (1 case). A total of 18 patients had stage IV carcinoma, which represented the majority. Of all patients, ~80% developed distant metastases within 2 years of initial diagnosis. Survival rates after the appearance of distant metastases were 43.5% at 5 years and 14.5% at 10 years. The results of the current study revealed that no factors significantly influenced long-term prognosis after the development of distant metastases. In future, it may be necessary to re-examine these results in a larger sample size and standardise treatment methods as a result.

A study of 17 cases for the identification of prognostic factors for anaplastic thyroid carcinoma.

Anaplastic thyroid cancer (ATC) has a poor prognosis. ATC accounts for only 1-2% of all thyroid carcinomas, yet it is one of the most lethal neoplasms in humans. Notably, there are no established treatment protocols for ATC. The present study investigated the prognostic and predictive factors of ATC. A retrospective analysis was conducted on 17 patients with histologically confirmed ATC. The median overall survival of all patients was 3.8 months. In patients under the age of 70 years, the statistically significant prognostic factors indicating longer survival were the absence of distant metastasis and treatment by radical resection. Furthermore, in contrast to previous findings, tumor size and white blood cell count were not associated with ATC prognosis in the present cohort. Importantly, tracheostomy did not contribute to improvement of prognosis and should perhaps not be considered, when unnecessary, to preserve the patient's quality of life. Prognostic factors for ATC are critical to clinicians to enable them to determine which patients will benefit from aggressive treatment strategies, as opposed to supportive care.

Optimization of therapeutic strategy for p16-positive oropharyngeal squamous cell carcinoma: Multi-institutional observational study based on the national Head and Neck Cancer Registry of Japan.

BACKGROUND: Although the American Joint Committee on Cancer TNM classification has been amended to include human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) as an independent entity, to the authors' knowledge the optimized de-escalating treatment modality has not been established to date. METHODS: The authors conducted a retrospective, nationwide, observational study in patients with HPV-related OPSCC who were treated from 2011 to 2014 in Japan to determine the best treatment modality. RESULTS: A total of 688 patients who were newly diagnosed with HPV-related OPSCC who were treated with curative intent at 35 institutions and had coherent clinical information and follow-up data available were included in the current study. In patients with T1-T2N0 disease (79 patients), both the 3-year recurrence-free survival and overall survival (OS) rates were 100% in the group treated with radiotherapy (RT) as well as the group receiving concurrent chemoradiotherapy (CCRT). The 3-year OS rates were 94.4% (for patients with T1N0 disease) and 92.9% (for patients with T2N0 disease) among the patients treated with upfront surgery. In patients with stage I to stage II HPV-related OPSCC, the 5-year recurrence-free survival and OS rates were 91.4% and 92%, respectively, in the patients treated with CCRT with relatively high-dose cisplatin (≥160 mg/m2 ; 114 patients) and 74.3% and 69.5%, respectively, in the patients treated with low-dose cisplatin (<160 mg/m2 ; 17 patients). CONCLUSIONS: Despite it being a retrospective observational trial with a lack of information regarding toxicity and morbidity, the results of the current study demonstrated that patients with T1-T2N0 HPV-related OPSCC could be treated with RT alone because of the equivalent outcomes of RT and CCRT, and patients with stage I to stage II HPV-related OPSCC other than those with T1-T2N0 disease could be treated with CCRT with cisplatin at a dose of ≥160 mg/m2 .

Evaluation of the 8th Edition TNM Classification for Anaplastic Thyroid Carcinoma.

BACKGROUND: The tumor-node-metastasis (TNM) classification system to categorized anaplastic thyroid cancer (ATC) was revised. METHODS: The revised system was evaluated using a large database of ATC patients. RESULTS: A total of 757 patients were analyzed. The proportion and median overall survival values (OS: months) for each T category were T1 (n = 8, 1.1%, 12.5), T2 (n = 43, 5.7%, 10.9), T3a (n = 117, 15.5%, 5.7), T3b (n = 438, 57.9%, 3.9), and T4 (n = 151, 19.9%, 5.0). The OS of the N0 and N1 patients were 5.9 and 4.3, respectively (log-rank p < 0.01). Sixty-three (58.3%) patients migrated from stage IV A to IV B by revision based on the existence of nodal involvement and 422 patients (55.7%) were stratified into stage IV B, without a worsening of their OS (6.1), leaving 45 patients (5.9%) in stage IV A with fair OS (15.8). The hazard ratios for the survival of the patients of stage IV B compared to stage IV A increased from 1.1 to 2.1 by the revision. No change was made for stage IV C (n = 290, 38.8%, 2.8). CONCLUSION: The revised TNM system clearly indicated the prognoses of ATC patients by extracting rare patients with fair prognoses as having stage IV A disease and categorized many heterogeneous patients in stage IV B.

Use of ALK Immunohistochemistry for Optimal Therapeutic Strategy of Pulmonary Large-cell Neuroendocrine Carcinoma and Identification of a Novel KIF5B-ALK Fusion Oncokinase.

BACKGROUND: Patients with adenocarcinoma of the lung are routinely screened for anaplastic lymphoma kinase (ALK) rearrangement because they can be treated by ALK-specific targeted therapy. The clinical and molecular characteristics of large-cell neuroendocrine carcinoma (LCNEC) associated with ALK rearrangement are still unclear. Herein, we assessed the ALK status in a series of patients with LCNEC by testing methods commonly used for adenocarcinoma. MATERIALS AND METHODS: ALK expression was first examined by immunohistochemistry. For a positively stained tumor, molecular analyses were then conducted. The ALK fusion partner found in a patient with ALK rearrangement was further identified by direct DNA sequencing. Patient clinicopathological features were also analyzed, focusing on the ALK rearrangement-positive case. RESULTS: Immunohistochemistry of seven patients identified strong ALK expression in one case of stage IV LCNEC. Molecular analysis identified a novel rearranged gene resulting from the fusion of kinesin family member 5B (KIF5B) exon 17 to ALK exon 20. The patient was treated with ALK-specific inhibitors, crizotinib and later, alectinib, and has remained alive for more than 24 months without disease progression. Three of the remaining six patients without ALK rearrangement had stage IV cancer and received cytotoxic chemotherapies. Their average overall survival was 5.4 months. CONCLUSION: To our knowledge, this is the first report of a KIF5B-ALK fusion gene in LCNEC. The patient was successfully treated with ALK inhibitors, suggesting that sensitivity to ALK inhibitor may define a specific LCNEC subtype. We propose that screening for ALK rearrangement in patients with LCNEC may assist in selecting potential candidates for targeted therapy.

Snail-induced EMT promotes cancer stem cell-like properties in head and neck cancer cells.

Epithelial-mesenchymal transition (EMT) is a key process involved in the invasion and metastasis of cancer cells. Furthermore, EMT can induce a cancer stem cell (CSC)-like phenotype in a number of tumor types. We demonstrated that Snail is one of the master regulators that promotes EMT and mediates cancer cell migration and invasion in many types of malignancies including head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated the role of Snail in inducing and maintaining CSC-like properties through EMT in HNSCC. We established HNSCC cell lines transfected with Snail. Stem cell markers were evaluated with real-time RT-PCR and western blot analysis. CSC properties were assessed using sphere formation and WST-8 assays as well as chemosensitivity and chick chorioallantoic membrane in vivo invasion assays. Introduction of Snail induced EMT properties in HNSCC cells. Moreover, Snail-induced EMT maintained the CSC-like phenotype, and enhanced sphere formation capability, chemoresistance and invasive ability. These data suggest that Snail could be one of the critical molecular targets for the development of therapeutic strategies for HNSCC.

Prognostic classification of thyroid follicular cell tumors using Ki-67 labeling index: risk stratification of thyroid follicular cell carcinomas.

This review emphasizes that the so-called high-risk thyroid carcinoma is not a distinct tumor entity, but a group of tumors with different histologies. High-grade histological features, such as tumor necrosis, increased mitoses, and nuclear pleomorphism, together with high Ki-67 labeling index (more than 10%), are good indicators of high-risk thyroid carcinoma and suggest a possible risk for anaplastic transformation. This review proposes the stratification of patients with thyroid carcinoma into low-, moderate-, and high-risk groups based on Ki-67 labeling index, which should be useful for the clinical management of patients, even after initial surgery. Currently, both the aggressive variant of papillary carcinoma and poorly differentiated carcinoma are aggressively treated by a completion of total thyroidectomy with prophylactic lymph node dissection followed by radioactive iodine treatment. Therefore, patients with moderate-risk or high-risk thyroid carcinoma based on Ki-67 labeling index should also be considered candidates for this treatment strategy.

A method of transoral finger dissection for a giant epiglottic lipoma.

Background. Subcutaneous lipomas that occur in the trunk and proximal extremities are commonly dissected by low-invasive method. However, a standard surgical method for lipomas of the epiglottis has been absent. Microscopic laryngeal surgery is appropriate to extirpate small epiglottic lipomas. However, microscopic laryngeal surgery may be insufficient for giant epiglottic lipomas because there is restricted visualization of the operating field of the tumor under the microscope. Furthermore, microscopic surgical instruments are very small to manipulate giant lipomas, and it would be excessive to approach these lipomas via external cervical incisions. Case Presentation. A 57-year-old female presented with a giant lipoma on the lingual surface of the epiglottis. Following a tracheotomy, microscopic surgery was inadequate to manipulate the epiglottic lipoma. Instead, we performed macroscopic surgery in which the epiglottic lipoma was pulled into the oral cavity with forceps and then separated from the surrounding tissues using the surgeon's finger to dissect the tumor en bloc. Conclusion. The low-invasive method of transoral finger dissection enabled the giant lipoma to be extirpated without leaving any remnants or causing excessive epiglottic damage.

Prevalence of human papillomavirus in oropharyngeal cancer: a multicenter study in Japan.

BACKGROUND: The incidence rates of oropharyngeal squamous cell carcinoma (OPSCC) have risen steadily in the USA and in northern Europe. These increases are thought to be a consequence of persistent infection with high-risk human papillomavirus (HPV) in OPSCC patients. HPV is an emerging etiologic factor in OPSCC. In Japan, the incidence of OPSCC has significantly increased over the last three decades. However, the population of HPV-positive OPSCC patients is currently unknown. We examined the nationwide trends with regard to HPV incidence in OPSCC patients at 21 specific sites, and examined the relationship between the presence of HPV and survival in OPSCC patients in Japan. METHODS: Tumor samples were obtained from patients with OPSCC prior to treatment, and HPV infection was investigated by polymerase chain reaction (PCR). Hybrid Capture 2 (HC2) was also adopted for swab examination on the surface of fresh tumors. RESULTS: HPV was detected by PCR in 79 (50.3%) out of 157 OPSCC patients. The clinical features of HPV-positive OPSCC were low differentiation, a tendency to involve the lateral wall, and high nodal staging. The sensitivity and specificity of HC2 were 93.7 and 96.2%, respectively, indicating its utility as a screening test. HPV-positive patients had significantly better overall survival and disease-free survival than HPV-negative patients.

Snail-induced epithelial-mesenchymal transition promotes cancer stem cell-like phenotype in head and neck cancer cells.

Head and neck squamous cell carcinoma (HNSCC) is known to have a poor prognosis. The resistance to treatment and distant metastasis are important clinical problems in HNSCC. The epithelial-mesenchymal transition (EMT) is a key process in successful execution of many steps such as the invasion and metastasis for cancer cells. Snail is one of the master regulators that promote EMT in many types of malignancies including HNSCC. Recently, it has been shown that Snail-induced EMT could induce a cancer stem cell (CSC)­like phenotype in a number of tumor types. In this study, we investigated the role of Snail in inducing EMT properties and CSC-like phenotype in HNSCC. We established HNSCC cell lines transfected with Snail. E-cadherin was analyzed using western blot analysis and immunofluorescence staining. Cell migration and invasion were assessed using wound-healing assay and modified Boyden chamber assay, respectively. CSC markers of HNSCC, CD44 and aldehyde dehydrogenase 1 (ALDH1), were also evaluated with western blot analysis, and chemosensitivity was assessed with WST-8 assay. Introduction of Snail induced EMT properties in HNSCC cells and enhanced cell migration and invasion. Moreover, Snail-induced EMT gained CSC-like phenotype and was associated with increased chemoresistance. These results suggest that Snail could be one of the attractive targets for the development of therapeutic strategies in HNSCC.

Heparin-binding EGF-like growth factor enhances the activity of invasion and metastasis in thyroid cancer cells.

Thyroid cancer sometimes contains poorly differentiated components, which have the potential of invasion and metastasis. We evaluated the possible roles of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, in cell growth and invasion of thyroid cancer cells, and demonstrated that HB-EGF is not only a potent mitogen but also a chemotactic factor in the thyroid cancer cells 8305C and SW579. The HB-EGF-mediated chemotaxis was inhibited by neutralizing antibody against the EGF receptor (EGFR/HER1/ErbB1) or tyrphostin AG1478, a specific inhibitor of the EGFR tyrosine kinase. The HB-EGF mRNA and protein expression was also analyzed using RT-PCR and immunofluorescence methods, respectively. In addition, in clinical immunohistochemical study, increased expression of HB-EGF and its receptors, HER1 and EGFR4 (HER4/ErbB4), was observed in thyroid carcinoma cells. Our findings suggest that HB-EGF acts as a potent paracrine and/or autocrine chemotactic factor as well as a mitogen that mediates HER1 and/or HER4 in the invasion and metastasis of thyroid carcinoma cells, including poorly differentiated papillary carcinomas or undifferentiated/anaplastic carcinomas. These data may aid in the development of novel therapeutic strategies for thyroid cancer.

Expression of REG III and prognosis in head and neck cancer.

Identification of a reliable biomarker for predicting prognosis in head and neck cancers is highly desirable and has long been sought. There have been several reports that members of the regenerating gene (REG) family are highly expressed in chronic inflammation and in tumors of the digestive organs. In addition, it has been described in several reports that REG expression is associated with the progression of digestive cancers. In the present study, we evaluated the effect of REG expression on the prognosis of hypopharyngeal cancer. We investigated 37 cases with hypopharyngeal squamous cell carcinoma, determined REG mRNA expression, which is easily detected in formalin-fixed paraffin-embedded tissues using the real-time reverse transcriptase-polymerase chain reaction method, and evaluated the survival rate using the Kaplan-Meier method. According to these results, REG III mRNA expression was significantly associated with prolonged survival. Therefore, we constructed hypopharyngeal cancer cell lines transfected with REG III and assessed the cell proliferation and chemosensitivity and/or radiosensitivity in vitro. Cells transfected with REG III exhibited significantly lower cell proliferation and higher chemosensitivity and/or radiosensitivity compared with the control cells. These data suggest that REG III may be a reliable biomarker of prognosis in hypopharyngeal cancer. This is the first report concerning the association of REG III expression and the prognosis of head and neck squamous cell carcinoma including hypopharyngeal cancer.

Esophageal perforation caused by a fish bone treated with surgically indwelling drainage and fibrin glue injection for fistula formation.

We herein report a case of thoracic esophageal perforation caused by a fish bone. The patient was a 68-year-old female who presented with a persistent sore throat after eating sea bream four days previously. She was diagnosed with an esophageal perforation and posterior mediastinal abscess formation by chest computed tomography and inflammatory findings in her blood test. Surgically indwelling drainage was able to effectively control the leakage of contaminants and infection. Endoscopic injection of fibrin glue into the long-standing thoracic-esophageal fistula promoted closure of the esophageal wall defect and enabled her to restart oral intake. This case report suggests that effective drainage and the use of fibrin glue sealant may be one of the treatment options for esophageal perforation.

Low-intensity ultrasound enhances the anticancer activity of cetuximab in human head and neck cancer cells.

The potential clinical use of ultrasound in inducing cell apoptosis and enhancing the effects of anticancer drugs in the treatment of cancers has previously been investigated. In this study, the combined effects of low-intensity ultrasound (LIU) and cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody, on cell killing and induction of apoptosis in HSC-3 and HSC-4 head and neck cancer cells, and its mechanisms were investigated. Experiments were divided into 4 groups: non-treated (CNTRL), cetuximab-treated (CETU), ultrasound-treated (UST) and the combination of cetuximab and US-treated (COMB). Cell viability was assessed by trypan blue staining assay and induction of apoptosis was detected by fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI) staining assay at 24 h after cetuximab and/or US treatment. To elucidate the effect of cetuximab and US on EGFR signaling and apoptosis in head and neck cancer cells after the treatments, the expression of EGFR, phospho-EGFR, and the activation of caspase-3 were evaluated with western blotting. More cell killing features were evident in the COMB group in HSC-3 and HSC-4 cells compared with the other groups. No differences in EGFR expression among the CETU, UST and COMB groups was observed, while the expression of phospho-EGFR in the CETU group was downregulated compared with that in the CNTRL group. Phospho-EGFR expression was much more downregulated in the COMB group compared with that in the other groups. In addition, the activation of caspase-3 in the UST group was upregulated compared with that in the CNTRL group. Caspase-3 activation was much more upregulated in the COMB group than that in the other groups. These data indicated that LIU was able to enhance the anticancer effect of cetuximab in HSC-3 and HSC-4 head and neck cancer cells.

Therapeutic strategies for head and neck cancer based on p53 status.

Squamous cell carcinomas of the head and neck (HNSCC) are one of the most common types of cancers worldwide, and despite advances in treatment, they still represent a clinical challenge. Inactivation of one or more components in the p53 signaling pathway is an extremely common event in human neoplasia, including HNSCC. The loss of p53 function is responsible for increased aggressiveness in cancers, while tumor chemoresistance and radioresistance can depend on deleted p53 expression, or on the expression of mutated-p53 proteins. Thus, consideration and manipulation of the p53 status during HNSCC cancer therapy should be considered. This review discusses the p53 signaling pathways activated by various cellular stresses, including exposure to cancer therapies. The recognition of the p53 status in cancer cells is a significant factor and could provide valuable assistance during the selection of an effective therapeutic approach.