Phase II study of trifluridine/tipiracil plus bevacizumab by RAS mutation status in patients with metastatic colorectal cancer refractory to standard therapies: JFMC51-1702-C7.

BACKGROUND: Although the efficacy of trifluridine/tipiracil (FTD/TPI) plus bevacizumab (BEV) against metastatic colorectal cancer (mCRC) has been demonstrated, little is known about its effectiveness upon disease stratification by RAS mutations. In this phase II study, we investigated the efficacy and safety profiles of FTD/TPI in mCRC according to RAS mutation status. PATIENTS AND METHODS: Eligible patients were mCRC refractory or intolerant to all standard therapies other than FTD/TPI and regorafenib. Patients received 4-week cycles of treatment with FTD/TPI (35 mg/m2, twice daily, days 1-5 and 8-12) and bevacizumab (5 mg/kg, days 1 and 15). The primary endpoint was disease control rate (DCR). The null hypothesis of DCR in both RAS wild-type (WT) and mutant (MUT) cohorts was 44%, assuming a one-sided significance level of 5.0%. The necessary sample size was estimated to be 49 patients (target sample size: 50 patients) for each cohort. RESULTS: Between January and September 2018, 102 patients were enrolled, and 97 patients fulfilled the eligibility criteria (48 in the RAS WT cohort and 49 in the RAS MUT cohort). DCRs in the RAS WT and MUT cohort were 66.7% [90% confidence interval (CI), 53.9%-77.8%, P = 0.0013] and 55.1% (90% CI, 42.4%-67.3%, P = 0.0780), respectively. The median progression-free survival (PFS) and overall survival (OS) were 3.8 and 9.3 months, respectively, in the RAS WT cohort and 3.5 and 8.4 months, respectively, in the RAS MUT cohort. The most common grade 3 or higher adverse event in both cohorts was neutropenia (46% in the RAS WT cohort and 62% in the RAS MUT cohort), without unexpected safety signals. CONCLUSIONS: FTD/TPI plus bevacizumab showed promising activity with an acceptable safety profile for pretreated mCRC, regardless of RAS mutation status, although the efficacy outcomes tended to be better in RAS WT.

[Problems and prospects for combined chemotherapy with 5-fluorouracil and low-dose cisplatin].

Recently, the efficacy of combined chemotherapy with 5-fluorouracil and low-dose cisplatin for advanced gastrointestinal cancer has been reported in Japan. However, this method has not become established, because the underlying logic is unclear and the quality of its clinical trials has been unsatisfactory. We suggest the mechanism for this depends not only on the action of CDDP as a modulator of 5-FU, but also on a mutual biochemical modulation in which 5-FU acts as a modulator of CDDP and enhances the effect of CDDP. We expect that numerous phase II studies and the accumulation of reliable data will lead to improvements.

Additional organizational features of the murine folylpolyglutamate synthetase gene. Two remotely situated exons encoding an alternate 5' end and proximal open reading frame under the control of a second promoter.

Nucleotide sequence analysis of independently isolated clones from a mouse liver cDNA library identified two additional splice variants of folylpolyglutamate synthetase (FPGS) mRNA with novel sequence at the 5' end. These variants incorporate two new alternatives (exons A1a and A1b) of exon 1 in the murine FPGS gene which are also spliced to exon 2. Exon A1a encodes most of the 5'-untranslated region. Exon A1b encodes a downstream segment of the 5'-untranslated region, two ATG start codons, and a unique mitochondrial leader peptide as well as 15 additional amino acids of cytosolic FPGS not encoded by all previously identified (Roy, K., Mitsugi, K., and Sirotnak, F. M. (1996) J. Biol. Chem., 271, 23820-23827) splice variants. It was also found by direct sequencing of genomic fragments that although exon A1b is spliced to exon 2, these new alternatives (i.e. exons A1a and A1b) to exon 1 are found approximately 9.5 kilobases upstream from exons B1a, B1b, and B1c. Exons A1a and A1b are separated from each other by a 124-nucleotide intron. Sequencing of the region 5' to exon A1a revealed a nucleotide sequence that was promoter-like and different from the downstream promoter region in the content of putative cis-acting elements. Primer extension analysis identified a number of potential transcription start sites within the more 3' end of this region. FPGS RNA transcripts incorporating exons A1a and A1b were detected in both normal mouse tissues, particularly, liver and kidney, and also to a varying extent in tumors; FPGS RNA transcripts incorporating exons B1a, B1b, and B1c were detected mainly in tumors. Thus, transcription of the FPGS gene in this tissue-specific manner appears to reflect the different usage of alternates to exon 1 under the control of different promoters. An unusual splice variant identified infrequently in a mouse liver cDNA library was 2.67 kilobases in size and incorporated exons A1a and A1b and a segment of the downstream promoter region along with exons B1c and B1b and exons 2-15.

Organization and alternate splicing of the murine folylpolyglutamate synthetase gene. Different splice variants in L1210 cells encode mitochondrial or cytosolic forms of the enzyme.

The organization of the murine folylpolyglutamate synthetase (FPGS) gene has been determined by sequence analysis that also revealed an interesting complexity in the case of exon 1. The entire nucleotide sequence of the L1210 FPGS cDNA, the 3'- and 5'-untranslated regions, the mitochondrial leader sequence, and the coding region were found to be distributed on 15 exons with an overall length of 10.358 kilobases. Two splice variants of exon 1 were identified by screening of an L1210 cell cDNA library. Variant I (exons 1a + 1b plus 2-15) incorporates all of the sequence homologous to the recently reported (Taylor, S. M., Freemantle, S. J., and Moran, R. G. (1995) Cancer Res. 55, 6030-6034) human exon 1, including two ATG start codons at positions +1 and +126, and encodes both mitochondrial and cytosolic form of FPGS. The most prevalent variant, Variant II (exons 1b plus 2 to 15), incorporates only a portion (92 nucleotides at the 3' end) of this sequence, incorporates only one ATG start codon at position +126, and encodes only cytosolic FPGS. The existence of this variant is consistent with the identification of an appropriately situated internal donor/acceptor site in what was believed to be exon 1. A third related variant (Variant III) with a novel 5' termini was originally identified by screening of a mouse liver cDNA library. This variant, which occurs at moderately low frequency in the L1210 cell cDNA library, incorporates an alternate to exon 1a (exon 1c) spliced to exon 1b plus exons 2-15 and encodes a different mitochondrial leader peptide than Variant I. The identification of these variants suggests another possible mechanism, i.e. at the level of precursor mRNA splicing, for regulating synthesis of mitochondrial versus cytosolic forms of FPGS in the cell. Exon 1c is positioned in the gene upstream of exon 1a separated by an intron of 56 nucleotides within a region of DNA sequence that like the homologous human sequence is distinctly promoter-like. However, the sequence of this region differs from the human sequence in terms of the number, position, and type of putative regulatory elements, particularly in regard to the number of SP-1 binding sites and the position of multiple transcription start sites as determined by enzymatic primer extension.

Different antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate synthetase gene expression at the level of mRNA transcription.

L1210 cell variants selected in the presence of the lipophilic dihydrofolate reductase inhibitor, metoprine, expressed increased levels of one-carbon, reduced folate transport inward (Sirotnak, F. M., Moccio, D. M., and Yang, C.-H. (1984) J. Biol. Chem. 259, 13139-13144). Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of 1,L5-CHO-folateH4 (natural diastereoisomer of 5-formyl-tetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in Km and the same increase in a 60-61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constitutively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.

Placental transfer, lacteal transfer and plasma protein binding of gemcitabine.

1. The placental transfer, lacteal transfer and plasma protein binding of gemcitabine have been studied in rat and dog after single intravenous administration of 10 mg/kg 14C-gemcitabine. 2. Radioactivity was distributed to the foetuses at 5 min after administration to rat on days 12 and 18 of gestation. The concentrations of radioactivity in the foetal liver, lung and kidney on day 18 of gestation were 4.0-6.4 times higher than in the maternal blood at 4 h after administration. Relatively high levels of radioactivity were noted in foetal tissues 24 h after administration, indicating slow elimination from the foetus. 3. The concentration of radioactivity in milk reached a maximum at 15 min after administration to the lactating rat on day 10 after delivery, then declined in a biphasic manner, and was below the detection limit at 48 h. The concentrations of radioactivity in milk were lower than plasma concentrations of radioactivity. 4. Plasma protein binding ratios were 10-16 and 1-7% between 5 min and 8 h after administration to the male rat and dog respectively. When fresh plasma from male rat, dog and adult man was spiked with 14C-gemcitabine at concentrations of 0.1-25 micrograms/ml, the plasma protein binding ratios were about 7% in both rat and dog, and 10% in man.

Disposition of gemcitabine in rat and dog after single and multiple dosings.

1. The disposition of radioactivity has been studied in rat and dog after intravenous administration of a single 10 mg/kg dose or multiple 1 mg/kg/day doses of 14C-gemcitabine. 2. Radioactivity was eliminated from the blood in a biphasic manner with half-lives of approximately 2 and 15 h in both the male and female rat. The concentration of radioactivity in the blood 24 h after the fifth dose was 4.4 times higher than that found after the first dose. In the male dog, the concentration of radioactivity in the blood showed a plateau during the first 2 h post-dose administration. 3. Radioactivity was rapidly and widely distributed throughout the body in both the male and female rat at 5 min after administration. Radioactivity was rapidly eliminated from the tissues with no evidence of accumulation. 4. After 120h, male rat excreted 95.2 and 1.9% of the dose in the urine and faeces respectively. Similar excretion patterns were observed in female rat and male dog. In rat, excretion of radioactivity in the urine 24 h after daily dosing was nearly constant, but excretion of radioactivity in the faeces slightly increased with increasing number of doses.

[A case of advanced gastric cancer complicated with multiple liver metastasis successfully treated with l-LV and 5-FU combination].

A 65-year-old man complaining of nausea and loss of appetite was diagnosed as having Borrmann type 3 gastric cancer with multiple liver metastasis. He was treated for 5 days with bolus injections of l-leucovorin (l-LV: 100 mg/m2/day) followed by 5-fluorouracil (5-FU; 370 mg/m2/day), and this was repeated every 4 weeks. The computed tomography scan after 3 cycles showed an approximately 70% decrease in the size of metastatic lesions, indicating a partial response. The primary gastric lesion also showed a partial response. There were modest but tolerable side effects such as diarrhea. After 3 cycles, the patient was discharged and was given oral 5-FU preparation. He died 9 months after initial chemotherapy with a response duration of 5 months. This l-LV and 5-FU combination therapy appears useful for advanced gastric cancer.

[Successful treatment of recurrent multiple lung metastasis from colon cancer with combination chemotherapy using methotrexate, 5-fluorouracil, and high-dose leucovorin: a case report].

A 66-year-old man, who had received sigmoidectomy for sigmoid cancer in 1985, was diagnosed as having multiple lung and liver tumors in September 1988. When celiac-angiography was performed, recurrent liver metastases from sigmoid cancer were suspected and he received a transarterial embolism with ADM 30 mg and MMC 20 mg. In addition, he was treated with a sequential chemotherapy with methotrexate (MTX), 1,200 mg intravenously (6 h-infusion) followed by 5-fluorouracil (5-FU), 600 mg/m2/day and leucovorin, 300 mg/body/day in continuous infusion for 5 days from day 2 with concomitant oral administration of dipyridamole (300 mg/day) over 14 days. Treatment was repeated every 28 days for two courses. For the third course, administration of only 5-FU, leucovorin and dipyridamole was performed. As a result, the size of pulmonary lesions was prominently reduced on computed tomography. Although mucositis, anal erosion, diarrhea and thrombocytopenia were noted, no severe side effects were observed. This sequential chemotherapy appears useful for metastatic lesions from colon cancer.

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 Chinese hamster cells.

A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed "growth inhibiting factor" (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity of GIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.

Production of anti-cardiolipin antibody in AKR/J mice with streptozocin-induced insulitis and diabetes.

We herein report that anti-cardiolipin antibodies (ACA) were detected in AKR/J mice treated with multiple low doses of streptozocin (STZ)-induced insulitis and diabetes. Daily intraperitoneal (i.p.) injections of 40 mg/kg body wt. of STZ for five consecutive days in the AKR/J mice resulted in hyperglycemia and mononuclear cell infiltrations of islets (insulitis). ACA appeared on day 14, when hyperglycemia began to occur, at a rate of 13.3% (4/30). The rate increased to 83.3% (25/30) on day 21, when diabetes developed, and then fell to 10% (3/30) on day 28. Neither the diabetic AKR/J mice treated with a single high dose of STZ (200 mg/kg body wt.) nor the non-diabetic insulitis free Balb/c mice and B10.S(9R) mice treated with multiple low doses of STZ (40 mg/kg body wt.) produced ACA. The IgG subclass of the ACA belonged mainly to IgG2a. These findings suggest that ACA are produced in association with the development of insulitis, but not induced by either hyperglycemia or STZ.

[5-fluorouracil + low-dose leucovorin and cisplatin sequential chemotherapy with dipyridamole for advanced nonresectable squamous cell carcinoma of the lung: a case report].

A 62-year-old man diagnosed as Stage IIIB advanced non-resectable squamous cell carcinoma of the lung was treated with a sequential combination of 5-fluorouracil (5-FU) and cisplatin (CDDP), with concurrent administration of leucovorin and dipyridamole as a biochemical modulator for 5-FU. After 3 cycles, the mass reduced in size more than 70% in CT scan and the patient underwent a thoracotomy. Histologically, the primary lesion was completely necrotized and of the 10 metastatic regional lymph nodes, only one lymph node contained a small amount of viable cells and 3 additional cycles were conducted. The patient is still alive 30 months after initial chemotherapy. This regimen appears to be potentially useful for non-small-cell lung cancer and warrants further clinical study.

Inhibition by 5-fluorouracil of cis-diamminedichloroplatinum(II)-induced DNA interstrand cross-link removal in a HST-1 human squamous carcinoma cell line.

To elucidate the mechanism of the synergistic cytotoxicity of 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum(II) (CDDP), we studied the interaction of these agents using a human squamous carcinoma cell line (HST-1). Exposure to 5-FU for 24 h and to CDDP for 1 h produced a 50% inhibitory concentration of 1.0 micrograms/ml (7.7 microM) and 2.5 micrograms/ml (8.3 microM), respectively. The cytotoxic action of CDDP was augmented, and a greater than additive effect was observed when the cells were exposed to 5-FU (1.0 micrograms/ml; 7.7 microM) for 24 h before the CDDP treatment. This synergistic activity was maximal when the interval between 5-FU and CDDP exceeded 24 h. In contrast, the cytotoxicity of CDDP was attenuated when it preceded the exposure to 5-FU. Thymidine did not alter the 5-FU-CDDP interaction. Evaluation of the kinetics of the removal of DNA interstrand cross-links, measured by alkaline elution, showed a significant reduction of this removal in the cells exposed to 5-FU followed by CDDP with a drug-free interval of 48 h, as compared with cells exposed to CDDP alone, or to 5-FU immediately followed by CDDP, although no differences were found in the formation of DNA interstrand cross-links by CDDP among these cells. No significant differences in the accumulation of intracellular platinum were detected by atomic absorption spectrophotometry. These findings suggest that 5-FU modulates the repair of platinum-DNA adducts, thereby potentiating the antitumor activity of CDDP.

Increase of peripheral B lymphocytes committed to the production of monoreactive and high affinity antibodies to HTLV-1 in patients with HAM/TSP.

We quantitated the frequency of B lymphocytes capable of producing antibodies to HTLV-1 in the peripheral blood from patients with HAM/TSP, non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. Epstein-Barr virus (EBV) was used as a polyclonal activator of B lymphocytes in a limiting dilution condition. We found that B lymphocytes committed to the production of monoreactive-IgG and -IgA antibodies to recombinant HTLV-1 (gag + env) hybrid protein were significantly increased in a number in patients with HAM/TSP as compared to non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. By transforming these B lymphocytes with EBV and fusing them with human-mouse heteromyeloma (F3B6), a stable hybridoma producing IgG monoclonal antibody (mAb) to HTLV-1 (gag + env) protein was generated from a patient with HAM/TSP. This mAb (IgG1, kappa), designated F31.1, specifically bound to the amino acid residues from 235 to 254 of HTLV-1 envelope glycoproteins (gp46) with high affinity (Kd = 4.0 x 10(-9) mol/l). These data indicate that the antigen-driven process of B lymphocytes maturation by HTLV-1 antigens is markedly increased in patients with HAM/TSP.

In vivo comparative therapeutic study of optimal administration of 5-fluorouracil and cisplatin using a newly established HST-1 human squamous-carcinoma cell line.

The efficacy and toxicity of 5-fluorouracil (5-FU) and cisplatin (CDDP) given in a sequential combination were evaluated in nude mice transplanted with HST-1, a newly established human squamous-carcinoma cell line. 5-FU and CDDP were given i.p. for 5 days and 1 day, respectively, either as single agents or in a sequential manner separated by a 24-h interval. The treatment was repeated every 30 days. Although inhibition of tumor growth was seen in all of the treated groups after two cycles, the sequence of 5-FU followed by CDDP significantly reduced the tumor burdens throughout all three courses and was more effective than the reverse sequence or either drug alone. Neither treatment-related death nor significant hematologic or nephrologic toxicities were seen, even following three cycles of therapy. Significant weight loss was observed only in mice treated with CDDP followed by 5-FU. This sequence dependence of the activity and toxicity of the 5-FU and CDDP combination should thus be incorporated into the design of a clinical trial.

Simple and efficient chemosensitivity assay for human primary tumor-cells using contact-sensitive microtiter plates.

A simple and efficient chemosensitivity assay for human primary tumors has been developed using 96-well microtiter plates covered totally with lethally irradiated 3T3 monolayers termed cell mats, on which the proliferation of normal human fibroblasts is preferentially inhibited. Two days after the inoculation of tumor cells into the microtiter cell mat plates, the cells were treated with various anticancer drugs, and the cell numbers were assessed by radioactivity using a 96-well automatic scintillation counter after subsequent radiolabeling with H-3-deoxyuridine for 24h. In this manner, complete dose response curves of many anticancer drugs were available from one plate within 5 days. Drugs tested in triplicate were adriamycin, mitomycin C, cisplatin, etoposide, and 5-fluorouracil at 0.01, 0.1, and Ix the peak tolerated drug concentrations in serum. Clinically, of the 39 primary tumor specimens of different types received, 4 were contaminated. The remaining 35 samples were successfully cultured, with 5 cultures being abandoned due to an insufficient cpm. As a result, cell survival curves were obtained from 30 (77%) specimens. This high evaluable rate might be due to the feeder effect brought by cell mats. Although optimization of the assay system has yet to be determined, the ability to have multiple drug sensitivity per plate with short-term duration would make this system an efficient assay for chemosensitivity test of human primary tumor cells.

[Successful treatment of a pretreated elderly case of small-cell lung carcinoma with continuous five-day intravenous infusion of cisplatin plus etoposide].

A 78-year-old man was admitted to our hospital with dyspnea in June 1988, and diagnosed as having small-cell lung carcinoma by cytological findings of pleural effusion. He was treated three times with CAV (cyclophosphamide, doxorubicin, vincristine) therapy and a partial response was achieved. In March 1989, he was again admitted complaining of right dull hypochondralgia accompanied by enlargement of primary tumor in the right lower lobe of the lung and metastases to mediastinal and intraabdominal lymph nodes. Because it was an aged and recurrent case, he was treated with continuous five-day infusion of etoposide, 30 mg/m2/day and CDDP, 18.5 mg/m2/day. After the second course, subjective symptoms clearly disappeared and swelling of mediastinal and intraabdominal lymph nodes was markedly reduced on computed tomography. No severe side effects except for moderate myelosuppression, alopecia and nausea were observed. This regimen appears useful in the treatment of small-cell lung carcinoma in elderly patients.

Establishment of Epstein-Barr virus-negative diffuse large cell lymphoma cell line with an 8;22 chromosomal translocation.

A new human B-cell lymphoma cell line was established from a pleural effusion of a patient with a diffuse large cell lymphoma which originated from an ileocecal tumor. The cell line, designated KAL-1, has been passaged 280 times over a period of 22 months. This cell line was successfully maintained in a chemically defined serum-free medium; its doubling time is approximately 24 h. Immunologically, the cells were demonstrated to express IgM lambda on the cell surface and to react with monoclonal antibodies to B-cell antigen including B1, B4, HLA-DR, and common acute lymphoblastic leukemic antigen but not with B2 and all the T-cell markers. Immunoglobulin gene analysis revealed rearrangements of both JH and C lambda. These data indicate that this cell line represents the B-cell lineage at the immature B-cell stage. This cell line was negative for Epstein-Barr virus nuclear antigen and had no detectable Epstein-Barr virus genome in cellular DNA. Chromosome analyses revealed that the cells carried an 8;22 chromosome translocation, reminiscent of variant type Burkitt's lymphoma. However, there was no histological evidence for Burkitt's lymphoma. Molecular studies showed that KAL-1 had deregulated high constitutive expression of c-myc. This cell line was demonstrated to be highly tumorigenic when injected into athymic nude mice. This tumor model should provide clues about the molecular mechanism involved in the pathogenesis of B-cell malignancy and appears to be a useful in vivo model for the study of molecular events during B-cell differentiation and therapeutic investigations.