Dynamic changes in RAS gene status in circulating tumour DNA: a phase II trial of first-line FOLFOXIRI plus bevacizumab for RAS-mutant metastatic colorectal cancer (JACCRO CC-11).

BACKGROUND: Few prospective studies have used liquid biopsy testing in RAS-mutant metastatic colorectal cancer (mCRC), and its clinical significance remains unknown. Therefore, this study aimed to carry out a biomarker analysis by liquid biopsy using updated data of the phase II trial of FOLFOXIRI plus bevacizumab as first-line chemotherapy for RAS-mutant mCRC. MATERIALS AND METHODS: A total of 64 patients who received modified FOLFOXIRI regimen (irinotecan 150 mg/m2, oxaliplatin 85 mg/m2, levofolinate 200 mg/m2, and fluorouracil 2400 mg/m2) plus bevacizumab biweekly were enrolled. The primary endpoint was the objective response rate (ORR). Plasma samples were collected at pre-treatment, 8 weeks after treatment, and progression in participants included in the biomarker study. The levels of circulating tumour DNA (ctDNA) and specific KRAS and NRAS variants were evaluated using real-time PCR assays. RESULTS: There were 62 patients (median age: 62.5 years, 92% performance status 0, 27% right side) who were assessable for efficacy and 51 for biomarker analysis. ORR was 75.8% (95% confidence interval 65.1% to 86.5%). The median progression-free survival was 12.1 months, and the median overall survival (OS) was 30.2 months. In 78% of patients, RAS mutations disappeared in the ctDNA at 8 weeks after treatment; these patients tended to have better outcomes than those with RAS mutations. Interestingly, RAS mutations remained undetectable during progression in 62% of patients. Survival analysis indicated that the median OS from progression was significantly longer in patients with RAS mutation clearance than in those with RAS mutation in the ctDNA at disease progression (15.1 versus 7.3 months, hazard ratio: 0.21, P = 0.0046). CONCLUSIONS: Our biomarker study demonstrated no RAS mutations in ctDNA at disease progression in 62% of patients with RAS-mutant mCRC. Both OS and post-progression survival were better in patients with clearance of RAS mutations in ctDNA after triplet-based chemotherapy.

Angularly-dispersed optical parametric amplification of optical pulses with one-octave bandwidth toward monocycle regime.

We demonstrated experimentally the generation of 65-microJ, 5.8-fs optical pulses with an ultrabroad bandwidth (540-1000 nm) by the use of a double-pass angularly-dispersed non-collinear optical parametric amplifier. We also confirmed up to the 95-microJ output from the amplifier when seed pulses were not pre-compensated for. Furthermore, we confirmed that the broadband pump pulses brought in the broader gain bandwidth (from 520 to 1080 nm) than numerical estimation based on CW-pump approximation. To the best of our knowledge, this is the system with the broadest gain bandwidth.

Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo.

The transcription factor E2F coordinately activates several cell cycle-regulatory genes. We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy oligodeoxynucleotide that contained consensus E2F binding site sequence (E2F-decoy) as a competitive inhibitor. The decoy's effect on human mesangial cell proliferation was evaluated by [3H]thymidine incorporation. The E2F decoy inhibited proliferation in a concentration-dependent manner, whereas a mismatch control oligodeoxynucleotide had little effect. Electrophoretic mobility shift assays demonstrated that the decoy's inhibitory effect was due to the binding of the decoy oligodeoxynucleotide to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy 1.1 glomerulonephritis model. The E2F decoy oligodeoxynucleotide was introduced into the left kidney 36 h after the induction of glomerulonephritis. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71%. Furthermore, treatment with the E2F decoy inhibited the glomerular expression of proliferating cell nuclear antigen at the protein level as well as the mRNA level. These findings indicate that decoy oligonucleotides can suppress the activity of the transcription factor E2F, and may thus have a potential in treating glomerulonephritis.

Phenotypic modulation of the mesangium reflected by contractile proteins in diabetes.

The phenotypic change of the mesangial cell is considered to play a pivotal role in the accumulation of extracellular matrix in diabetic nephropathy. This investigation was undertaken to evaluate the expression of the various isoforms of contractile proteins in the streptozocin (STZ)-induced diabetic rat kidney and in renal biopsy specimens from patients with diabetic nephropathy. Specific antibodies to myosin heavy chain isoforms (SM1, SM2, SMemb), caldesmon, and alpha-smooth muscle actin and cDNAs for SMemb were used. Increased expression of SMemb at the mRNA and protein levels was demonstrated at 1 week after STZ administration in the rat. Both levels were increased at 4 weeks. Mesangial staining of caldesmon was observed at 4 weeks and that of alpha-smooth muscle actin at 24 weeks. Immunohistochemical mesangial staining of the contractile proteins was pronounced in patients with diabetic nephropathy in contrast to the trace mesangial staining in normal control subjects. These results indicate that the phenotypic change in mesangial cells occurs in the early stages of diabetes and that several stages in phenotypic changes may exist. Expression of the contractile protein isoforms, especially SMemb, should serve as a new marker for the subsequent glomerular hypertrophy and sclerosis.

Effects of nicorandil on the recovery of reflex potentials after spinal cord ischaemia in cats.

1. The pathophysiological significance of ATP-sensitive K+ (KATP) channels in the central nervous system is not fully understood. In this study the effects of nicorandil (a hybrid vasodilator having a dual mechanism of action as a K+ channel opener and a nitrate) on the recovery of the spinal cord reflex potentials after spinal cord ischaemia were examined and compared with those of pinacidil and nitroprusside in anaesthetized spinal cats. 2. Spinal cord ischaemia was produced by occlusion of the thoracic aorta and the bilateral internal mammary arteries for 10 min. Regional blood flow in the spinal cord was continuously measured with a laser-Doppler flow meter. The monosynaptic (MSR) and polysynaptic reflex (PSR) potentials, elicited by electrical stimulation of the tibial nerve, were recorded from the lumbo-sacral ventral root. The recovery process of spinal reflex potentials was reproducible when the occlusion was repeated twice at an interval of 120 min. 3. Pretreatment with nicorandil (30-100 micrograms kg-1) accelerated the recovery of PSR potentials after spinal cord ischaemia. Such an accelerating effect on the recovery of PSR potentials was also shared by pinacidil (100 micrograms kg-1), another K+ channel opener. In addition, the accelerating effect of nicorandil (100 micrograms kg-1) on the recovery of PSR potentials was abolished by co-administration of glibenclamide (3 mg kg-1), a sulphonylurea KATP channel blocker. Nitroprusside (8 micrograms kg-1min-1) retarded rather than improved the recovery of PSR potentials after spinal cord ischaemia. All of these drugs failed to improve the spinal cord blood flow during ischaemia and reperfusion. 4 These results suggest that nicorandil promotes the recovery of polysynaptic reflex potentials after spinal cord ischaemia by opening the KATP channels of neurones rather than by increasing local bloodflow. K+ channel openers may exert a salutary effect on the functional recovery of the ischaemic spinal cord.

All-solid-state 5-kHz 0.2-TW Ti:sapphire laser system.

We have developed an all-solid-state 5-kHz Ti:sapphire laser system, which produces 22-fs, 0.2-TW pulses. An average power of 22.2 W is the highest ever obtained in ultrashort laser sources. The serious thermal lensing due to high power pumping in a small area of the Ti:sapphire crystal is controlled successfully by a stable quasi-cavity with two concave mirrors.

Unique action determinants of double acting topoisomerase inhibitor, TAS-103.

O6-methylguanine-DNA methyltransferase (MGMT), gamma-glutamylcysteine synthetase (gamma-GCS), and glutathione (GSH) are found to participate in resistance to TAS-103, a topoisomerase I/II inhibitor. In 13 human cancer cell lines, MGMT expression correlated with IC50 for TAS-103, whereas gamma-GCS expression inversely correlated with the IC50 value, suggesting MGMT may work to decrease TAS-103 activity but gamma-GCS may increase it. A reduced gamma-GCS and GSH, and an increased MGMT were associated with the development of resistance in A549 and DLD cells, and gamma-GCS inhibition by buthionine sulphoximine increased the TAS-103 resistance, whereas MGMT inhibition by both O6-benzyl-guanine and MGMT-antisense transfection sensitized cells to TAS-103.

Herbimycin A down-regulates messages of cyclin D1 and c-myc during erythroid differentiation of K562 cells.

The ansamycin antibiotic, herbimycin A, is a potent tyrosine kinase inhibitor, and induces the erythroid differentiation of bcr-abl-possessing K562 cells. The growth of K562 cells was cytostatically reduced to less than 50% of the control level at 48 h by 0.5 microgram/ml of herbimycin A treatment. A total of 12% and 53% of the treated cells were benzidine-positive at 24 h and 48 h, respectively. The percentage of cells in the S phase decreased rapidly from 60% to 15% after 12 h of treatment. The reduction of S phase cells persisted until 24 h, whereas the G1 population conversely increased. Then underphosphorylated retinoblastoma gene product increased from 6 h to 24 h, but returned to baseline at 48 h. Most cell cycle controlling genes were unchanged by herbimycin A treatment. However, both cyclin D1 and c-myc were prominently down-regulated in the early phase of treatment, corresponding to the decline of the S phase population. Cyclin D1 was initially down-regulated to an undetectable level at 6 h, although its expression recovered gradually from 12 h and returned to baseline at 24 h. c-myc was also down-regulated from 1 h to 6 h. These data suggest that signals originating from bcr-abl kinase are at least partly transduced through both c-myc and cyclin D1, and that herbimycin A-induced erythroid differentiation occurs during or after the cessation of growth due to interference with these signals.

A novel variant of acute myelomonocytic leukemia carrying t(3;12)(q26;p13) with characteristics of 3q21q26 syndrome.

Chromosomal translocation often results in aberrant activation of the genes with oncogenic potential and, thus, plays an important role in leukemogenesis. We report a unique case of acute myelomonocytic leukemia carrying a rare reciprocal translocation, t(3;12)(q26;p13). This patient displayed typical clinical features of 3q21q26 syndrome such as abnormal thrombopoiesis and rapid disease progression. Blastic cells from the patient strongly expressed the EVI1 gene, which is located on 3q26 and is normally suppressed in bone marrow cells. Expression of the TEL gene, located on 12p13, was also observed, but fusion transcript between two genes was not found. No structural alterations of the EVI1 and TEL genes were detected by Southern blot and PCR analyses. We reviewed previous literature and found 10 other cases with t(3;12)(q26;p13). These patients comprise a unique disease group with features including dyshematopoiesis and poor prognosis. However, characteristics related to 3q21q26 syndrome were observed only in the present case. Further investigation is required to elucidate the molecular basis of this particular entity.

A case of arteriovenous malformation in the submucosal layer of the stomach.

Arteriovenous malformation (AVM) of the stomach is extremely rare. We report a patient with asymptomatic gastric AVM detected during mass screening of the upper gastrointestinal tract. The patient, a 69-year-old female, had no history of gastrointestinal bleeding. Endoscopy revealed a gastric submucosal tumor (3 cm in diameter) at the posterior wall below the esophago-cardiac junction. Endoscopic ultrasonography (EUS) showed a hypoechoic structure in the third layer of the stomach, suggesting gastric AVM in the submucosal layer. Complete resection of the AVM tissue was performed by strip biopsy with surgical resection. Histological examination showed AVM in the submucosal layer. EUS examination was useful for the diagnosis of gastric AVM in this case.

Inhibition of nitric oxide synthesis accelerates the recovery of polysynaptic reflex potentials after transient spinal cord ischemia in cats.

Nitric Oxide (NO) has been implicated as a mediator of neuronal injury in vascular stroke. On the other hand, NO is suggested to play a neuroprotective role by increasing blood flow during cerebral ischemia. In order to evaluate the role of NO in the spinal cord ischemia, effects of nitric oxide synthase (NOS) inhibition on the recovery of reflex potentials after a transient spinal cord ischemia were examined in urethane-chloralose anesthetized spinal cats. Spinal cord ischemia was produced by occlusion of the thoracic aorta and the both internal mammary arteries for 10 min. Regional blood flow (RBF) in the spinal cord was continuously measured with a laser-Doppler flow meter. The monosynaptic (MSR) and polysynaptic reflex (PSR) potentials elicited by electrical stimulation of the tibial nerve, were recorded from the L7 or S1 ventral root. The recovery process of spinal reflex potentials was reproducible when the oclusion was repeated twice at an interval of 120 min. Pretreatment with N(G)-monomethyl-L-arginine (L-NMMA, 10 mg/kg), a NOS inhibitor significantly accelerated the recovery of PSR potentials after spinal cord ischemia. The accelerating effect of L-NMMA on the recovery of PSR potentials was abolished by co-administration of L-arginine (1 mg/kg/min) but not by that of D-arginine (1 mg/kg/min). L-NMMA failed to improve RBF in the spinal cord during ischemia and reperfusion. Nitroprusside (10 microg/kg/min), a NO donor, retarded the recovery of PSR potentials after spinal cord ischemia. These results suggest that NO production has a significant influence on the functional recovery after transient spinal cord ischemia.

Beneficial effects of nitric oxide synthase inhibition on the recovery of neurological function after spinal cord injury in rats.

We have demonstrated recently that treatment with N(G)-monomethyl-L-arginine (L-NMMA) accelerates electrophysiological recovery after transient spinal cord ischaemia in anaesthetized cats. To determine whether nitric oxide synthase (NOS) inhibition in the acute phase of spinal cord injury results in better functional recovery in the chronic phase, we evaluated the influence of L-NMMA on the time course of changes of neurological function and the histopathological changes after spinal cord compression in rats. Experimental spinal cord injury was produced in anaesthetized rats by short-term (5 min) compression with a thread placed around the spinal cord at T13. The recovery of motor function was assessed by a treadmill test 10, 20 and 30 days after spinal cord compression. The latency of potentials evoked by hindlimb stimulation was measured at the funiculus posterior at C1 10 days after the spinal cord injury in anaesthetized rats. Histological examinations were also performed at the same time. The compression-induced spinal cord injury resulted in motor dysfunction of hindlimbs, an increase in the latency of the evoked potentials and neuronal degeneration in funiculus posterior at T13. Repeated administration of L-NMMA for 1 day significantly accelerated the recovery of the motor function, shortened the latency of the evoked potentials and attenuated the myelin vacuolization in the spinal cord. These beneficial effects of L-NMMA on neurological function and histopathological changes were abolished by coadministration of L- but not D-arginine. These results suggest that NOS inhibition during the early stage of spinal cord injury has beneficial effects on the recovery of neurological function and the histopathological changes in the chronic stage.

Septic sequelae after splenectomy for trauma in adults.

Of 619 trauma patients who underwent splenectomy, 503 lived more than 10 days postoperatively. In this group systemic sepsis developed in 114 patients (22.7 percent). The most frequent septic focus was intraabdominal abscess (59 patients). The incidence of postoperative septic complications increased with the severity of trauma. Of the 41 patients who died after the 10th postoperative day, 36 died from sepsis. There were no septic complications or deaths in the 13 patients under age 15 years. Septic morbidity and mortality rates in splenectomized patients were significantly (p less than 0.01) greater than those in 2,368 consecutive trauma patients treated from 1978 to 1979. Long-term follow-up information was obtained in 242 patients. Follow-up encompassed 1,046 patient-years, with a mean patient follow-up interval of 4.4 years. Severe bacterial infections have occurred in six patients (2.5 percent). Thus far there have been no deaths from overwhelming sepsis. Interestingly, 11.5 percent of the patients complained of more severe viral infections after splenectomy. Our data support the concept of preserving the traumatized spleen whenever possible.

Phenotypic changes of the mesangium in diabetic nephropathy.

In diabetic nephropathy there is accumulation of matrix proteins. Overproduction of these matrix proteins considered to be due to the phenotypic change of mesangial cell. In order to detect the phenotypic change of the mesangial cell, renal biopsy specimens from patients with diabetic nephropathy were stained with antibodies against various types of collagens and contractile-associated protein, caldesmon. Type III collagen was not stained in the glomerulus and type VI collagen showed mesangial pattern from normal controls. In diabetes, mesangial staining of type III collagen and increases in type VI collagen were observed in the mesangium. Increased mesangial staining of caldesmon was noted in the glomerulus from diabetic nephropathy in contrast to only vessel staining from normal controls. These results indicate that phenotypic changes are noted in the mesangium in diabetes. Expression of contractile-associated protein such as caldesmon, would serve as a useful marker to predict glomerulosclerosis.

Development of a new membrane-type heated humidifier for laparoscopic surgery.

BACKGROUND: Recently, several animal studies showed that the core body temperature falls during pneumoperitoneum, and this hypothermia could be prevented by using heated and humidified gas insufflation. However, there are no satisfactory heated humidifiers to meet this purpose. Therefore, we developed a new membrane-type heated humidifier. MATERIALS AND METHODS: The newly developed heated humidifier employs an ion-exchange membrane (Nafion: Du Pont Co. Ltd) tube that passes water selectively in molecular form and a gas compartment is completely separated from distilled water to prevent infection. This humidifier consists of a Nafion tube assembly and a case that includes the heater. A perforated protecting tube is located outside the Nafion tube to prevent direct contact with the Nafion tube when it is assembled. The Nafion tube assembly is inserted in the case, and dry gas flows inside of the Nafion tube. The space between the case and the Nafion tube assembly is filled with distilled water. A heater raises the temperature of the distilled water, and heat and water vapor are transferred to cold and dry gas through the Nafion membrane. Four different types of insufflators were involved in this performance comparison study: a Nafion-based heated and humidified insufflator, a conventional heated insufflator, a conventional heated and humidified insufflator, and a conventional cold and dry insufflator. Temperature and relative humidity were measured once each minute for 15 min, which was repeated four times. Each insufflator was operated at two rates of flow: 5 L/min and 10 L/min. RESULTS: Temperature and humidity of the conventional cold and dry insufflator, the Nafion membrane-type heated and humidified insufflator, the conventional heated insufflator, and the conventional heated and humidified insufflator measured at the distal end of circuit reached 22.0 +/- 0.2 degrees C and 0%, 36.7 +/- 1.1 degrees C and 100%, 29.0 +/- 0.4 degrees C and 0%, and 31.3 +/- 0.4 degrees C and 70.5 +/- 5.3% in 15 min at 5 L/min flow rates. At 10 L/min flow rates, temperature and humidity were almost the same as those at 5 L/min. The membrane-type heated humidifier demonstrated statistically significant improvement in both the temperature ( p < 0.0001) and relative humidity ( p < 0.0001) parameters in comparison to the conventional normal, heated, or heated and humidified insufflators at 5 L/min and 10 L/min continuous flow rate in statistics using repeated-measure ANOVA. CONCLUSION: This newly developed heated humidifier offers the great advantages of maintaining intraabdominal temperature and humidity in comparison to conventional insufflators for laparoscopic surgery.

[The study of cases with infection by methicillin-resistant Staphylococcus aureus].

The purpose of this study was to evaluate the risk factors which lead to death due to highly critical staphylococcic enteritis manifested by high fever, large amount of watery diarrhea and gastric secretion with leukopenia and lymphocytopenia after surgical procedures. We experienced eight cases of severe staphylococcic enteritis by methicillin-resistant Staphylococcus aureus (MRSA) occurred in the early days after operation from 1986 to 1990. Seven out of eight cases underwent gastrectomy due to gastric cancer. Oral antibiotics were administered in five out of eight cases as the preparation of gastrointestinal tract. Five had been injected the third-generation cephalosporin (CZX) as prophylactic antibiotic administration. In hematological examination, leukopenia and lymphocytopenia were observed in five cases (62.5%) who died soon after operation. So as to prevent the occurrence of MRSA enteritis, it is important to avoid using third-generation cephalosporin after gastrectomy.

[A case of aggressive myeloma with abnormal plasmocytes in pleural effusion and cerebrospinal fluid].

Extramedullary involvement of myelomas is common but invasion of myeloma cells into the pleural cavity and cerebrospinal fluid (CSF) is rare. We report an aggressive case of multiple myelomas (Bence Jones lambda type) with pleural and meningeal infiltration. A 66-year-old man was referred to our hospital because of anemia, thrombocytopenia, and dyspnea. His peripheral blood contained 2% bizarre plasma cells. Bone marrow biopsy specimens and immunoelectrophoresis confirmed the diagnosis. A chest radiograph disclosed pleural effusion in both lungs containing M-protein and numerous abnormal cells. The patient also suffered from disorientation, speech disorder, and muscle weakness. A lumbar puncture revealed atypical plasma cells in CSF. Four courses of chemotherapy (cyclophosphamide, doxorubicin, and prednisolone) and the intrathecal administration of methotrexate and cytarabine at 3-week intervals were effective in decreasing the pleural effusions and eliminating plasma cells from CSF. Nonetheless a chest wall tumor, pelvic mass, and pneumonia developed, and the patient died 5 months after initial presentation. Pleural infiltration of myeloma cells and multiple lesions with plasma cell involvement were discovered at autopsy.

[Nontuberculous atypical mycobacterial infection with progressive pancytopenia in a patient with myelodysplastic syndrome].

We describe a patient with myelodysplastic syndrome (MDS) who developed disseminated infection due to nontuberculous mycobacteria (NTM). A 64-year-old man was admitted because of persistent fever that had been unresponsive to antibiotics. Bone marrow aspiration specimens showed myelodysplasia (RA), but the origin of the fever was unclear. Cytopenia worsened to a level that required transfusion of red blood cells and platelets. Repeated bone marrow examination revealed hypoplasia with hemophagocytosis. Several weeks later, photochromogenic NTM was isolated from bone marrow specimens, sputum and broncho-alveolar lavage (BAL) fluid which had been obtained on admission. Antituberculosis treatment with clarithromycin markedly improved the patient's general condition and hematological abnormalities. Three months after resolution of the NTM infection, the peripheral blood monocyte count increased, the fever recurred, and the patient suddenly died of myocardial infarction. Disseminated infection with NTM has gained attention as a frequent complication of AIDS, and NTM can also be one of the pathogens causing disseminated infection in patients with MDS. In the present case, infection with mycobacteria that normally would have been digested by macrophages and would not have caused disseminated infection in a healthy individual, was probably related to the clinical features including high fever, severe pancytopenia and hemophagocytosis.

[Disseminated MALT lymphoma associated with macroglobulinemia].

We report a case of disseminated MALT lymphoma with macroglobulinemia in an 80-year-old man who presented with a persistent fever. A radiograph of the chest showed infiltration of the left lung and pleural effusion of the right lung. The fluid contained numerous atypical lymphoid cells, which were positive for CD19, CD20, and HLA-DR, and negative for CD5 and CD10. Analysis of a pleural biopsy sample demonstrated no abnormality. A CT scan of the abdomen showed extensive thickening of the wall of the stomach adjacent to a peritoneal mass. Endoscopic examination disclosed antral ulceration. Histopathological examination of gastric samples revealed infiltration by centrocyte-like cells and lymphoepithelial lesions. Serum electrophoresis detected a macroglobulin peak at 34.5 g/l, and immunoelectrophoresis revealed an IgM kappa component. A bone marrow aspirate showed infiltration by the same lymphoid cells as those in the pleural fluid. A chromosome study of the lymphoid cells from both the bone marrow and pleural fluid showed a normal karyotype. The final diagnosis was MALT lymphoma involving the stomach, lungs and bone marrow. The patient did not consent to chemotherapy, and instead was given oral prednisolone. He died of respiratory distress one year and four months after diagnosis. Autopsy revealed wide dissemination of the tumor cells.

[A case of metastatic splenic tumor treated with intra-arterial immunochemotherapy to induce endogenous LAK cell].

The patient was an 82-year-old female with a 32-month clinical course after right hemicolectomy for ascending colon cancer. Serum carcino embryonic antigen (S-CEA) increased about 38 ng/ml, and metastatic spleen tumor was recognized by abdominal CT and MRI. To induce endogenous LAK, intra-arterial immunochemotherapy with cyclophosphamide (CPM) and OK-432 was performed. Three days after CPM administration, peripheral white blood cells decreased. Recovery from this CPM side effect was achieved with granulocyte colony-stimulating factor (G-CSF). LAK activity of peripheral blood lymphocytes increased from 3% to 17%. Then intra-arterial immunochemotherapy was continued for 7 months, after which the tumor size increased but the S-CEA level remained under 40 ng/ml.