Combination gemcitabine plus S-1 versus gemcitabine plus cisplatin for advanced/recurrent biliary tract cancer: the FUGA-BT (JCOG1113) randomized phase III clinical trial.

BACKGROUND: Gemcitabine plus cisplatin (GC) is the standard treatment of advanced biliary tract cancer (BTC); however, it causes nausea, vomiting, and anorexia, and requires hydration. Gemcitabine plus S-1 (GS) reportedly has equal to, or better, efficacy and an acceptable toxicity profile. We aimed to confirm the non-inferiority of GS to GC for patients with advanced/recurrent BTC in terms of overall survival (OS). PATIENTS AND METHODS: We undertook a phase III randomized trial in 33 institutions in Japan. Eligibility criteria included chemotherapy-naïve patients with recurrent or unresectable BTC, an Eastern Cooperative Oncology Group Performance Status of 0 - 1, and adequate organ function. The calculated sample size was 350 with a one-sided α of 5%, a power of 80%, and non-inferiority margin hazard ratio (HR) of 1.155. The primary end point was OS, while the secondary end points included progression-free survival (PFS), response rate (RR), adverse events (AEs), and clinically significant AEs defined as grade ≥2 fatigue, anorexia, nausea, vomiting, oral mucositis, or diarrhea. RESULTS: Between May 2013 and March 2016, 354 patients were enrolled. GS was found to be non-inferior to GC [median OS: 13.4 months with GC and 15.1 months with GS, HR, 0.945; 90% confidence interval (CI), 0.78-1.15; P = 0.046 for non-inferiority]. The median PFS was 5.8 months with GC and 6.8 months with GS (HR 0.86; 95% CI 0.70-1.07). The RR was 32.4% with GC and 29.8% with GS. Both treatments were generally well-tolerated. Clinically significant AEs were observed in 35.1% of patients in the GC arm and 29.9% in the GS arm. CONCLUSIONS: GS, which does not require hydration, should be considered a new, convenient standard of care option for patients with advanced/recurrent BTC. CLINICAL TRIAL NUMBER: This trial has been registered with the UMIN Clinical Trials Registry (http://www.umin.ac.jp/ctr/index.htm), number UMIN000010667.

Phase II trial of combination treatment with paclitaxel, carboplatin and cetuximab (PCE) as first-line treatment in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (CSPOR-HN02).

Background: The standard of care for first-line treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) is combination treatment with platinum, 5-FU and cetuximab (PFE). However, this regimen requires hospitalization to ensure proper hydration and continuous infusion of 5-FU, and causes severe nausea and anorexia. We evaluated the efficacy and safety of paclitaxel, carboplatin and cetuximab (PCE) as first-line treatment in patients with R/M SCCHN. Patients and methods: Eligibility criteria included recurrent and/or metastatic, histologically proven SCC of the oropharynx, oral cavity, hypopharynx or larynx; PS 0-1; adequate organ function; no suitable local therapy for R/M SCCHN; and no prior systemic chemotherapy for R/M SCCHN. Chemotherapy consisted of paclitaxel 100 mg/m2 on days 1, 8; carboplatin area under the blood concentration-time curve 2.5 on days 1, 8, repeated every 3 weeks for up to 6 cycles; and cetuximab at an initial dose of 400 mg/m2, followed by 250 mg/m2 weekly until disease progression or unacceptable toxicities. Primary end point was overall response rate. Secondary end points were safety, treatment completion rate, progression-free survival, overall survival, and clinical benefit rate. Planned sample size was 45 patients. Results: Forty-seven subjects were accrued from July 2013 to October 2014. Of 45 evaluable, 40 were male; median age was 63 years; Eastern Cooperative Oncology Group Performance Status was 0/1 in 23/22 cases; site was the hypopharynx/oropharynx/oral cavity/larynx in 17/11/10/7 cases; and 36/9 cases were smokers/nonsmokers, respectively. Overall response rate, the primary end point, was 40%. Median overall survival was 14.7 months and progression-free survival was 5.2 months. Grade 3/4 adverse events included neutropenia (68%), skin reaction (15%), fatigue (9%) and febrile neutropenia (9%). A potentially treatment-related death occurred in one patient with intestinal pneumonia. Conclusions: The PCE regimen shows promising activity with acceptable toxicity in the outpatient clinic. Further studies are needed to compare PCE with PFE in this population. Registered clinical trial number: UMIN000010507.

Establishment of estrogen receptor-positive transplantable rat thyroid tumor cell lines in vivo.

We established 17 transplantable rat thyroid tumor cell lines from the primary thyroid tumor of rats induced by N-bis(2-hydroxypropyl)nitrosamine. Among the 17 tumor cell lines established, only two of them (D1 and G1) were estrogen receptor (ER) positive. These two cell lines were characterized with respect to transplantability, histological features, ER contents and cellular localization, and expression of ER message. The ER contents, determined by dextran-coated charcoal assay, were 13.3 and 20.7 fmol/mg protein for D1 and G1 cell lines, respectively. Scatchard plot analysis indicates that the dissociation constants (Kd) were 0.17 and 0.4 nM, respectively, for D1 and G1 cell lines. Sucrose density centrifugation analysis detected a hormone-receptor complex which sedimented at the 4S region, characteristic for ER. Immunohistological staining revealed that the ER was localized in the nuclei. The presence of ER in D1 and G1 cell lines was further confirmed by reverse transcriptase-polymerase chain reaction to detect the ER mRNA. These results demonstrated that ER is expressed in some thyroid tumors. The ER-positive transplantable tumor cell lines are useful for studying the direct effect of estrogen on thyroid tumors in vitro and in vivo.

Transfection with mutant p53 gene inhibits heat-induced apoptosis in a head and neck cell line of human squamous cell carcinoma.

PURPOSE: To confirm that human cancer cells show p53-dependent heat sensitivity through an apoptosis-related mechanism, we examined the heat sensitivity and Bax-mediated apoptosis after heating in a human squamous cell carcinoma cell line, SAS, with identical genetic backgrounds except for the p53 status. MATERIALS AND METHODS: We performed colony formation assay, Western blotting and analyses of apoptosis, using the SAS cells transfected with pC53-248 vector with mutant p53 gene (SAS/Trp248 cells) or the cells transfected with pCMV-Neo-Bam vector (SAS/neo cells) as a control. RESULTS: SAS/Trp248 cells showed heat resistance due to the dominant negative nature of mp53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly after heating in SAS/neo cells, but increased very little in SAS/Trp248 cells. CONCLUSION: These results suggest that heat resistance brought by mp53-transfection is p53-dependent and closely correlates with the induction of apoptosis in human squamous cell carcinomas.

Different ras gene mutational frequencies in thyroid papillary carcinomas in Japan and Thailand.

The incidence and pattern of ras oncogene mutations in human malignancies demonstrate geographic and racial differences. For example, specificity of alterations is found in cholangiocellular carcinomas in Thai patients with a different etiology from those in Japanese patients. In the present study, a comparison of ras gene mutations in thyroid papillary carcinomas from Japanese and Thai patients was performed using single-strand conformation polymorphism and direct sequencing analyses. The incidence of ras mutation differed markedly in Japanese (two of 24 carcinomas, 8.3%) and Thai (five of 10 carcinomas, 50%) patients. In addition, all but one ras mutation occurred at codon 12 of the K-ras gene in the Thai cases. These results suggest that thyroid cancers in Thailand may be due to specific genetic and/or environmental factors.

Lack of p16/CDKN2 alterations in thyroid carcinomas.

Exons 1-3 of the p16/CDKN2 gene and exons 4-9 of the p53 gene were screened for mutations by single-strand conformation polymorphism (SSCP) analysis and direct sequencing of PCR-amplified DNA from human primary thyroid carcinomas and thyroid carcinoma cell lines. The samples included 21 papillary carcinomas, 2 undifferentiated carcinomas, 1 follicular carcinoma, 1 medullary carcinoma and 2 cell lines originating from thyroid undifferentiated carcinomas. No homozygous deletions and mutations in the p16/CDKN2 were observed in any of the primary tumors or cell lined. In contrast, one of the two undifferentiated carcinomas an both cell lines demonstrated point mutations in the p53 gene. These results that p16/CDKN2 gene alteration is not required for malignant transformation in the thyroid, while p53 gene mutations may play a role in the progression from differentiated to undifferentiated carcinoma.

Ki-ras oncogene activation in transplantable rat thyroid carcinoma induced by N-bis(2-hydroxypropyl)nitrosamine.

We have established 17 transplantable rat thyroid carcinoma cell lines from primary thyroid tumors of rats induced by N-bis(2-hydroxypropyl)nitrosamine (DHPN) (Cancer Res. (1993) 53, 4408-4412). The present study was designed to evaluate point mutations in the murine c-Ki-ras gene of these carcinoma cell lines. Using PCR amplification and direct sequencing, we found that the activated form of the Ki-ras oncogene was present in 4 (23%) of a total of 17 cell lines, all the Ki-ras gene mutations being GC-->AT transitions. In three of the cell lines, the mutations occurred in codon 12 (GTP-binding domain), and in the remaining one the first nucleotide of codon 63 was affected. Histologically, three of the carcinomas with Ki-ras mutation were diagnosed as well-differentiated carcinomas, and the other as poorly differentiated carcinoma. Mutations of the ras gene are relatively uncommon in tumors of these histological types. From these experimental results, we suggest that the mutation induced by DHPN is due to damage to guanine in cellular DNA. In addition, Ki-ras activation may play an important role in the initiation of thyroid carcinogenesis.

Expression of the estrogen receptor in human thyroid neoplasms.

The expression and quantitation of the estrogen receptor (ER) in human thyroid tumors were examined by biochemical, immunohistochemical, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. For this study, neoplasms, adenomatous goiters and adjacent normal thyroid tissues were obtained from 35 patients which included 10 cases of papillary carcinomas, 17 cases of adenomas and 8 cases of adenomatous goiters. Regardless of the histopathological subtype, ER was detected in 19% (5/27) of the neoplastic tissues with the mean value of ER content of 5.0 +/- 1.3 fmol/mg protein and the mean Kd value of 0.38 +/- 0.28 nM. ER was also detected, but at a lower concentration (2.8 +/- 1.6 fmol/mg protein), in the surrounding normal tissues. There was no significant difference between the neoplasms and adenomatous goiters with respect to the incidence of ER positivity and ER content. Furthermore, ER-positive specimens, as determined by both biochemical and immunohistochemical techniques, also showed the expression of ER mRNA detected by RT-PCR method. These results demonstrate that both ER mRNA as well as ER protein are expressed in thyroid neoplasms. This suggests the possibility that estrogen may affect the tumorigenesis or the progression of some thyroid neoplasms.

Focal distribution of p16/CDKN2 gene mutations within individual prostate carcinomas.

To examine the potential role of pl6/CDKN2 gene mutations in prostate tumorigenesis, focal areas within individual tumors were investigated. Eleven cases of histologically heterogeneous prostate carcinomas obtained by radical prostatectomy were subjected to analysis of p16/CDKN2 gene mutations. DNA was extracted from 5 to 10 separate areas of each tumor with different growth or histological patterns. Exons 1 through 3 of the p16/CDKN2 gene were amplified using the polymerase chain reaction (PCR) and screened for homozygous deletions and mutations of this gene by single-strand conformation polymorphism (SSCP) analysis. No homozygous deletions were observed in any of the prostate carcinomas, but two of the eleven tumors demonstrated mutations in exon 2 of p16/CDKN2 gene. Missense mutations were detected in only one and two foci, respectively, out of six ana ten selected tumor areas. The present results suggest that p16/CDKN2 gene mutations, although they occur at a low incidence, are involved in prostate tumorigenesis, indicating a mutational heterogeneity in addition to morphological heterogeneity.

p53 tumor suppressor gene and ras oncogene mutations in hypopharyngeal squamous cell carcinomas.

To examine the potential role of p53 and ras gene mutations in hypopharyngeal tumorigenesis, twenty-eight primary hypopharyngeal carcinomas, obtained at biopsy or total pharyngolaryngectomy, were investigated. Exons 5 through 9 of the p53 gene and exons 1 and 2 of the H-, K-, N-ras gene were screened using a combination of immunohistochemistry and single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP). The targeted DNA sequences coding for p53 and ras were confirmed by direct DNA sequencing. Point mutations of p53 were found in 9 (32.1%) of the 28 cases, including one with a double mutation, 3 in exon 5, 1 in exon 6, 2 in exon 7 and 4 in exon 8. Positive nuclear immunostaining for p53 was evident in 14 (50.0%) lesions. Seven (25.0%) of the 28 demonstrated point mutations in the H-rns gene, and 11 (39.3%) showed positive cytoplasmic staining for I as. The 5-year survival rate was worse with than without p53 overexpression (p <0.05). The present results suggest that gene mutations, although they occur at a relatively low incidence, are involved in hypopharyngeal tumorigenesis with p53 expression being a prognostic factor.

Genetic alterations in thyroid carcinomas and adenomas detected by restriction landmark genomic scanning.

Alterations of genomic DNAs in 9 papillary thyroid carcinomas and 3 follicular thyroid adenomas were examined by restriction landmark genomic scanning, a 2-dimensional gel analysis that allows detection of deletions, amplifications and other rearrangements of genomic DNA. DNAs from both thyroid tumors and associated non-tumorous glandular tissues were cleaved with the restriction enzyme NotI end-labeled with P-32 and size-fractionated by 2-dimensional electrophoresis using HinfI in a second digestion. The altered spots in carcinomas and adenomas were compared with those in nontumorous samples. Five and 4 spots were commonly amplified in carcinomas and adenomas, respectively. One amplified spot was apparently specific only for carcinoma and was not detected in any of adenomas examined. In contrast, 12 spots reduced in intensity were frequently observed in tumors, although a subset of 5 were more sporadically affected in adenomas. The results indicate both common and distinct genetic abnormalities occurring in thyroid tumors, which may relate to the different biological behaviors of malignant and benign neoplasms.

CDDP induces p53-dependent apoptosis in tongue cancer cells.

We have investigated the CDDP sensitivities of two tongue cancer cell lines with differing p53 genetic status, one with wild-type p53 (SAS) and the other with mutant-type p53 (HSC-4). SAS was about 2 times more sensitive at the D10 dose and demonstrated increased p53 and Bax protein levels at 10 h after CDDP treatment on Western blot analysis. On the other hand, overexpression of p53 in HSC-4 was observed without CDDP treatment and no elevation of Bax could be detected. Apoptosis was observed after CDDP treatment in SAS but not in HSC-4 by Hoechst 33342-staining and electrophoresis methods. These findings indicate that p53 plays an important role in apoptosis as a positive regulator of Bax expression. It is suggested that p53 status may have predictive potential with regard to response to CDDP therapy.

Amplification of int-2 and bcl-1 genes in squamous cell carcinoma of the larynx.

We studied int-2 and bcl-1 gene amplification in 21 operated patients with cancer of the larynx. In 9 cases, the int-2 gene was amplified (42.9%) and in 4 cases. the bcl-1 gene was amplified (25.0%). Co-amplification of int-2 and bcl-1 was observed in three cases (18.8%), all of which were glottic carcinomas. Other amplified cases were supraglottic carcinomas. Tumor specimens with int-2 amplification were associated with a significantly worse prognosis. We suggest that int-2 amplification is one of the prognostic factors in laryngectomized patients with cancer of the larynx.

Tissue transmigration of CZON (Cosmosin) to middle ear mucosa, maxillary sinus mucosa, and palatine tonsils.

The concentration of CZON was determined by HPLC in surgical patients with chronic otitis media, sinusitis, and tonsillitis. One gram of CZON was injected intravenously prior to surgery. The time course of the mean tissue CZON level was as follows: In the middle ear mucosa, 3.7 micrograms/g at 15 min, 7.2 micrograms/g at 30 min, and 2.9 micrograms/g at 1 hr (the half life: 21.3 min). In the maxillary sinus mucosa, 10.5 micrograms/g at 15 min, 11.8 micrograms/g at 30 min, and 2.8 micrograms/g at 1 hr (the half life: 17.5 min). In the tonsils, 14.9 micrograms/g at 15 min, 9.3 micrograms/g at 30 min, and 2.0 micrograms/g at 1 hr (the half life: 13.2 min). The concentration was high in the maxillary sinus mucosa and the tonsils, but was low in the middle ear mucosa. In the formers the transfer ratio reached its peak 15 to 30 min after administration, but in the latter the peak was reached 30 to 60 min after administration. The order of the transfer ratio at each region was above 25%. The tissue concentration exceeded the MIC80s of frequent isolates from these infections. CZON is considered to be a highly useful drug in the treatment of these infections.

[Aberrant expression of carbohydrate antigens in squamous cell carcinoma of the tongue].

An immunohistochemical study was performed to observe the aberrant expression of blood-related group antigens in squamous cell carcinomas of the tongue from 10 patients and 13 normal tongue tissues using seven monoclonal antibodies, A, B, H, X001, H001, Lea and Leb, and four lectins, PNA, VVA-B4, UEA-1 and LTA. In SCC, Leb antigens were expressed in all cases examined (10 cases). T and Tn antigens which were examined with PNA and VVA-B4 stains, were expressed in seven out of 10 and six out of 10 cases, respectively, while these two antigens were not found in normal tissues. A, B and H antigens were expressed in most normal tongue epithelial cells, strictly dependent on the patient blood type. But in SCC, A and B antigens were not detected in five out of seven cancer cells from blood group A, B and AB individuals, whereas H antigen was expressed in 5 out of 7 cancer cells from blood group A, B and AB individuals. These results suggest that the expression of blood group-related antigens in suppressed to that of more immature carbohydrate chains in the setting of tongue cancer.

[Evaluation of blood-group-related carbohydrate antigens in thyroid neoplasms].

An immunohistochemical study using a panel of monoclonal antibodies (mAbs) against blood group A, B, H and Lewis antigens Le(a), Le(b), Le(x) and Le(y) and lectins, such as PNA, VVA-B4 and UEA -1, was carried out to investigate the aberrant expression of carbohydrate antigens in formalin-fixed, paraffin-embedded tissue sections of thyroid neoplasms. The tissues examined consisted of 26 papillary carcinomas, seven follicular adenomas, seven follicular carcinomas, one anaplastic carcinoma and one medullary carcinoma. MAbs against A, B and H antigens reacted strongly with papillary carcinomas from most of the individuals studied (22 of 26 individuals) and their reactivity corresponded well to the blood groups of individuals. In follicular adenomas and carcinomas, these mAbs reacted weakly with a small number of neoplastic cells from two of seven, and four of seven individuals, respectively. Positive staining with mAbs against Le(a) and Le(b) antigens was found in almost all papillary carcinomas. On the contrary, these mAbs rarely reacted with cells from follicular adenomas and carcinomas. T and Tn antigens which were recognized by PNA and VVA-B4, respectively, were only weakly expressed in a limited number of cells from the thyroid neoplasms. Normal cells adjacent to malignant cells were not stained by any of the stains used in this study. These results are discussed in light of our recent findings that polylactosamine structures produced in papillary carcinomas are quite different from those expressed in other types of neoplasms.

[Immunohistochemical study of epidermal growth factor receptor and estrogen receptor in human thyroid tumors].

Expressions of epidermal growth factor receptor (EGFR) and estrogen receptor (ER) in human thyroid tumors were examined immunohistochemically in frozen sections. Forty-eight fresh surgical specimens of thyroid tissues, 19 carcinomas, 12 adenomas, 7 adenomatous goiters and 10 normal thyroid tissues were studied. All cases, including normal thyroid tissues, expressed the EGFR immunoreaction in the cytoplasm. Incidences of stronger than (+) staining intensity of EGFR were 84% (16/19) in carcinoma, 67% (8/12) in adenoma, 57% (4/7) in adenomatous goiter and 30% (3/10) in normal thyroid tissue. This result suggests that the staining intensity of EGFR expression is associated with malignant potential. The frequency of ER positive cases was 11% (2/19) in carcinoma, 17% (2/12) in adenoma and 0% in both adenomatous goiter and normal thyroid tissue. No relationship between EGFR and ER expression was recognized. However, a case of papillary carcinoma with strong ER immunoreactive intensity expressed weak EGFR intensity, suggesting an inverse relation in immunoreaction between EGFR and ER in papillary carcinoma.