Irinotecan and mitomycin C in 5-fluorouracil-refractory colorectal cancer patients. A phase I/II study of the Southern Italy Cooperative Oncology Group.

PURPOSE: To define the maximum tolerated dose (MTD) of irinotecan (CPT-11) given on days 1 and 8 with mitomycin C (MMC) given on day 1 in a monthly cycle, and to assess the toxicity and activity of this regimen in patients with previously treated colorectal carcinoma. METHODS: Fifty-two patients, all pretreated with adjuvant 5-fluorouracil (20 patients) and/or one (35 patients) or two (8 patients) lines of chemotherapy, were entered in this study. Escalating doses of CPT-11 (starting from 150 mg/m2) were administered on days 1 and 8, with escalating doses of MMC (starting from 8 mg/m2) given on day 1, recycling every 28 days. At least 3 patients were treated at each dose level. Escalation proceeded unless 2 out of 3 or 4 out of 6 patients experienced a dose-limiting toxicity (DLT) after the first cycle. RESULTS: Twelve patients were entered in the phase I study, and 4 consecutive dose levels were tested. At the last dose level (CPT-11 200 mg/m2 plus MMC 10 mg/m2) 4 of 6 patients experienced a DLT (i.e., grade 4 neutropenia in 2 patients and grade 3 diarrhea in 2 patients). Therefore, this dose level was considered as the MTD. Forty patients were treated at the previous dose level (CPT-11, 175 mg/m2 plus MMC 10 mg/m2). One complete, 4 partial, 3 minor responses and 11 cases of stable disease were registered, giving a response rate of 12% [95% confidence interval (CI), 4-27%] and an overall control of tumor growth in 47% (95% CI, 31-64%) of patients. The median time to treatment failure was 6 months (range 1-19+). The median survival time was 14.5 months, and the 1-year and 2-year probability of survival were 56 and 43%. Neutropenia and diarrhea affected 62 and 58% of patients, grade 3 or 4 being registered in 26 and 23% of them, respectively. One episode of neutropenic fever was reported. Other acute toxicities were usually mild and manageable. CONCLUSIONS: CPT-11 175 mg/m2 on days 1 and 8 associated with MMC 10 mg/m2 on day 1, every 4 weeks, is a safe and moderately active regimen in heavily pretreated patients with advanced colorectal carcinoma. The role of MMC in this combination is doubtful, and further attempts with other new agents should be made to improve the outcome in these patients.

Phase I study of mitoxantrone, raltitrexed, levofolinic acid and 5-fluorouracil in advanced solid tumours.

PURPOSE: We have recently evaluated the combination of raltitrexed, levofolinic acid (LFA) and 5-fluorouracil (5-FU) in advanced head and neck and colorectal cancer, and we have shown that this combination is well tolerated and has clinical activity. Clinical combination studies have shown that raltitrexed and anthracyclines can be combined at full doses without unexpected toxicities. Based on these observations, we started a phase I study of mitoxantrone plus raltitrexed administered on day 1, followed by LFA and 5-FU on day 2 in patients with advanced solid tumors. PATIENTS AND METHODS: Mitoxantrone was given at a starting dose of 6 mg/m2, raltitrexed at a fixed dose of 3 mg/m2, LFA at a fixed dose of 250 mg/m2, and 5-FU at a starting dose of 750 mg/m2. Mitoxantrone and 5-FU doses were subsequently escalated alternately up to dose-limiting toxicity. Treatment was repeated every 14 days. RESULTS: Four dose levels were tested in 18 patients. All three patients treated at the fourth dose level had grade 4 neutropenia after the first cycle. Therefore, this level was defined as the maximum tolerated dose and the dose level immediately below (mitoxantrone 7 mg/m2 and 5-FU 900 mg/m2) was selected for further evaluation. Neutropenia was the main toxic effect. Nonhaematologic side effects were mild. One complete response and five partial responses (all but one in patients with head and neck cancer) were observed, for an overall response rate of 33% (95% confidence interval, 13% to 59%). CONCLUSIONS: Mitoxantrone, raltitrexed and 5-FU can be combined at doses which are close to those used in monotherapy. The observed activity is encouraging, especially in the subset of patients with head and neck cancer.

Cisplatin-epirubicin-paclitaxel weekly administration with G-CSF support in advanced breast cancer. A Southern Italy Cooperative Oncology Group (SICOG) phase II study.

PURPOSE: It has been shown in vitro that both cisplatin and epirubicin increase the antitumor activity of paclitaxel. Weekly administration could give a substantial improvement in the therapeutic index of cisplatin and paclitaxel. This study was aimed at defining the antitumor activity of a weekly cisplatin-epirubicin-paclitaxel (PET) administration in locally advanced or metastatic breast cancer patients. PATIENTS AND METHODS: Sixty-eight breast cancer patients with advanced disease, who had not received prior chemotherapy (except adjuvant), received weekly cisplatin 30 mg/sqm, paclitaxel 120 mg/sqm and epirubicin 50 mg/sqm plus G-CSF (day 3-5), for a maximum of 12 cycles. Thirty-five patients had stage IIIB and 33 stage IV disease (14 with visceral metastases). RESULTS: All patients were evaluable for response on an intent to treat basis. Overall, 21 complete and 38 partial responses have been recorded for an 87% ORR (95% CI = 76-94%). Fourteen CRs and 19 PRs have been registered in the 35 patients with locally advanced disease for a 94% ORR (95% CI = 81-99%) while 7 CRs and 19 PRs were observed in the 33 patients with metastatic disease for a 79% ORR (95% CI-61-91%). Surgery was performed in 33/35 women with locally advanced disease. Four of these patients (11%) showed no invasive cancer on pathologic examination, and in an additional 8 patients tumor < 1 cm was found in the breast. Only 4/33 patients who underwent surgery relapsed. The projected one-year RFS was greater than 80%. At an 11-month median follow-up (range, 3-19), 11 patients had progressed and 5 had died among the 33 patients with metastatic disease, the median progression-free survival in this group being 14 months. Severe hematologic toxicity was uncommon, grade 3-4 neutropenia and thrombocytopenia occurring in 32% and 4% of patients, respectively. Only 2 episodes of neutropenic sepsis were registered. Packed red blood cell transfusions were required in 7 patients. Vomiting, diarrhoea, mucositis and skin toxicity were severe in 6%, 9%, 10%, and 9% of patients, respectively. Peripheral neuropathy was observed in 47% of patients. CONCLUSIONS: The weekly PET administration is a well tolerated and very effective approach in advanced breast cancer patients. It can produce a 40% clinical complete response rate, with a more than 10% pCR rate in patients with T4 disease, and an about 80% ORR in those with distant metastases. A phase III trial comparing PET with a standard every 3 weeks epirubicin-taxol administration is underway.

Cisplatin, raltitrexed, levofolinic acid and 5-fluorouracil in locally advanced or metastatic squamous cell carcinoma of the head and neck: a phase I-II trial of the Southern Italy Cooperative Oncology Group (SICOG).

BACKGROUND: The combination of cisplatin (CDDP) and 5-fluorouracil (5-FU) can be regarded as a reference regimen in squamous cell carcinoma of the head and neck (SCCHN). Raltitrexed (Tomudex) is a direct and specific thymidilate synthase (TS) inhibitor, which has shown clinical activity against SCCHN in a previous phase I study, when combined with 5-FU and levo-folinic acid (LFA). Preclinical data support the combination of CDDP and raltitrexed. The aim of the present study was to evaluate the combination of cisplatin, raltitrexed. LFA and 5-FU in a phase I-II study. PATIENTS AND METHODS: Patients with locally advanced or metastatic SCCHN were treated with a combination of cisplatin at the starting dose of 40 mg/m2. followed by raltitrexed at the starting dose of 2.5 mg/m2 on day 1; levo-folinic acid at fixed dose of 250 mg/m2, followed by 5-fluorouracil at the starting dose of 750 mg/m2 on day 2. Doses of the three cytotoxic agents were alternately escalated up to dose-limiting toxicity (DLT). Treatment was recycled every two weeks and given up to a maximum of eight courses; after chemotherapy, patients with locally advanced disease received a locoregional treatment. RESULTS: Forty-five patients were entered into the study. Six dose levels were tested. At CDDP 50 mg/m2, raltitrexed 3 mg/m2, 5-FU 900 mg/m2, four out of six patients showed DLT, which was in all cases grade 4 neutropenia. Therefore, this dose level was defined as maximum tolerated dose (MTD). CDDP 60 mg/m2, raltitrexed 2.5 mg/m2, LFA 250 mg/m2, 5-FU 900 mg/m2 was the dose level recommended for phase II. CDDP, Raltitrexed and 5-FU mean actually delivered dose intensities at the selected dose level were 26, 1.05, and 378 mg/m2/week, respectively. Neutropenia was the main side effect and was observed even at the lowest dose levels. Nonhematologic side effects were mild. Nine complete responses (20%) and twenty-one partial responses (47%) were observed, for an overall response rate of 67% (95% confidence interval (95% CI): 51%-80%), according to intention to treat analysis. Fifteen of fifteen patients (100%) treated at the dose level selected for phase II had an objective response (5 complete responses, 10 partial responses). CONCLUSIONS: The results of our dose escalation clearly demonstrate that it is possible to combine CDDP, raltitrexed, and modulated 5-FU at effective doses, without unexpected toxicities. The response data point to an impressive clinical activity, which will be better defined by an ongoing large phase II study.

Oxaliplatin and raltitrexed combined with leucovorin-modulated 5-fluorouracil i.v. bolus every two weeks: a dose finding study in advanced previously treated colorectal carcinoma. Southern Italy Cooperative Oncology Group.

PURPOSE: To determine the maximum tolerated dose of oxaliplatin (L-OHP) given as a two-hour infusion followed by raltitrexed (Tomudex [TOM]) administered as a 15-min infusion on day 1, and bolus 5-fluorouracil (5-FU) modulated by a fixed dose of levo-folinic acid (LFA) 250 mg/m2 on day 2, recycling every two weeks, and to have preliminary evidence of activity of this combination in pretreated advanced colorectal cancer patients. PATIENTS AND METHODS: Fifty-two patients with advanced colorectal carcinoma previously treated with one (25 cases) or two or more lines of chemotherapy, including irinotecan (26 cases), and/or modulated 5-FU (40 cases) entered this study. Starting doses of L-OHP, TOM, and 5-FU were 85, 2.5 and 750 mg/m2, respectively. RESULTS: Seven dose levels were tested. Neutropenia was the main dose limiting toxicity of the dose escalation (8 of 13 cases). The recommended doses were 130 mg/m2 of L-OHP, and 3.0 mg/m2 of TOM on day 1, followed by 250 mg/m2 of LFA, and 1050 mg/m2 of 5-FU on day 2, every two weeks. Severe diarrhoea and stomatitis were rarely reported. Most patients complained of mild peripheral sensitive aeurotoxicity, which was related to the cumulative dose of L-OHP. Twelve patients were considered as having a major responses (one complete), and an additional eight patients showed a minor response; the median time to treatment failure was twenty-four weeks. CONCLUSIONS: With this regimen it is possible to give full doses of all three cytotoxic drugs every two weeks. Its activity and its manageable toxicity profile deserve further evaluation in pretreated advanced colorectal cancer patients.

Ribonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA.

The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized rne-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.

Alternative patterns of his operon transcription and mRNA processing generated by metabolic perturbation.

Previous studies have shown that the expression of the his operon of Salmonella typhimurium is regulated at the level of transcription initiation, transcription elongation and RNA processing. We have analyzed his RNA in both prototrophic strains or strains harboring regulatory and auxotrophic mutations grown under a variety of metabolic conditions that lead to differential expression of the operon. Under some of these conditions, there is an increase in the amount of prematurely released his-specific RNA, resulting in modulation of the relative amount of full-length transcripts. Under the same metabolic conditions, there is also a modulation of RNA processing events that generate a very stable RNA species comprising the five distal cistrons. These effects appear to be due to perturbation of the translation process caused by alterations in the intracellular pool of initiator transfer RNA.

A tRNA gene mapping within the chloroplast rDNA cluster is differentially expressed during the development of Daucus carota.

In vivo analysis of expression of the chloroplast rDNA cluster during somatic embryogenesis of Daucus carota (D.carota) was performed by Northern-blot analysis with different DNA probes, spanning both the 16S rRNA gene, the 16S-23S rRNA spacer, which contains the two mosaic tRNA genes tRNA(Ile) and tRNA(Ala), and the region upstream of the 16S rRNA gene, where a tRNA(Val) maps. We show that expression both of the spacer tRNAs tRNA(Ile) and tRNA(Ala) is not significantly regulated during development whereas the amount of the transcript corresponding to tRNA(Val) is not detectable during early embryonic stages and progressively accumulates during late phases. Multiple transcription start sites have been identified upstream of the tRNA(Val) gene by S1 mapping analysis, which are activated late during the embryogenesis. These data indicate that developmental control mechanisms act on plastid gene expression during embryogenesis in carrot.

Characterization of the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium.

We have cloned and sequenced the genomic regions encompassing the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium. Rho factor of S. typhimurium has only three amino acid differences with respect to the Escherichia coli homolog. Northern (RNA) blots and primer extension experiments were used to characterize the N. gonorrhoeae rho transcript and to identify the transcription initiation and termination elements of this cistron. The function of the Rho factor of N. gonorrhoeae was investigated by complementation assays of rho mutants of E. coli and S. typhimurium and by in vivo transcription assays in polar mutants of S. typhimurium.

The histidine operon of Azospirillum brasilense: organization, nucleotide sequence and functional analysis.

A 3457-base pair fragment of Azospirillum brasilense DNA which complemented mutations in the hisA and hisF genes of Escherichia coli was sequenced. The sequence analysis revealed the presence of six major contiguous open reading frames (ORF). The comparison of the predicted amino acid sequence of these ORF with those encoded by the eubacterial, archaebacterial and eukaryotic his genes sequenced thus far revealed that four of them have a significant degree of homology with the E. coli hisH, hisA, hisF and the C-terminal domain of the hisI gene products. S1 mapping experiments indicated that the putative transcription start site coincided with the AUG translational initiation codon of the hisBd gene, the first gene of the A. brasilense his operon. Downstream from the last ORF, a sequence was identified which functions as a Rho-independent transcription terminator. Comparison of amino acid sequences, gene order and organization and evolutionary aspects of the A. brasilense his cluster are discussed.

Processing of a polycistronic mRNA requires a 5' cis element and active translation.

We have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild-type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis-acting element and translation of the cistron immediately downstream of the 5' end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter.

A cytosine- over guanosine-rich sequence in RNA activates rho-dependent transcription termination.

We have constructed an expression vector carrying the Escherichia coli his operon control region to study the ability of defined segments of DNA to cause rho factor-mediated transcription termination both in vivo and in vitro. We have previously identified a consensus motif consisting of a region of high cytosine over guanosine content common to several cryptic intracistronic transcription termination elements unmasked by polar mutations. We show that a DNA fragment possessing features similar to the ones previously identified is capable of causing rho-mediated mediated release of transcripts in vivo and in vitro. The efficiency of termination depends on the length and efficiency of termination depends on the length and relative cytosine over guanosine ratio of the element.

A consensus motif common to all Rho-dependent prokaryotic transcription terminators.

We have characterized at the molecular level several polar mutations in four different cistrons of the his operon of S. typhimurium. An analysis of the his-specific transcripts produced in vivo in the mutant strains, together with in vitro transcription assays, led to the identification of several cryptic Rho-dependent transcription termination elements within the his operon that are activated by the uncoupling of transcription and translation. Common features of these elements were sought and found with a computer program. We have identified a consensus motif, consisting of a cytosine-rich and guanosine-poor region, that is located upstream of the heterogeneous 3' endpoints of the prematurely terminated in vivo transcripts and that is present in all the Rho-dependent transcription terminators described thus far.