[Romidepsin (Istodax® for intravenous injection 10†mg): pharmacokinetics, pharmacodynamics and clinical study outcome].

Romidepsin (Brand name: ISTODAX® for Injection 10 mg) is a novel antitumor drug that inhibits histone deacetylase (HDAC). Romidepsin strongly inhibited class I HDAC activity in vitro and demonstrated a strong antitumor activity against human tumor cell line xenograft in vivo. Based on its demonstrated efficacy against T-cell lymphoma in early clinical studies, multicenter phase II clinical studies in overseas with romidepsin were conducted in patients with cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL), followed by approval for the treatment of CTCL and PTCL in the U.S. and other countries. Thereafter, domestic phase I/II studies were planned. The phase I study was designed to evaluate the tolerability of romidepsin in Japanese patients with relapsed or refractory PTCL/CTCL and thereby determine the recommended dose, as patients were administered romidepsin by intravenous infusion at a dose of 9 or 14 mg/m2 over 4 hours on days 1, 8 and 15 of each 28-day cycle, and 14 mg/m2 was determined as the recommended dose for phase II. While the phase II study was designed to include 40 Japanese patients with relapsed or refractory PTCL to evaluate the efficacy and safety of romidepsin. Treatment response was 42.5% and the most common AEs of Grade ≥ 3 were lymphopenia (74.0%), neutropenia (54.0%), leukocytopenia (46.0%) and thrombocytopenia (38.0%). The overall safety profile was considered to be within the acceptable range. On the basis of these result, romidepsin was approved in July 2017 for the treatment of relapsed or refractory PTCL in Japan.

LKB1 gene mutations in Japanese lung cancer patients.

Mutation of the LKB1 gene (also known as STK11) is regarded as a cause of Peutz-Jeghers syndrome. In Caucasian patients, LKB1 somatic mutations occur in approximately one-third of lung adenocarcinomas. The aim of the present study was to examine the LKB1 gene in Japanese patients with lung cancer and to evaluate its clinical and pathological implications. We sequenced the LKB1 gene in 22 lung cancer cell lines and 100 Japanese patients with lung cancer (including 81 adenocarcinomas, 14 squamous cell carcinomas and five other histological types) who had undergone curative pulmonary resection. We also determined expression levels of the LKB1 gene by quantitative real-time reverse transcription-polymerase chain reaction and correlated these results with the clinical and pathological features of patients. Among the 22 cell lines, four had mutations and three of these were in adenocarcinoma cells. Of 100 primary lung cancers, only three had LKB1 gene mutations (3%). All of them were male smokers with adenocarcinomas. Hence, when confined to this subset of patients, the mutation frequency was 9% (3/33). No significant correlation was observed between the expression level of LKB1 and patient clinicopathological features. In conclusion, LKB1 gene mutations were relatively rare in Japanese patients with lung cancer compared with Caucasian patients. LKB1 gene mutations appear to be frequent in male, smoking patients of Caucasian origin, in contrast to EGFR or HER2 mutations that are frequent in non-smoking, female patients of Asian origin.

The absence of human equilibrative nucleoside transporter 1 expression predicts nonresponse to gemcitabine-containing chemotherapy in non-small cell lung cancer.

We report here the development of a polyclonal antibody for human equilibrative nucleoside transporter 1 (hENT1) and assess the expression of hENT1 in non-small cell lung cancer (NSCLC) patients who were treated with gemcitabine-containing chemotherapy. hENT1 expression was analyzed by immunohistochemical staining in 24 NSCLC biopsy samples of formalin-fixed, paraffin-embedded tissues. The hENT1-positive staining in NSCLC samples was significantly associated with response to gemcitabine-containing chemotherapy. Responses to gemcitabine-containing chemotherapy were evident in none of the seven patients with no hENT1 expression. These results indicate that the absence of hENT1 expression may be useful to predict NSCLC patients who will not respond to gemcitabine-containing chemotherapy.

MRP8/ABCC11 directly confers resistance to 5-fluorouracil.

Multidrug-resistance-associated protein, MRP8/ABCC11 (ABCC11), is an efflux pump for nucleotide analogues and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). To test whether ABCC11 directly confers 5-fluorouracil (5-FU) resistance, we used the 5-FU-resistant subline PC-6/FU23-26 selected from PC-6 human small-cell lung cancer cells by 5-FU and found that it increases the resistance by approximately 25-fold. The intracellular FdUMP accumulation was reduced in PC-6/FU23-26 cells concomitant with the overexpression of the ABCC11 gene. These findings suggest that ABCC11 confers 5-FU resistance in the sublines by enhancing the efflux for the active metabolite FdUMP. Previously, methotrexate also increased the efflux by ABCC11, and we found cross-resistance to methotrexate in PC-6/FU23-26 cells. To confirm our hypothesis, we examined whether decreasing the expression of ABCC11 in PC-6/FU23-26 cells by small interfering RNA altered the cytotoxicity to 5-FU and methotrexate and found that this enhanced 5-FU and methotrexate cytotoxicity in PC-6/FU23-26 cells. These data indicate that expression of the ABCC11 gene is induced by 5-FU, and that ABCC11 is directly involved in 5-FU resistance by the efflux transport of the active metabolite FdUMP.

PRL-1 tyrosine phosphatase regulates c-Src levels, adherence, and invasion in human lung cancer cells.

Phosphatases of regenerating liver (PRL) constitute a subfamily of the protein tyrosine phosphatases that are implicated in oncogenic and metastatic phenotypes. In this study, we evaluated the role of PRL-1 in cell proliferation and metastatic processes in human lung cancer cells. We stably transfected human A549 lung cancer cells with several short hairpin RNAs for PRL-1 and found decreased invasive activity in the resulting clones compared with control cells. In addition, cells with suppressed PRL-1 exhibited greater adherence and cell spreading on fibronectin and a decreased proliferation rate compared with control cells. To address possible mechanisms for the altered phenotypes, we examined known biochemical regulators of adhesion and invasion. Inhibition of PRL-1 decreased c-Src and p130Cas expression and Rac1 and Cdc42 activation without any apparent modification of focal adhesion kinase (FAK) expression. Total tyrosine FAK phosphorylation and Tyr397 phosphorylation levels were continuously elevated in PRL-1 knockdown cells plated on fibronectin. In immunofluorescence studies, reduction in PRL-1 seemed to decrease cell membrane protrusions with a reduction in actin fiber extensions in spite of continuous phosphorylation of Tyr397 FAK, which could reflect reduced adhesion turnover. Our data implicate PRL-1 in the fundamental process of cell adhesion and migration in human lung cancer cells by affecting Rac1, Cdc42, and c-Src activation. These results support the hypothesis that PRL-1 plays an important role in maintaining the malignant phenotype by exploiting Src activation processes, and that PRL-1 could be a promising therapeutic target for cancer metastasis and cell growth.

Effects of FOXP3 gene polymorphism in sarcoidosis patients.

BACKGROUND: FOXP3 is a critical regulator of the development and function of regulatory T (Treg) cells, which are capable of suppressing immune responses. A recent study showed that the presence of the (GT) n microsatellite polymorphism in the FOXP3 gene was associated with enhancer activity, resulting in an effect on type I diabetes mellitus. Furthermore, sarcoidosis reportedly increases the prevalence of Treg cells in bronchoalveolar lavage fluid and peripheral blood. Because Treg cells may play roles in immune responses in sarcoidosis, in this study we investigated whether the FOXP3 gene polymorphism affects sarcoidosis. METHODS: One hundred and eight sarcoidosis patients and 100 healthy control subjects were studied. PCR-based fragment analysis combined with fluorescent technology were used to determine the FOXP3 (GT)n genotype. RESULTS: We found that the genotype distribution did not differ between sarcoidosis patients and healthy controls. Among sarcoidosis patients, the prevalence of the (GT)15 allele was higher in patients without skin lesions than in patients with skin lesions (p = 0.037, odds ratio = 2.96, 95% confidence interval: 1.07-8.24). CONCLUSIONS: Although the FOXP3 gene polymorphism has no effect on susceptibility to sarcoidosis, the (GT)15 allele may exert protective effects against skin involvement.

The determinants of sensitivity and acquired resistance to gemcitabine differ in non-small cell lung cancer: a role of ABCC5 in gemcitabine sensitivity.

We examined the expression levels of the multidrug resistance protein 5 (ABCC5) gene in non-small cell lung cancer (NSCLC) cell lines to clarify the relationship with the sensitivity to gemcitabine. The expression levels of ABCC5 were inversely correlated with gemcitabine sensitivity significantly (r = 0.628; P < 0.01) in 17 NSCLC cells, whereas the expression of ABCC5 in the gemcitabine-resistant NSCLC cell line H23/GEM-R was the same as that in parental NCI-H23 cells. Treatment with the ABCC5 inhibitor zaprinast altered the sensitivity to gemcitabine in ABCC5-expressing NSCLC cells. In addition, decreasing the expression of ABCC5 by small interfering RNA altered the cytotoxicity to gemcitabine. These results indicate that modulation of ABCC5 activity could be used to increase the gemcitabine sensitivity in NSCLC. Previously, we found a decreased expression of deoxycytidine kinase in H23/GEM-R cells, and further investigation in this study showed an increased expression of ribonucleotide reductase subunit 1 in H23/GEM-R cells. We therefore also examined the effect of modifying the expression of both genes on gemcitabine resistance. We found that using small interfering RNA to decrease the expression of ribonucleotide reductase subunit 1 resulted in a decreased resistance to gemcitabine in H23/GEM-R cells. Furthermore, pretreatment with pemetrexed resulted in an increased deoxycytidine kinase expression concomitant with the alteration of the resistance to gemcitabine in H23/GEM-R cells. The determinants for sensitivity and the acquired resistance in gemcitabine are quite different; nonetheless, modification of these factors may increase the efficacy of gemcitabine in the treatment of NSCLC.

[A case of pulmonary dirofilariasis that required differentiation from nontuberculous mycobacteriosis].

A 48-year-old man was admitted with an abnormal shadow on a chest X ray. Chest X ray and CT revealed a solitary nodule in the right lung field. Mycobacterium gordonae was cultured from sputum. Tumorectomy by video-assisted thoracoscopic surgery was done for histological diagnosis, and Dirofilaria worms accompanied with epithelioid cell granuloma with necrosis were found, while no bacteria were cultured from biopsy specimen, thus we diagnosed dirofilariasis. Because a solitary nodule can be caused by either Dirofilaria species or nontuberculous mycobacteria, this case was of interest with regard to the differential diagnosis of a lung solitary nodule.

Role of ABCG2 as a biomarker for predicting resistance to CPT-11/SN-38 in lung cancer.

To examine the mechanism of resistance to 7-ethyl-10-hydroxycamptothecin (SN-38) in lung cancer, we continuously exposed the non-small-cell lung cancer (NSCLC) cell line NCI-H23 to SN-38 and selected the SN-38-resistant clone H23/SN-38. After 2 months of culturing in SN-38-free conditions, H23/SN-38 cells recovered their sensitivity to SN-38 and were subsequently established as the revertant H23/SN-38REV cell line. Because H23/SN-38 cells show cross resistance to certain anticancer drugs, such as topotecan, etoposide, doxorubicin and mitoxantrone, we examined the gene and protein expression levels of drug efflux transporters of the ATP-binding cassette (ABC) family. We found that both gene and protein expression of ABCG2/BCRP (ABCG2) in H23/SN-38 cells was increased compared with that in NCI-H23 cells and H23/SN-38REV cells. The cellular accumulation of topotecan in H23/SN-38 cells was decreased compared with that in NCI-H23 and H23/SN-38REV cells, and treatment with reserpine (an inhibitor of ABCG2) increased the cellular accumulation of topotecan in H23/SN-38 cells. Furthermore, treatment with reserpine also altered the sensitivity of H23/SN-38 cells to SN-38. These results indicate that the upregulation of ABCG2 was functional, and related to the resistance of H23/SN-38 cells to SN-38. Moreover, we found that gene expression levels of ABCG2 were significantly correlated with the concentration of SN-38 for 50% cell survival in 13 NSCLC cells (r=0.592, P<0.05). The present results indicate that the induction of ABCG2 by SN-38 does confer acquired resistance to CPT-11/SN-38, but the induction of ABCG2 and subsequent drug resistance are reversible. However, the expression level of ABCG2 may be a useful indicator of CPT-11/SN-38 activity in lung cancer.

The role of thymidylate synthase and dihydropyrimidine dehydrogenase in resistance to 5-fluorouracil in human lung cancer cells.

The expressions of thymidylate synthase (TS) and intracellular metabolic enzymes have been reported to be associated with the sensitivity and/or resistance to 5-fluorouracil (5-FU). However, since the role of these enzymes in the mechanism of resistance to 5-FU has not been fully examined in lung cancer, in the present study we measured the expression levels of TS, dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyltransferase (OPRT) genes in lung cancer cell lines by real-time PCR, and the sensitivity to 5-FU using the MTS assay. The expression of DPD was significantly correlated with the concentration of 5-FU for 50% cell survival in 15 non-small-cell lung cancer (NSCLC) cell lines (p<0.05), but the expressions of TS, TP, and OPRT were not. Treatment with 5-chloro-2,4-dihydroxypyridine, an inhibitor of DPD, altered the sensitivity to 5-FU in DPD-expressing RERF-LC-MT cells, indicating that modulation of DPD activity could increase the 5-FU sensitivity in lung cancer. In contrast, TS expression was dramatically higher in a 5-FU-resistant small-cell lung cancer cell line than in the parent cell line, whereas the expressions of DPD, TP, and OPRT genes were not markedly different. In order to examine the effect of other cytotoxic agents on TS and DPD expression, we compared the expressions of both genes between cisplatin-, paclitaxel-, gemcitabine-, or 7-ethyl-10-hydroxycamptothecin-resistant lung cancer cells and their respective parent cells, but found no differences between any pair of resistant subline and the corresponding parent cell line. Our results indicate that degradation of 5-FU due to DPD is an important determinant in 5-FU sensitivity, while induction of TS contributes to acquired resistance against 5-FU in lung cancer. Therefore, the expression levels of TS and DPD genes may be useful indicators of 5-FU activity in lung cancer.

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms in sarcoidosis patients.

BACKGROUND AND AIM: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a co-stimulatory molecule that is expressed on activated T-cells, and is an important regulator of T-cell activation in T-cell-dependent immune responses. CTLA-4 signals lead to the downregulation of T-cell proliferation and activation. Sarcoidosis is a systemic granulomatous disorder of unknown etiology, which shows abnormal regulation of T cell activation. Polymorphisms of CTLA-4 have been reported to alter CTLA-4 expression, and are associated with many diseases. In the present study we investigated CTLA-4 polymorphisms of promoter -318(C/T) and exon 1 +49(A/G) in sarcoidosis patients and control subjects to determine whether these genetic variations affect sarcoidosis. METHODS: One hundred and six sarcoidosis patients and 100 healthy control subjects were studied. The polymerase chain reaction technique and direct genomic sequencing were used to determine the genotypes of promoter -318(C/T) and exon 1 +49(A/G) in the CTLA-4 gene. RESULTS: We found no difference in the distribution of either genotype between the healthy control subjects and sarcoidosis patients. Among the sarcoidosis patients, the -318(C/T) CC genotype (p = 0.011) and AG or GG genotype at position +49(A/G) (p = 0.004) were increased with ocular involvement compared with those patients without ocular involvement. Furthermore, the +49(A/G) GG genotype was increased in patients with three or more organs affected compared with patients with fewer organs affected (p = 0.019). CONCLUSIONS: The CTLA-4 polymorphisms are not associated with disease susceptibility of sarcoidosis, but these genetic variations significantly influence phenotypes of sarcoidosis.

[A case of thymic carcinoma responding to combination chemotherapy with nedaplatin, etoposide, and ifosfamide].

We described a case of thymic carcinoma that responded remarkably to combined chemotherapy with etoposide (ETP), ifosfamide (IFO) and nedaplatin. A 68-year-old woman was admitted to our hospital because of hoarseness and dysphasia. Chest computed tomographic (CT) scans showed an anterior mediastinal tumor. Using CT-guided needle biopsy, the diagnosis was squamous cell type of thymic carcinoma. We gave her modified VIP therapy with ETP, IFO and nedaplatin. The tumor contraction response was evident after 3 courses of treatment. This case suggested that nedaplatin may be one of the promising agents for thymic carcinoma chemotherapy.

Spontaneous hepatic rupture due to metastatic tumor of lung adenocarcinoma.

A 64-year-old man diagnosed as lung adenocarcinoma with hepatic tumor was admitted to our hospital. He carried the hepatitis B virus but was negative for PIVKA-II and alpha-fetoprotein, and hence we diagnosed a case of stage IV lung adenocarcinoma. We planned to administer systemic chemotherapy, but he experienced sudden-onset abdominal discomfort accompanied with decreased blood pressure. We diagnosed hemorrhagic ascites due to spontaneous rupture of the liver tumor. Emergency angiography and therapeutic embolization stabilized his clinical condition. Hemorrhagic ascites due to metastatic liver tumor is rare and the sudden onset of abdominal symptoms is an indicator of rupture.

Efficacy of docetaxel as a second-line chemotherapy for thymic carcinoma.

Thymic carcinoma is a rare and aggressive tumor, and the efficacy of second-line chemotherapy is still unclear. Here, we reported a case of thymic carcinoma that responded well to the administration of docetaxel alone as a second-line chemotherapy. A 64-year-old woman was diagnosed with thymic carcinoma (squamous cell type) with bone metastasis, and she, therefore, received nedaplatin combined with etoposide and ifosfamide. She responded partially, after which she received irradiation for bone metastasis. Two months after chemotherapy, the thymic carcinoma exhibited gradual regrowth and she experienced shoulder pain. We treated this with docetaxel alone (60 mg/m2 every 4 weeks). After three courses of docetaxel, we observed a partial response and her shoulder pain disappeared. This case demonstrated that docetaxel is effective as a second-line chemotherapy for thymic carcinoma.

Determinants of sensitivity and resistance to gemcitabine: the roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non-small cell lung cancer.

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT-PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non-small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r =-0.6769, P = 0.0005), whereas dCK expression levels were not. In a highly gemcitabine-sensitive cell line, NCI-H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI-H23 cells to gemcitabine and subsequently established the drug-resistant clone H23/GEM-R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.

Matrix metalloproteinase-1 polymorphism of promoter region in sarcoidosis and tuberculosis patients.

BACKGROUND AND AIM OF THE WORK: Matrix metalloproteinases (MMPs) are associated with several diseases. MMP-1 is one of the interstitial collagenases and the most highly expressed. Recent studies have found that a single nucleotide polymorphism located in the promoter region of the MMP-1 gene affects transcriptional activity. This polymorphism, 1G/2G, has been reported to associate with several malignant tumors and lung diseases. In this study, we investigated whether this polymorphism is associated with sarcoidosis or tuberculosis. METHODS: Polymerase chain reaction-restriction fragment length polymorphism was used to determine the MMP-1 genotypes of 103 sarcoidosis patients, 105 tuberculosis patients and 106 healthy control subjects. RESULTS: We found no differences in genotype distributions and allele frequency between sarcoidosis or tuberculosis patients and healthy control subjects. In sarcoidosis patients with ocular involvement, a significant increase in 1G/1G or 1G/2G genotype was observed compared with patients without (p = 0.009, odds ratio (OR) = 3.22, 95% confidence interval (CI): 1.34-7.71). In sarcoidosis patients with three or more organs involved, 1G/1G type tended to increase compared with patients without (p = 0.035, OR = 5.17, 95% CI: 1.12-23.9). In tuberculosis patients with cavity formation, an increasing trend of 1G/1G type was observed compared with patients without (p = 0.064, OR = 7.69, 95% CI: 0.89-66.3). CONCLUSIONS: Although MMP-1 polymorphism was not associated with onset risk of sarcoidosis and tuberculosis, the clinical characteristics of both diseases were affected by this polymorphism.

Analysis of beta-tubulin gene alteration in human lung cancer cell lines.

We examined lung cancer cell lines for the presence of beta-tubulin gene alteration based on a previous report of a relationship between frequent beta-tubulin gene mutation in non-small-cell lung cancer and clinical response to taxanes as well as the prognosis. The mutation was defined by analyzing genomic DNA from 31 lung cancer cell lines by direct genomic sequencing using specific primers for the beta-tubulin class I gene. We detected only three genetic alterations at nonsplice sites in two introns, and a silent genetic alteration at codon 217 in exon 4. The mutation of the beta-tubulin gene was rare; moreover, these genetic alterations were predicted to evoke no biological alteration of the cancer cells. Our data suggest that the beta-tubulin gene mutation does not play a major role in the genetic mechanism of resistance to taxanes. In addition, the presence of a closely related family of beta-tubulins or pseudogenes was thought to hinder accurate evaluation of the beta-tubulin gene.

[A case of sarcoidosis with rheumatic features (Löfgren's syndrome)].

Rheumatoid arthritis was diagnosed in a 30-year-old woman with erythema nodosum and arthritic symptoms since 1994, and she was treated with anti-rheumatic agents. Mediastinal and bilateral hilar lymphadenopathy and abnormal pulmonary shadows were detected in 1996, and she was admitted to our hospital in 1997. We also recognized the elevation of ACE and lysozyme, and found granulomas in a transbronchial lung biopsy and an arthrosis synovia biopsy. From these findings, sarcoidosis was diagnosed. Sarcoidosis demonstrating erythema nodosum, arthritis, and bilateral hilar lymphadenopathy is called Löfgren's syndrome. In Caucasians, Löfgren's syndrome is frequently encountered, but it is rare in Japanese. Our case had coexisting arthrosis symptoms, and satisfied the diagnosis criteria of rheumatic arthritis. Therefore, the differential diagnosis was important. We emphasize that it is necessary to consider Löfgren's syndrome when diagnosing patients with rheumatic features, even in Japan.

p185(HER-2/neu) and p21(CIP1/WAF1) expression in primary tumors and lymph node metastases in non-small cell lung cancer.

p185(HER-2/neu), a tyrosine kinase receptor, is one of the target molecules for cancer therapy, and its expression may reduce the sensitivity of tumor cells to anti-cancer drugs. p21(CIP1/WAF1) is a cyclin-dependent kinase inhibitor, and its expression may also be involved in chemoresistance. Non-small cell lung cancer (NSCLC) is a potentially systemic disease, and systemic therapies play an important role in its treatment. However, there have been no studies comparing the expression of these molecules between primary and metastatic tumors. We investigated the expression of p185(HER-2/neu) and p21(CIP1/WAF1) in 57 paired samples of primary NSCLC tumors and corresponding lymph node metastases by immunohistochemistry. Expression of each of p185(HER-2/neu) and p21(CIP1/WAF1) was highly correlated between primary tumors and lymph node metastases, and similar correlations were also obtained when adenocarcinoma and squamous cell carcinoma cases were analyzed individually. However we failed to detect any correlation between p185(HER-2/neu) and p21(CIP1/WAF1) expression. Our results suggested that expression of both p185(HER-2/neu) and p21(CIP1/WAF1) is concordant between primary and metastatic tumors.