A genetic variant in Rassf1a predicts outcome in mCRC patients treated with cetuximab plus chemotherapy: results from FIRE-3 and JACCRO 05 and 06 trials.

The Hippo pathway is involved in colorectal cancer (CRC) development and progression. The Hippo regulator Rassf1a is also involved in the Ras signaling cascade. In this work, we tested single nucleotide polymorphisms within Hippo components and their association with outcome in CRC patients treated with cetuximab. Two cohorts treated with cetuximab plus chemotherapy were evaluated (198 RAS wild-type (WT) patients treated with first-line FOLFIRI plus Cetuximab within the FIRE-3 trial and 67 Ras WT patients treated either with first-line mFOLFOX6 or SOX plus Cetuximab). In these two populations, Rassf1a rs2236947 was associated with overall survival (OS), as patients with a CC genotype had significantly longer OS compared with those with CA or AA genotypes. This association was stronger in patients with left-side CRC (hazard ratio (HR): 1.79 (1.01-3.14); P=0.044 and HR: 2.83 (1.14-7.03); P=0.025, for Fire 3 and JACCRO cohorts, respectively). Rassf1a rs2236947 is a promising biomarker for patients treated with cetuximab plus chemotherapy.

Genetic variants of DNA repair-related genes predict efficacy of TAS-102 in patients with refractory metastatic colorectal cancer.

Background: Tri-phosphorylated trifluridine (FTD) incorporation into DNA is TAS-102's main anti-tumor action. We tested whether genetic polymorphisms in homologous recombination (HR) and cell cycle checkpoint pathway for DNA repair is associated with outcomes in refractory metastatic colorectal cancer (mCRC) patients treated with TAS-102. Patients and methods: We analyzed genomic DNA extracted from 233 samples of three cohorts: an evaluation cohort of 52 patients receiving TAS-102, a validation cohort of 129 patients receiving TAS-102 and a control cohort of 52 patients receiving regorafenib. Single nucleotide polymorphisms of genes involved in HR (ATM, BRCA1, BRCA2, XRCC3, FANCD2, H2AX, RAD51) and cell cycle checkpoint (ATR, CHEK1, CHEK2, CDKN1A, TP53, CHE1, PIN1, PCNA) were analyzed by PCR-based direct sequencing. Results: In univariate analysis for the evaluation cohort, patients with any G allele in ATM rs609429 had longer overall survival (OS) than those with the C/C variant (8.7 vs. 4.4 months, HR 0.37, 95% CI: 0.14-0.99, P = 0.022). Patients carrying any A allele in XRCC3 rs861539 had significantly longer progression-free survival (PFS) (3.8 vs. 2.3 months, HR 0.44, 95% CI: 0.21-0.92, P = 0.024) and OS (15.6 vs. 6.3 months, HR 0.25, 95% CI: 0.08-0.79, P = 0.012) than those with the G/G variant. In multivariable analysis, ATM rs609429 remained significant for OS (P = 0.020). In the validation cohort, patients having ATM rs609429 with any G allele showed longer OS and PFS; the G/A variant in XRCC3 rs861539 showed longer OS, though without statistical significance. Conclusion: Genetic variants in the HR pathway may predict clinical outcome in mCRC patients receiving TAS-102.

Association of variants in genes encoding for macrophage-related functions with clinical outcome in patients with locoregional gastric cancer.

BACKGROUND: Nuclear factor-kappaB (NF-κB) and CCL2/CCR2 chemokine axis play a central role in tumor progression such as stimulation of angiogenesis, acceleration of tumor invasion and migration, and suppression of innate immunosurveillance in the macrophage-related functions. There have been few reports regarding association of the macrophage function-related genes with the clinical outcome in gastric cancer. We hypothesized that variants in genes encoding for NF-κB and CCL2/CCR2 axis may predict prognosis in gastric cancer and tested whether the functional single-nucleotide polymorphisms (SNPs) will be associated with clinical outcome in patients with gastric cancer across two independent groups. PATIENTS AND METHODS: This study enrolled two cohorts which consisted of 160 Japanese patients and 104 US patients with locoregional gastric cancer. Genomic DNA was analyzed for association of 11 SNPs in NFKB1, RELA, CCL2, and CCR2 with clinical outcome using PCR-based direct DNA sequencing. RESULTS: The univariable analysis showed four SNPs had significant association with clinical outcome in the Japanese cohort, NFKB1 rs230510 remained significant upon multivariable analysis. The patients with the A allele of the NFKB1 rs230510 had significantly longer overall survival (OS) compared with those with the T/T genotype in both the Japanese and US cohort in the univariable analysis. In contrast, genotypes with the T allele of CCL2 rs4586 were significantly associated with shorter OS compared with the C/C genotype in the US cohort [hazard ratio (HR) 2.43; P = 0.015] but longer OS in the Japanese cohort (HR 0.58; P = 0.021), resulting in the statistically significant opposite impact on OS (P = 0.001). CONCLUSIONS: Our study provides the first evidence that the NFKB1 rs230510 and CCL2 rs4586 are significantly associated with the clinical outcome in patients with locoregional gastric cancer. These results also suggest that the genetic predisposition of the host may dictate the immune-related component of the tumor for progression in gastric cancer.

Variations in genes involved in dormancy associated with outcome in patients with resected colorectal liver metastases.

BACKGROUND: Tumor dormancy has been described as a state of hibernation. Dormancy can be switched to proliferation by different pathways, which may play a critical role in tumor recurrence. In this study, we investigated genetic variations within genes involved in tumor dormancy and their association with recurrence and outcome in patients with colorectal liver metastases (CLM) who underwent neoadjuvant bevacizumab-based chemotherapy. PATIENTS AND METHODS: Genomic DNA was extracted from resected CLM (FFPE) from 149 patients. Single-nucleotide polymorphisms (SNPs) in 14 genes associated with dormancy were analyzed by direct Sanger DNA sequencing and evaluated for response, recurrence-free survival (RFS), overall survival (OS) and recurrence patterns. RESULTS: NME1 rs34214448 C>A was significantly associated with RFS in univariable analysis (P = 0.039) and with intrahepatic recurrence (P = 0.014). NOTCH3 rs1044009 T>C and CD44 rs8193 C>T showed a significant difference in 3-year OS rates (P = 0.004 and P = 0.042, respectively). With respect to radiological response, CD44 rs8193 C>T variant genotypes were associated with a significantly higher response rate (P = 0.033). Recursive partitioning analyses revealed that Dll4 rs12441495 C>G, NME1 rs34214448 C>A and NOTCH3 rs1044009 T>C were the dominant SNPs predicting histological response, RFS and OS, respectively. CONCLUSION: Our data suggest that gene variations within genes involved in tumor dormancy pathways are associated with response and outcome in patients with resected CLM. These data may lead to new and more effective treatment strategies targeting tumor dormancy.

Distinct gene expression profiles of proximal and distal colorectal cancer: implications for cytotoxic and targeted therapy.

Colorectal cancer (CRC) is a heterogeneous disease with genetic profiles and clinical outcomes dependent on the anatomic location of the primary tumor. How location has an impact on the molecular makeup of a tumor and how prognostic and predictive biomarkers differ between proximal versus distal colon cancers is not well established. We investigated the associations between tumor location, KRAS and BRAF mutation status, and the messenger RNA (mRNA) expression of proteins involved in major signaling pathways, including tumor growth (epidermal growth factor receptor (EGFR)), angiogenesis (vascular endothelial growth factor receptor 2 (VEGFR2)), DNA repair (excision repair cross complement group 1 (ERCC1)) and fluoropyrimidine metabolism (thymidylate synthase (TS)). Formalin-fixed paraffin-embedded tumor specimens from 431 advanced CRC patients were analyzed. The presence of seven different KRAS base substitutions and the BRAF V600E mutation was determined. ERCC1, TS, EGFR and VEGFR2 mRNA expression levels were detected by reverse transcriptase-PCR. BRAF mutations were significantly more common in the proximal colon (P<0.001), whereas KRAS mutations occurred at similar frequencies throughout the colorectum. Rectal cancers had significantly higher ERCC1 and VEGFR2 mRNA levels compared with distal and proximal colon tumors (P=0.001), and increased TS levels compared with distal colon cancers (P=0.02). Mutant KRAS status was associated with lower ERCC1, TS, EGFR and VEGFR2 gene expression in multivariate analysis. In a subgroup analysis, this association remained significant for all genes in the proximal colon and for VEGFR2 expression in rectal cancers. The mRNA expression patterns of predictive and prognostic biomarkers, as well as associations with KRAS and BRAF mutation status depend on primary tumor location. Prospective studies are warranted to confirm these findings and determine the underlying mechanisms.

2,2'-Bipyridyl-6-carbothioamide and its ferrous complex: their in vitro antitumoral activity related to the inhibition of ribonucleotide reductase R2 subunit.

2,2'-Bipyridyl-6-carbothioamide (BPYTA), a synthetic compound with antitumoral activity, is characterized by chelating properties because of the N*-N*-S* tridentate ligand system and is therefore comparable to alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazones which are potent inhibitors of ribonucleotide reductase (RR). Electron paramagnetic resonance studies on the small subunit of mouse recombinant RR (R2) demonstrated that BPYTA can destroy the R2 tyrosyl radical only if Fe(II) is present (73% destruction at 50 microM, after 20 min of contact). The R2 inhibition was reversible and time dependent. Studies on tumoral lines confirmed that the main cell target of BPYTA is RR and demonstrated that the iron-complexed form compared to the nonchelated form has some difficulty in crossing the cell membrane. Spectrophotometric and electron paramagnetic resonance studies clearly indicated that BPYTA chelates iron only when this is reduced and that the BPYTA-Fe(II) complex is stable in the presence of oxygen. From reported results we conclude that BPYTA is a powerful RR inhibitor (R2 subunit) which has a different mechanism of action from that of Desferal. It has some properties in common with alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazones, but they are not identical. It would be interesting to do further studies on the BPYTA mechanism of action and evaluate the in vivo antitumoral activity of the preformed complex.

A viral immunodepressive factor associated with experimental mouse tumors.

An immunodepressive factor (IDF) capable of inhibiting the rejection of allogeneic lymphomas in mice was detected in a number of transplantable mouse tumors. The allograft reactivity of the host was impaired for at least 10 months after IDF administration. No clinical evidence of bacterial or viral infection was detected in IDF-treated animals. Sera collected from mice at various intervals after injection of IDF showed a great increase in the IDF-titer. The IDF persisted in mouse sera for at least 3 months, whereas no IDF activity was found in sera of rats given injections of the factor. IDF was capable of replicating in vitro in mouse embryo cells, but not in rat embryo or HeLa cell cultures. IDF was inactivated in vitro by heat (65 degrees for 30 min), ultraviolet light or ether, but not by ethyl alcohol. These studies indicate that IDF is a virus capable of producing a long-lasting asymptomatic infection specifically interfering with the host's allograft reactivity. In several instances a close association between the lactic dehydrogenase virus and IDF was found. Nevertheless no conclusion was reached on the identity or nonidentity between the two viruses.

Prostatectomy at high-volume centers improves outcomes and lowers the costs of care for prostate cancer.

BACKGROUND: High-volume surgeons with ⩾250 radical prostatectomies provide superior oncological outcomes as evidenced by a lower rate of PSA recurrence (PSAR). The financial benefits of performing prostatectomies at high-volume centers (HVC) are unexplored. METHODS: A base case--referent scenario--where the share of prostatectomies at high- and low-volume centers were evenly divided at 50% was defined. Additional scenarios with increasing shares of prostatectomies at HVC with 10% increments were also modeled. Using a lower probability of PSAR as the only advantage of more experienced surgeons, the savings that would result from fewer recurrences, avoidance of salvage radiation therapy (SRT) and management of fewer men with metastatic cancer were calculated. RESULTS: The savings associated with performing 80% of radical prostatectomy at HVC were $177, $357 and $559 per prostatectomy at 5, 10 and 20 years, respectively. These savings would offset referral costs of up to $1833 per prostatectomy referral at no additional total societal costs. Given the longer average biochemical failure-free survival with prostatectomies at HVC, referral costs of more than $1833 may be cost effective. CONCLUSIONS: Under the conservative assumption of accounting for lower rates of PSAR as the only benefit of surgery in an HVC, performing prostatectomies at an HVC was associated with savings that may offset part of the initial referral costs.

Design and synthesis of modified quinolones as antitumoral acridones.

The bacterial topoisomerase II (DNA gyrase) and the mammalian topoisomerase II represent the cellular targets for quinolone antibacterials and a wide variety of anticancer drugs, respectively. In view of the mechanistic similarities and sequence homologies exhibited by the two enzymes, tentative efforts to selectively shift from an antibacterial to an antitumoral activity was made by synthesizing a series of modified tricyclic quinolones, in which the essential 3-carboxylic function is surrogated by phenolic OH and the classic C-6 fluorine atom is replaced by a NH2 group. The resulting 7-amino-9-acridone derivatives were assayed for their antibacterial as well as cytotoxic activities. No antibacterial activity was found. On the other hand, many derivatives showed significant cytotoxic activity against both HL-60 and P388 leukemias and a wide panel of human and rodent solid tumor cells, derivatives 25 and 26 displaying the best overall antiproliferative activity. Against the LoVo cell line, derivative 25 exhibited higher cytotoxic effects than etoposide.

Furanfurin and thiophenfurin: two novel tiazofurin analogues. Synthesis, structure, antitumor activity, and interactions with inosine monophosphate dehydrogenase.

The syntheses of furan and thiophene analogues of tiazofurin (furanfurin and thiophenfurin, respectively) are described. Direct stannic chloride-catalyzed C-glycosylation of ethyl 3-furan-carboxylate (6) or ethyl 3-thiophencarboxylate (18) with 1,2,3,5-tetra-O-acetyl-D-ribofuranose gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivatives. Deprotection of ethyl 5-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)furan-3-carboxylate (9 beta) and ethyl 5-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)thiophene-3-carboxylate (20 beta) with sodium ethoxide afforded ethyl 5-beta-D-ribofuranosylfuran-3-carboxylate (12 beta) and ethyl 5-beta-D-ribofuranosylthiophene-3-carboxylate (23 beta) which were converted into 5-beta-D-ribofuranosylfuran-3-carboxamide (furanfurin, 4) and 5-beta-D-ribofuranosylthiophene-3-carboxamide (thiophenfurin, 5) by reaction with ammonium hydroxide. The anomeric configuration and the site of glycosylation were established by 1H-NMR and proton-proton nuclear Overhauser effect difference spectroscopy. The structure of compound 23 beta was confirmed by X-ray crystallography. Thiophenfurin was found to be cytotoxic in vitro toward murine lymphocytic leukemia P388 and L1210, human myelogenous leukemia K562, human promyelocytic leukemia HL-60, human colon adenocarcinoma LoVo, and B16 melanoma at concentrations similar to that of tiazofurin. In the same test furanfurin proved to be inactive. Thiophenfurin was found active in vivo in BD2F1 mice inoculated with L1210 cells with a % T/C of 168 at 25 mg/kg. K562 cells incubation with thiophenfurin resulted in inhibition of inosine monophosphate (IMP) dehydrogenase (63%) and an increase in IMP pools (6-fold) with a concurrent decrease in GTP levels (42%). Incubation of adenosine-labeled K562 cells with tiazofurin, thiophenfurin, and furanfurin resulted in a 2-fold higher NAD analogue formulation by thiophenfurin than by tiazofurin. Furanfurin was converted to the NAD analogue with only 10% efficiency. The results obtained support the hypothesis that the presence of S in the heterocycle in position 2 with respect to the glycosidic bond is essential for the cytotoxicity and IMP dehydrogenase activity of tiazofurin, while the N atom is not.

The immunodepressive and hematotoxic activity of imidazole-4-carboxamide,5-(3,3-dimethyl-1-triazeno) in mice.

The immunodepressive and bone marrow stem cell-reducing activities of imidazole-4-carboxamide,5-(3,3-dimethyl-1-triazeno) (DTIC), an antitumoral agent reported to possess little immunodepressive capacity in man, have been investigated in mice and compared with those displayed by cyclophosphamide (Cy). In this species, single doses of DTIC could profoundly depress antilymphoma allograft resistance as well as the number of antibody-producing cells after primary and secondary stimulation with sheep erythrocytes. The highest immunodepressive activity was observed when DTIC was given before antigen and the effect observed was substantially more long lasting than seen with Cy, which was, however significantly more active on a milligram per kilogram basis. In contrast, both agents displayed a quantitatively equal activity in reducing bone marrow stem cells. It is concluded that DTIC can be an effective immunodepressant and that this activity may have clinical implications.

The 2,2'-bipyridyl-6-carbothioamide copper (II) complex differs from the iron (II) complex in its biochemical effects in tumor cells, suggesting possible differences in the mechanism leading to cytotoxicity.

2,2'-bipyridyl-6-carbothioamide (BPYTA) is an antitumor agent with chelating properties. It has previously been shown that the R2 subunit of ribonucleotide reductase (RR) is its major cellular target, but RR inhibition is observed only in the presence of ferrous iron (BPYTA-Fe, molar ratio 2:1). Because the copper (II) complex of BPYTA (BPYTA-Cu, molar ratio 1:1)) has in vitro antitumor activity comparable to that of BPYTA-Fe, we studied the mechanism of action of this new metal complex. Spectorphotometric and HPLC studies demonstrated that, at pH 7.5, BPYTA-Cu is stable at molar ratio 2:1 and copper is in its favored oxidized form [BPYTA-Cu(II)]. Electron paramagnetic resonance (EPR) studies with mouse recombinant R2 demonstrated that BPYTA-Cu destroys the R2 tyrosyl radical at the same concentration at which BPYTA-Fe does (78% vs 73% destruction at 200 microM, with 5 min of contact), but R2 inhibition is not time-dependent. Studies of the metabolism of [14C] cytidine suggest that the cytotoxic activity of BPYTA-Cu can be explained in terms of RR inhibition. However, the significant inhibition of RNA synthesis and the lack of cross-resistance to BPYTA-Cu of cell lines resistant to other RR inhibitors suggest that BPYTA-Cu may have more than one cellular target. Moreover, cell proliferation studies suggest that, unlike BPYTA-Fe, BPYTA-Cu displays its activity immediately after contact with the target cells. Our study demonstrates that significant differences in the biochemical effects of BPYTA and, perhaps, also its mechanism of action are due solely to the bonded transition metalloelement. This might also be the case with other chelators that demonstrate cytotoxic activity following metalloelement chelation.

Implications and problems in analysing cytotoxic activity of hydroxyurea in combination with a potential inhibitor of ribonucleotide reductase.

The cytotoxicity of hydroxyurea in combination with 2.2'-bipyridyl-6-carbothioamide (a potential inhibitor of ribonucleotide reductase) on P388 murine leukemia is reported. Synergistic activity was studied using various interpretations of the isobologram method and the combination index method. We evaluated the pros and cons of these methods and their overall usefulness. In our opinion, to obtain all possible information from a compound association, it is important to choose a formally correct method that (a) can quantitatively evaluate synergism or antagonism, (b) may offer the possibility of averaging final results, (c) needs a minimal amount of experimental data, and (d) is rapid. Moreover, we emphasize both the utility of testing at least three molar ratios of compound association and the importance of carefully choosing the fractional inhibition used in calculating the combination effect. Such evaluation of drug combinations gives information essential to the preparation of new anticancer drug regimens and to the early assessment of biochemical interactions.

In vitro chemosensitivity testing in acute leukemias. Results of a retrospective study.

A retrospective study was carried out on 67 patients with ANLL and ALL to assess the capacity of a rapid in vitro chemosensitivity assay to predict clinical response and patient survival. The test evaluates the extent to which a drug inhibits DNA synthesis by measuring the incorporation of 125Iododeoxyuridine into leukemic cell DNA. Correlations between the in vitro chemosensitivity data and clinical outcome showed that this test predicts the clinical response to remission-inducing drugs with a good level of accuracy (sensitivity = 0.82, specificity = 0.71 but that it is unable to predict length of survival.

Correlation between lowered PgE2 levels and alkaline reflux gastritis in patients subjected to Billroth II. Pylorogastrectomy: possible improvement with sulglycotide.

In patients subjected to pylorogastrectomy with anterior gastroenterostomy (Billroth's operation II) the remaining gastric mucosa is exposed to the damaging action of bile with consequent reduction of locally generated prostaglandins. The purpose of the present study was to explore the correlation between PgE2 levels in such postgastrectomy patients before and after treatment with sulglycotide. Twelve patients with clinical, endoscopic and histological evidence of alkaline reflux gastritis were treated with sulglycotide in daily doses of 1600 mg for 30 days. At termination there was a definite clinical, endoscopic and histological improvement with significant (p less than 0.001) increase of PgE2 levels.

Immunologic reactivity in patients bearing solid tumors.

The immune reactivity of 50 patients with solid tumors before therapeutic treatment and 26 healthy controls was assessed by both in vitro and in vivo tests. No significant differences between controls and patients were found in peripheral lymphocyte counts or in delayed hypersensitivity to recall antigens. However, impairment of lymphocyte stimulation with phytohemagglutinin (PHA) and a decrease in the number of E-rosette forming cells (E RFC) were detected in cancer-bearing patients irrespective of tumor type. The results of the present study further confirm the usefulness of the PHA and E-RFC tests in monitoring the immune competence of cancer patients.

In vitro chemosensitivity of newly diagnosed and relapsing acute non-lymphoid leukemia patients.

We evaluated the in vitro sensitivity of circulating blasts from 25 newly diagnosed and 7 relapsing ANLL patients to drugs employed in vivo for inducing remission. Ten of the 14 newly diagnosed complete responders were in vitro sensitive to cytosine arabinoside and daunorubicin, whereas 10/11 non-responders were resistant to both agents. Although cells from all 7 relapsing patients were in vitro sensitive to the remission inducing agents, only 4 entered complete remission. Even if only indicative, these findings suggest that the poor prognosis of relapsing patients may be due, at least in part, to factors other than drug resistance. Moreover, the chemosensitivity test adopted is a better predictor in newly diagnosed than relapsing patients, as indicated by the concordance between in vitro and in vivo results.

Combination chemotherapy with 5-fluorouracil and methyl-CCNU for the treatment of advanced gastrointestinal cancer.

Sixteen patients with advanced gastrointestinal cancer (colorectal 12/16, gastric 4/16) were treated with a combination of 5-fluorouracil (5-FU) plus 1-(2-chlorethyl)-3(4-methyl-cycloexyl)-1-nitrosourea (Me-CCNU). The therapeutic program consisted of orally administered Me-CCNU (140 mg/m2) and intravenous 5-FU (9.5 mg/kg by bolus injection for 5 days). The cycles were repeated at 6-week intervals. At the beginning of the therapy, 11/16 patients were in performance status (PS) 0-1 and 5 patients in PS 2-3. Eight patients developed early progressive disease between the 1st and 2nd course of therapy. Only a minor tumor response was observed in the remaining 50% of the patients. However, the patients with stabilized disease lived longer (11.8 months) than non-responders (3.5 months).

[Pirenzepine parenterally administered in high doses in the treatment of upper digestive hemorrhage]. / La pirenzepina per via parenterale ad alto dosaggio nel trattamento delle emorragie digestive alte.

22 patients with moderate and severe haemorrhages of the UAC were given endovenous doses of Pirenzepine (100 mg/48 hrs). The drug was effective in 86% of the cases and its effect on pH values was demonstrated by testing before and after administration (p less than 0.01). Better results were observed in the treatment of patients with moderate and severe DUS.

An in vitro assay for evaluating chemosensitivity of leukaemia cells: preclinical studies.

The present study was aimed at defining the standard conditions for predictive chemosensitivity testing of human leukaemias. The in vitro cytotoxic potential of 11 anticancer drugs against L1210 murine leukaemia was comparatively determined 3 and 48 h after in vitro exposure of leukaemia cells to these agents. In preliminary testing, drug-induced damage was estimated in terms of percent inhibition of: tumour cell proliferation, by microscopic determination of viable cell numbers; and DNA synthesis, by measuring the extent of incorporation of the labelled nucleoside 125IUdR. Since the two tests appeared to yield comparable results, all subsequent experiments on the various murine tumours were performed using the radiolabel incorporation technique. The results obtained pointed out that this metabolic assay performed 48 hours after drug exposure appears to be capable of predicting both "spontaneous" and "induced" in vivo resistance of murine leukaemias.