Phase I Study Evaluating the Combination of Afatinib with Carboplatin and Pemetrexed After First-line EGFR-TKIs.

BACKGROUND/AIM: Promising reports have described the combination of first-generation epidermal growth factor receptor tyrosine-kinase inhibitors (EGFR-TKIs) with carboplatin plus pemetrexed or bevacizumab. However, no analysis of afatinib with platinum-doublet chemotherapies has been performed. PATIENTS AND METHODS: We evaluated the safety and antitumor efficacy of afatinib combined with carboplatin and pemetrexed in EGFR-mutated non-small-cell lung cancer (NSCLC) patients who progressed during first-generation EGFR-TKIs. RESULTS: Ten patients received 20 or 30 mg/day afatinib with carboplatin (area under the curve, 5) and pemetrexed (500 mg/m2). Dose-limiting toxicities included delay of afatinib ≥14 days, grade 3 diarrhea, grade 3 hypokalemia, grade 3 serum amylase increase and grade 4 thrombocytopenia. The recommended dose of afatinib was 20 mg/day in this combination therapy. Overall response rate was 30% and median progression-free survival was 13.7 months. CONCLUSION: This is the first study to investigate the combination of afatinib, carboplatin and pemetrexed. At the recommended dose, this combination was well tolerated and had a good clinical efficacy.

Different profiles of circulating angiogenic factors and adipocytokines between early- and late-onset pre-eclampsia.

OBJECTIVE: Circulating angiogenic factors have been shown to be important in the pathophysiology of pre-eclampsia. Blood levels of adipocytokines differ in pre-eclampsia relative to controls and may also play an important role in disease pathogenesis. Differences in the circulating levels of these molecules were compared between matched normotensive controls and women with pre-eclampsia with onset before or at/after 32 weeks, and according to whether the women were of normal weight (18.5 < body mass index < 25) or overweight. DESIGN: A cross-sectional study of 110 pregnant Japanese women who visited the Department of Obstetrics and Gynecology, Okayama University Hospital, Okayama, Japan. SETTING: Tertiary referral centre serving 2000 births. METHODS: Serum concentrations of soluble fms-like tyrosine kinase 1 (sFlt-1), placental growth factor (PlGF), soluble endoglin (sEng), adiponectin and leptin were measured in women with pre-eclampsia and in normotensive controls matched for age, gestational week, parity and body mass index. Main outcome measures Serum levels of sFlt-1, PlGF, the sFlt-1/PlGF ratio, sEng, adiponectin and leptin. RESULTS: The sFlt-1/PlGF ratio in early-onset pre-eclampsia was significantly higher than that in late-onset pre-eclampsia (112.0 +/- 30.2 versus 45.4 +/- 43.8, P = 0.037). There was a significant elevation of leptin in both subtypes relative to controls (early: 58.6 +/- 18.3 ng/ml versus 26.0 +/- 6.7 ng/ml, P = 0.001; late: 39.5 +/- 9.2 ng/ml versus 22.0 +/- 4.3 ng/ml, P = 0.005), but adiponectin was increased only in late-onset pre-eclampsia (36.5 +/- 13.4 microg/ml versus 12.0 +/- 4.3 microg/ml, P = 0.003). Significant differences in angiogenic factors and adiponectin were found between normal and overweight women only in late-onset pre-eclampsia. CONCLUSIONS: These data suggest that there are different profiles of angiogenic factors and adipocytokines between women who develop early- and late-onset pre-eclampsia.

Expression and potential role of peroxisome proliferator-activated receptor gamma in the placenta of diabetic pregnancy.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed predominantly in adipose tissue and is known to be involved in adipocyte differentiation and insulin sensitivity. Recent reports indicated that PPARgamma-deficient mice were embryonic lethal due to abnormal placental development, suggesting that PPARgamma plays an important role in normal development of placenta. On the other hand, expression of vascular endothelial growth factor (VEGF), the other important factor in placental development, has been demonstrated to be regulated by PPARgamma in vascular smooth muscle cells. Also, diabetic pregnancy is often associated with defective placental functions. In order to investigate physiological roles of PPARgamma and VEGF in placental development during diabetic pregnancy, we examined the expressions of PPARgamma and VEGF in placentas, which were obtained from normal and streptozotocin-induced diabetic pregnant mouse, and studied in vitro effects of hyperglycemic condition and PPARgamma ligands (rosiglitazone and 15-deoxy-delta(12,14)prostaglandin J(2)) on trophoblasts using human choriocarcinoma cell lines. In diabetic mouse placentas (n=5), expressions of PPARgamma and VEGF proteins significantly increased as compared with these in normal placenta (n=3 or 4). In vitro studies indicated that hyperglycemic condition (42 mM) significantly enhanced the PPARgamma expression and hCG production, and significantly suppressed cell proliferation, however these effects were attenuated by PPARgamma ligands that accompanied with increased VEGF production. These data suggest that the PPARgamma pathway might be involved in the impairment of placental development induced by high glucose conditions, and that VEGF might play some roles in this pathway.

Three-year follow-up of the Japanese patients with microvascular angina attributable to coronary microvascular spasm.

BACKGROUND: We recently reported that coronary microvascular spasm could cause angina in patients with chest pain and normal coronary arteriograms. However, the long-term prognosis of these patients or the effect of calcium channel blockers is not known. METHODS: Of consecutive 283 patients who underwent acetylcholine testing for the evaluation of chest pain, we identified 68 patients with microvascular angina attributable to coronary microvascular spasm. All patients were discharged on calcium channel blockers and followed up for an average period of 3.3 years. RESULTS: As compared with those having epicardial spasm (n=169), there was a female predominance in the microvascular spasm group (P<0.01), and 81% of the female patients were postmenopausal. During the follow-up, no patient died and one patient (1%) developed non-Q wave myocardial infarction. The frequency of chest pain was unchanged or increased in 24 patients (36%) and decreased or disappeared in 42 patients (64%). The angina status was improved only in 16 of 33 patients treated with calcium channel blockers alone. By contrast, it was improved in 18 of 21 patients on the combination of calcium channel blockers and angiotensin converting enzyme inhibitors (P<0.05). CONCLUSIONS: Patients with microvascular angina in the present study were more women and had a different risk factor profile as compared with those having epicardial spasm. Long-term prognosis was excellent with regard to mortality, but angina persisted in many patients even on calcium channel blockers. The result warrants prospective studies to evaluate the efficacy of angiotensin converting enzyme inhibitors as adjunct to calcium channel blockers in this population.

Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans.

Rho-kinase plays an important role in modulating Ca(2+) sensitivity of vascular smooth muscle and has been suggested to be involved in the increased systemic vascular resistance in hypertensive animals. However, it remains to be examined whether this is also the case in patients with essential hypertension. Recently, it has been shown that fasudil is a specific Rho-kinase inhibitor. The aim of this study was to examine whether Rho-kinase is involved in the pathogenesis of hypertension in humans by using this Rho-kinase inhibitor. Studies were performed in hypertensive patients (HT group, n=14) and age-matched normotensive subjects (NT group, n=12). Forearm blood flow was measured by a strain-gauge plethysmograph during intra-arterial infusion of graded doses of fasudil (3.2, 6.4, 12.8, and 25.6 microg/min) or sodium nitroprusside (0.4, 0.8, 1.6, and 3.2 microg/min). Resting forearm vascular resistance was significantly higher in the HT group than in the NT group (22+/-4 versus 17+/-5 U, respectively; P<0.05). The extent of the increase in forearm blood flow evoked by fasudil was significantly greater in the HT group than in the NT group (12.3+/-1.4 versus 6.0+/-0.6 mL. min(-1). 100 mL(-1), respectively; P<0.01). The percent decrease in forearm vascular resistance was significantly greater in the HT group than in the NT group (63.6+/-4.7% versus 29.6+/-3.9%, respectively; P<0.01). By contrast, forearm vasodilator response evoked by sodium nitroprusside was comparable between the 2 groups. These results provide the first evidence that Rho-kinase may be involved in the pathogenesis of the increased peripheral vascular resistance in hypertension in humans.

Plasma nitrite/nitrate concentrations as a tumor marker for hepatocellular carcinoma.

Since plasma concentrations of nitrite/nitrate, the stable end-products of nitric oxide, increase in patients with hepatocellular carcinoma (HCC) correlatively to tumor volume, we examined the ability of plasma nitrite/nitrate to discriminate between those patients with HCC and those without and compared the diagnostic performance of the parameter with that of serum alpha-fetoprotein (AFP) concentrations. Plasma nitrite/nitrate and serum AFP concentrations were measured using a Griess reaction and a solid phase enzyme immunoassay, respectively. Eighty-nine patients with chronic liver diseases (CLD) with (n=39) or without HCC (n=50) and 50 healthy control subjects participated in the study. A receiver operating characteristic (ROC) curve was used to determine the optimal cut-off value and accuracy. The areas under ROC curves for nitrite/nitrate and AFP were calculated to be 0.758 and 0.812, respectively, which were not significantly different. There was no correlation between the concentrations of plasma nitrite/nitrate and serum AFP. The sensitivity, the specificity, and diagnostic efficiency were 79.5, 72.0, and 75.3%, respectively, for nitrite/nitrate, and 74.4, 76.0, and 75.3%, respectively, for AFP. Based on a partial ROC curve, the clinical utility of plasma nitrite/nitrate as a tumor marker approximated that of serum AFP, but exceeded in AFP-negative patients. Indeed, nitrite/nitrate was positive in 70% of AFP-negative HCC patients. The simultaneous determinations of serum AFP and plasma nitrite/nitrate concentrations gave significant improvement in detection of HCC in CLD patients compared with that of serum AFP alone.

Beta1 integrins play an essential role in adhesion and invasion of pancreatic carcinoma cells.

To investigate the role of beta1 integrins in pancreatic carcinoma invasion, we analyzed the relationship between the activity of beta1 integrins and the invasive ability of human pancreatic carcinoma cell lines. AsPC1, BxPC3, PANC1, SU8686, KP1NL, KP2, and H48N cells had high expression of beta1 and alpha6 subunits, and various levels of alpha2, alpha3, and alpha5 expression as determined by flow cytometry. Cell adhesion assay revealed that alpha2beta1, alpha5beta1, and alpha6beta1 integrins were the predominant adhesion receptors for collagen, fibronectin, and laminin, respectively. Beta1 integrins on different cell types showed a wide range of constitutive activity. Anti-beta1 monoclonal antibody (MAB) TS2/16 rapidly activated beta1 integrins, and thus TS2/16 requirement in cell adhesion represented the levels of constitutive activity of beta1 integrins. Notably, as the result of in vitro chemoinvasion assay, the levels of constitutive activity of beta1 integrins correlated with the invasive ability of pancreatic carcinoma cells. The inhibitory anti-beta1 MAB 13 completely blocked the invasion of these cell lines. Alternatively, the stimulatory anti-beta1 MAB TS2/16 strongly inhibited the invasion. These results show an essential role of beta1 integrins in invasion of pancreatic carcinoma cells and also suggest subtle regulatory mechanisms of cell invasion.

Recurrent subcutaneous abscess of the sternal region in ulcerative colitis.

An 18-yr-old female patient with extensive ulcerative colitis suffered from several episodes of recurrent aseptic subcutaneous abscesses of the sternal region with a course paralleling that of her colitis. The abscess seemed to occur secondarily to osteomyelitis of the sternum, which is a manifestation of the synovitis, acne, pustulosis, hyperostosis, and osteomyelitis (SAPHO) syndrome.

Bcl-2 protein expression and gut neurohormonal polypeptide/amine production in colorectal carcinomas and tumor-neighboring mucosa, which closely correlate to the occurrence of tumor.

To clarify whether advanced colorectal carcinomas and tumor-neighboring mucosa simultaneously produce both Bcl-2 protein and gut neurohormonal polypeptides and/or amines, and the interrelationship of these phenomenon, we studied retrospective analysis of Bcl-2 protein production and neuroendocrine characteristics in 52 cases of advanced colorectal carcinoma and surrounding mucosa. All of the tumor-neighboring mucosa presented hyperplasia. The rates of enhanced immunoreactivity of the tumor-neighboring mucosa and of positive immunoreactivity of the carcinomas against human Bcl-2 protein and against human vasoactive intestinal polypeptide, pancreatic polypeptide and somatostatin were 78.8% and 94.2%, 82.7% and 59.6%, 78.8% and 67.3%, and 88.5% and 84.6% respectively. Double immunostaining for Bcl-2 protein and each peptide hormone revealed simultaneous expression. In contrast, that of tumor-neighboring mucosa and carcinomas to serotonin and chromogranin-A and to argyrophilia were 11.5% and 1.9%, 32.7% and 17.3%, and 26.9% and 21.2%, respectively. We concluded that tumor-neighboring crypt cells displayed not only hyperplasia but also neuroendocrine characteristics and that enhanced Bcl-2 protein immunoreactivity correlated with tumor occurrence in the wall of the colorectum. The production of Bcl-2 protein by tumor cells and tumor-neighboring crypt cells indicates that the bcl-2 protooncogene may act not only as an inhibitor of apoptosis but also as an inducer of neuroendocrine differentiation from the latent characteristics of the endodermal stem cell.

Role of beta1 integrins in adhesion and invasion of hepatocellular carcinoma cells.

To investigate the role of integrins in hepatocellular carcinoma (HCC) invasion, we analyzed the relationship between the expression and activity of beta1 integrins and the invasive ability of multiple HCC cell lines. Human HCC cell lines, PLC/PRF/5, Hep3B, HepG2, HLE, HuH7, and C3A cells, had high expression of beta1 and alpha6 subunits, and various levels of alpha1, alpha2, alpha3, and alpha5 expression as determined by cell surface flow cytometry. Activity of beta1 integrins was evaluated by cell adhesion to collagen, fibronectin, and laminin in the presence or absence of the stimulatory anti-beta1 monoclonal antibody (mAb) TS2/16. Different types of HCC cells showed various levels of constitutive activity of beta1 integrins as assessed by the TS2/16 requirement in cell adhesion. TS2/16 rapidly stimulated constitutively inactive or partially active beta1 integrins to fully active states, and as the result, the levels of cell adhesion to each ligand correlated with the expression levels of corresponding beta1 integrins. Thus, in the presence of TS2/16 stimulation, the levels of cell adhesion to collagen, fibronectin, and laminin correlated predominantly with the expression levels of alpha2, alpha5, and alpha6, respectively. Remarkably, as a result of in vitro chemoinvasion assay, the levels of constitutive activity of beta1 integrins correlated with the invasive ability of HCC cells. The inhibitory anti-beta1 mAb 13 almost completely blocked the invasion of PLC/PRF/5 and Hep3B cells that are the most invasive HCC cell lines. Alternatively, the stimulatory anti-beta1 mAb TS2/16 strongly inhibited the invasion. These results not only show an essential role of beta1 integrins in invasion of HCC cells but also suggest subtle regulatory mechanisms of cell invasion.

[Cure by THP-COP therapy in patients with perforated T-cell type malignant lymphoma of the jejunum].

A 52-year-old man had suffered abdominal pain from Dec. 15, 1992. On Jan. 1993, he was admitted to our hospital for a diagnosis of T-cell malignant lymphoma of stomach of diffuse large cell type by gastroendoscopical biopsy. On the following day, he underwent emergency an operation with a diagnosis of panperitonitis. A perforation site had been found at the jejunum 60 cm distant from the Treitz ligament. It was resected and sutured concomitant with omental patch. The pathological diagnosis was the same. After the operation, we started THP-COP therapy on Jan. 25, 1993. During the admission, he was given THP-COP therapy 6 times, and had a complete remission. He was discharged Feb. 26, 1994, and shows no evidence of disease at this writing.

Defining extracellular integrin alpha-chain sites that affect cell adhesion and adhesion strengthening without altering soluble ligand binding.

It was previously shown that mutations of integrin alpha4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in alpha4beta1-dependent cell adhesion. Some reports have suggested that alpha-chain "EF-hand" sites may interact directly with ligands. However, we show here that mutations of three different alpha4 "EF-hand" sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on alpha4beta1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin alpha4 "EF-hand-like" sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.

High plasma concentrations of nitrite/nitrate in patients with hepatocellular carcinoma.

OBJECTIVES: Nitric oxide (NO) is considered to play a central role in macrophage or Kupffer cell-induced tumor cytotoxicity. Hepatocytes also produce NO in response to several inflammatory stimuli. Thus, there is a possibility that NO production by hepatic tissue is accelerated in patients with hepatocellular carcinoma (HCC). We, therefore, measured plasma nitrite/nitrate levels as an index of in vivo NO production in patients with HCC. METHODS: Plasma nitrite/nitrate levels were measured using Griess reaction in 95 patients with chronic hepatitis (CH) and compensated liver cirrhosis (LC) with (n = 48) or without HCC (n = 47), as well as 45 healthy control subjects. Possible factors related to nitrite/nitrate levels were evaluated for each subject. RESULTS: Plasma nitrite/nitrate levels in patients with HCC based on CH (mean +/- SD, 71.7 +/- 23.1 microM) and LC (52.4 +/- 20.2 microM) were significantly higher than those without HCC (CH, 31.1 +/- 15.0 microM; LC, 34.6 +/- 16.1 microM) (p < 0.01). Plasma nitrite/nitrate levels in patients with HCC based on CH were significantly higher than those in patients with HCC based on LC (p < 0.05). Simple regression analysis showed that plasma nitrite/nitrate levels significantly correlated with both tumor surface area (r = 0.577, p = 0.001) and tumor volume (r = 0.532, p = 0.003). CONCLUSION: Patients with HCC have elevated plasma nitrite/nitrate levels correlating to tumor volume as well as tumor surface area.

Analysis of intramedullary cell density by MRI using the multiple spin-echo technique.

Analysis of the intramedullary cell distribution by magnetic resonance imaging (MRI) using conventional techniques involves subjectively interpreting images and estimating the cell distribution on the basis of signal intensity characteristics. In recent years, attempts have been made to achieve more precise analysis by new techniques, including chemical shift imaging. The multiple spin-echo (MSE) technique offers some advantages over conventional MRI. Since it allows measurement of the transverse magnetization decay curve at 32 or more points, it is capable of separating several tissue components with different relaxation times. In addition, this technique can be used with MRI instruments having a static magnetic field as low as 1.0 Tesla. In the present study, the intramedullary cell density was assessed by MRI using the MSE technique in 4 patients with aplastic anemia (AA), 4 patients with myelodysplastic syndrome (MDS), and 5 normal subjects. The water component of the marrow (with a short relaxation time) and the fat component (with a long relaxation time) were separated from each other by analyzing MR images obtained using the MSE technique, and the signal intensity ratio of the 2 components was calculated. The ratio was significantly higher in the AA group than in the other groups (AA vs. MDS, P = 0.0209, AA vs. normal controls, P = 0.0143). The present technique appears promising for quantitative assessment of the intramedullary cell density.

[Hypereosinophilia after allogeneic bone marrow transplantation. A possible role of IL-5 overproduction by donor T-cells chronic GVHD].

Chronic graft-versus-host disease (GVHD) is thought to result from abnormalities of several cytokine regulations in vivo. We analyzed interleukin-5 (IL-5) production by peripheral lymphocytes in a patient showing hypereosinophilia associated with chronic GVHD after allogeneic bone marrow transplantation. IL-5 production by activated T-lymphocytes which are known to play a major role in chronic GVHD was upregulated when stimulated by PMA + ionomycin. Therefore, hypereosinophilia observed in our patient may be correlated with IL-5 production in donor T-lymphocytes.

Case Report: haemothorax due to hepatocellular carcinoma rupture successfully controlled by transcatheter arterial embolization.

A 64-year-old man was admitted to our hospital with multiple hepatocellular carcinoma (HCC) lesions in the liver and lung. On the seventh hospital day, a chest radiograph showed a marked increase in right pleural effusion. A thoracentesis revealed a haemothorax. Despite repeated pleural taps and blood transfusions, the patient's clinical status worsened and he developed severe dyspnoea. An inferior phrenic arteriography on the 19th hospital day showed a tumour growing over the diaphragm into the right thoracic cavity, suggesting a tumour rupture. A transcatheter arterial embolization (TAE) of the inferior phrenic artery successfully controlled the bleeding and improved the haemothorax. There was no rebleeding; however, the patient died of advanced HCC 3 months later. To our knowledge, this is the first case of a haemothorax secondary to a ruptured HCC that was treated successfully with TAE.

Hematologic response in patients with aplastic anemia after long-term administration of recombinant human granulocyte colony-stimulating factor and erythropoietin.

Twenty patients with aplastic anemia underwent long-term administration (10 weeks) of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in doses from 50 to 800 micrograms/m2 per day by intravenous infusion or 50 to 100 micrograms/m2 per day by subcutaneous injection and re-combinant human erythropoietin (rhEPO) in doses ranging from 2000 to 8000 IU/m2 per day by intravenous injection three times a week for at least 4 weeks. The goal was to evaluate whether therapy ameliorated pancytopenia in these patients as well as to determine its safety. All assessable patients showed a substantial increase in absolute neutrophil count, with a recovery of myeloid components (granulocyte series) in the bone marrow, after 2 to 10 weeks of treatment. An increase > 1.5 g/dL in hemoglobin (Hb) concentration was observed in 2 patients (10%). A decrease > 50% in red cell transfusion requirement was observed in 2 patients (10%). Seven patients showed recovery of neutropenia, anemia, and platelet count. In addition, there was no serious infection before or during therapy, and side effects were mild. Of the 20 patients, 3 showed a dramatic improvement in severe anemia after 10 weeks of treatment accompanying a recovery of erythroid components in the bone marrow. They no longer require red cell transfusions and have had normal Hb concentrations and normal ferrokinetics. These results indicate that long-term administration of rhG-CSF and rhEPO may benefit some patients with aplastic anemia. Further studies will be necessary to elucidate the mechanism by which rhGCSF and rhEPO stimulate hematopoiesis and improve hematologic abnormalities in these patients.