Amivantamab plus lazertinib versus osimertinib in first-line EGFR-mutant advanced non-small-cell lung cancer with biomarkers of high-risk disease: a secondary analysis from MARIPOSA.

BACKGROUND: Amivantamab-lazertinib significantly prolonged progression-free survival (PFS) versus osimertinib in patients with epidermal growth factor receptor (EGFR)-mutant advanced non-small-cell lung cancer [NSCLC; hazard ratio (HR) 0.70; P < 0.001], including those with a history of brain metastases (HR 0.69). Patients with TP53 co-mutations, detectable circulating tumor DNA (ctDNA), baseline liver metastases, and those without ctDNA clearance on treatment have poor prognoses. We evaluated outcomes in these high-risk subgroups. PATIENTS AND METHODS: This analysis included patients with treatment-naive, EGFR-mutant advanced NSCLC randomized to amivantamab-lazertinib (n = 429) or osimertinib (n = 429) in MARIPOSA. Pathogenic alterations were identified by next-generation sequencing (NGS) of baseline blood ctDNA with Guardant360 CDx. Ex19del and L858R ctDNA in blood was analyzed at baseline and cycle 3 day 1 (C3D1) with Biodesix droplet digital polymerase chain reaction (ddPCR). RESULTS: Baseline ctDNA for NGS of pathogenic alterations was available for 636 patients (amivantamab-lazertinib, n = 320; osimertinib, n = 316). Amivantamab-lazertinib improved median PFS (mPFS) versus osimertinib for patients with TP53 co-mutations {18.2 versus 12.9 months; HR 0.65 [95% confidence interval (CI) 0.48-0.87]; P = 0.003} and for patients with wild-type TP53 [22.1 versus 19.9 months; HR 0.75 (95% CI 0.52-1.07)]. In patients with EGFR-mutant, ddPCR-detectable baseline ctDNA, amivantamab-lazertinib significantly prolonged mPFS versus osimertinib [20.3 versus 14.8 months; HR 0.68 (95% CI 0.53-0.86); P = 0.002]. Amivantamab-lazertinib significantly improved mPFS versus osimertinib in patients without ctDNA clearance at C3D1 [16.5 versus 9.1 months; HR 0.49 (95% CI 0.27-0.87); P = 0.015] and with clearance [24.0 versus 16.5 months; HR 0.64 (95% CI 0.48-0.87); P = 0.004]. Amivantamab-lazertinib significantly prolonged mPFS versus osimertinib among randomized patients with [18.2 versus 11.0 months; HR 0.58 (95% CI 0.37-0.91); P = 0.017] and without baseline liver metastases [24.0 versus 18.3 months; HR 0.74 (95% CI 0.60-0.91); P = 0.004]. CONCLUSIONS: Amivantamab-lazertinib effectively overcomes the effect of high-risk features and represents a promising new standard of care for patients with EGFR-mutant advanced NSCLC.

Morphological and immunohistochemical study of the midgut and fat body of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: noctuidae) treated with essential oils of the genus Piper.

Essential oils are a promising alternative to insecticides. We investigated the LD50 of oils extracted from Piper corcovadensis, P. marginatum, and P. arboreum after 48 h topical contact with Spodoptera frugiperda larvae using morphometry, histochemistry and immunohistochemistry of the midgut and fat body. Chromatography revealed that E-caryophyllene was the principal compound common to the Piper species. The essential oils of P. corcovadensis, P. marginatum and P. arboreum caused deleterious changes in the midgut of S. frugiperda larvae. P. corcovadensis oil produced the lowest LD50 and significant histopathological alterations including elongation of the columnar cells, formation of cytoplasmic protrusions, reduction in carbohydrate, increased apoptotic index and decreased cell proliferation. P. arboreum oil caused histopathological alterations similar to P. corcovadensis, but caused the highest rate of cell proliferation and increased regenerative cells, which indicated rapid regeneration of the epithelium. Our findings demonstrated the insecticidal potential of P. corcovadensis for control of S. frugiperda owing to the significant damage it inflicted on S. frugiperda midgut.

Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer.

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

Exploiting HPV-Induced Carcinogenesis for a Rational Drug Development in Cervical Cancer.

Cervical carcinomas are almost universally associated with high-risk human papillomavirus (HPV) infections, and are a leading cause of cancer death in women worldwide. Since the late 1990s, when a spate of studies reported the benefit of cisplatin-based chemotherapy, there had been a dearth of clinical trials in cervical cancer (CC). More effective therapies in locally advanced and recurrent or metastatic CC are an urgent clinical need. In the era of molecular oncology one should look beyond conventional chemoradiation and chemotherapy for locally advanced and advanced CC. The fact that the initiating oncogenic insult, infection with a high-risk HPV and viral oncoprotein expression is common to almost all CC offers unique opportunities for disease control. Diverse biologic pathways with an implication in the development and progression of CC are being explored. For the first time, increase in overall survival has recently been obtained for advanced CC patients with a target drug, the antiangiogenic agent bevacizumab, and durable complete responses after HPV-targeted adoptive T cell therapy in metastatic CC patients were achieved. In this review, we will summarize molecular aspects of HPV infection focusing on potential targets to stop the carcinogenic process, present updated drug development data, and discuss challenges and prospects for the future.

Nonoperative management of rectal cancer after chemoradiation opposed to resection after complete clinical response. A comparative study.

INTRODUCTION: Surgery is the standard treatment of rectal cancer after neoadjuvant therapy. Some authors advocate a nonoperative management (NOM) after complete clinical response (cCR) following chemoradiotherapy (CRT). We compare our results with NOM to standard resection in a retrospective analysis. METHODS: Rectal adenocarcinomas submitted to NOM after CRT between September 2002 and December 2013 were compared to surgical patients that had pathological complete response (pCR) during the same period. Endpoints were Overall Survival (OS), Disease Free Survival (DFS), Local Relapse (LR) and Distant Relapse (DR). RESULTS: Forty-two NOM patients compared to 69 pCR patients operated after a median interval of 35 weeks after CRT. NOM tumors were distal (83.3% vs 59.4%, p = 0.011), less obstructive (26.2% vs 54.4%, p = 0.005) and had a lower digital rectal score (p = 0.024). Twelve (28.0%) recurrences in NOM group and eight (11.5%) in the surgical group occurred after a follow-up of 47.7 and 46.7 months respectively. Isolated LR occurred in five (11%) NOM patients and one (1.4%) in the surgical group. Four (80%) LR were surgically salvaged in NOM group. No difference in OS was found (71.6% vs 89.9%, p = 0.316) but there was a higher DFS favoring surgical group (60.9% vs 82.8%, p = 0.011). Distal tumors had worse OS compared to proximal tumors in surgical group (5-year OS of 85.5% vs 96.2%, p = 0.038). CONCLUSION: The NOM achieved OS comparable to surgical treatment and spared patients from surgical morbidity but it resulted in more recurrences. This approach cannot be advocated routinely and controlled trials are warranted.

Different Therapeutic Outcomes of Benznidazole and VNI Treatments in Different Genders in Mouse Experimental Models of Trypanosoma cruzi Infection.

The lack of translation between preclinical assays and clinical trials for novel therapies for Chagas disease (CD) indicates a need for more feasible and standardized protocols and experimental models. Here, we investigated the effects of treatment with benznidazole (Bz) and with the potent experimental T. cruzi CYP51 inhibitor VNI in mouse models of Chagas disease by using different animal genders and parasite strains and employing distinct types of therapeutic schemes. Our findings confirm that female mice are less vulnerable to the infection than males, show that male models are less susceptible to treatment with both Bz and VNI, and thus suggest that male models are much more suitable for selection of the most promising antichagasic agents. Additionally, we have found that preventive protocols (compound given at 1 dpi) result in higher treatment success rates, which also should be avoided during advanced steps of in vivo trials of novel anti-T. cruzi drug candidates. Another consideration is the relevance of immunosuppression methods in order to verify the therapeutic profile of novel compounds, besides the usefulness of molecular diagnostic tools (quantitative PCR) to ascertain compound efficacy in experimental animals. Our study aims to contribute to the development of more reliable methods and decision gates for in vivo assays of novel antiparasitic compounds in order to move them from preclinical to clinical trials for CD.

Use of (99m)Tc-doxorubicin scintigraphy in females with breast cancer: a pilot study.

OBJECTIVE: Doxorubicin (Eurofarma, São Paulo, Brazil) is an antitumour agent widely used in the treatment of breast cancer and can be used for tumour tracking when labelled with a radionuclide. Here, we present the results obtained with technetium-99m ((99m)Tc)-doxorubicin, using the direct method, to evaluate its uptake in breast cancer. METHODS: Four females with confirmed breast carcinoma diagnosis and breast image reporting and data system Category 5 on mammography underwent whole-body and thorax single-photon emission CT/CT imaging 1 and 3 h after (99m)Tc-doxorubicin administration. RESULTS: We observed increased uptake in breast carcinoma lesions and elimination via renal and hepatic pathways. CONCLUSION: These preliminary results suggest that (99m)Tc-doxorubicin may be a promising radiopharmaceutical for the evaluation of patients with breast cancer. Further studies are ongoing. ADVANCES IN KNOWLEDGE: To our knowledge, this is the first study to evaluate the use of a directly labelled doxorubicin tracer in humans. (99m)Tc-doxorubicin could provide information on the response of tumours to doxorubicin.

Use of 99mTc-doxorubicin scintigraphy in females with breast cancer: a pilot study.

OBJECTIVE:: Doxorubicin (Eurofarma, São Paulo, Brazil) is an antitumour agent widely used in the treatment of breast cancer and can be used for tumour tracking when labelled with a radionuclide. Here, we present the results obtained with technetium-99m (99mTc)-doxorubicin, using the direct method, to evaluate its uptake in breast cancer. METHODS:: Four females with confirmed breast carcinoma diagnosis and breast image reporting and data system Category 5 on mammography underwent whole-body and thorax single-photon emission CT/CT imaging 1 and 3 h after 99mTc-doxorubicin administration. RESULTS:: We observed increased uptake in breast carcinoma lesions and elimination via renal and hepatic pathways. CONCLUSION:: These preliminary results suggest that 99mTc-doxorubicin may be a promising radiopharmaceutical for the evaluation of patients with breast cancer. Further studies are ongoing. ADVANCES IN KNOWLEDGE:: To our knowledge, this is the first study to evaluate the use of a directly labelled doxorubicin tracer in humans. 99mTc-doxorubicin could provide information on the response of tumours to doxorubicin.

A time-compressed simulated geomagnetic storm influences the nest-exiting flight angles of the stingless bee Tetragonisca angustula.

Insects have been used as models for understanding animal orientation. It is well accepted that social insects such as honeybees and ants use different natural cues in their orientation mechanism. A magnetic sensitivity was suggested for the stingless bee Schwarziana quadripunctata, based on the observation of a surprising effect of a geomagnetic storm on the nest-exiting flight angles. Stimulated by this result, in this paper, the effects of a time-compressed simulated geomagnetic storm (TC-SGS) on the nest-exiting flight angles of another stingless bee, Tetragonisca angustula, are presented. Under an applied SGS, either on the horizontal or vertical component of the geomagnetic field, both nest-exiting flight angles, dip and azimuth, are statistically different from those under geomagnetic conditions. The angular dependence of ferromagnetic resonance (FMR) spectra of whole stingless bees shows the presence of organized magnetic nanoparticles in their bodies, which indicates this material as a possible magnetic detector.

Delineación tumoral basada en 18F-FDG-PET en el cáncer cervical: delimitación del contorno umbral y volúmenes de la lesión / 18F-FDG-PET-based tumor delineation in cervical cancer: Threshold contouring and lesion volumes

Evaluar el algoritmo de segmentación tumoral de la imagen PET semiautomatizada para delinear el volumen tumoral grueso (GTV) en pacientes con cáncer cervical localmente avanzado. Material y métodos. Se evaluó retrospectivamente a 32 pacientes con cáncer cervical localmente avanzado. Se utilizó la delineación GTV basada en imagen PET semiautomatizada, utilizando un algoritmo previamente establecido (GTV2SD) y 2 métodos basados en umbrales fijos (GTV40% y GTV50%). Se calculó GTV2SD, como el píxel con el valor medio más 2 desviaciones estándar de la intensidad del hígado, y GTV40% y GTV50% con el 40% y el 50% de intensidad tumoral máxima (Tmáx), respectivamente. A continuación se compararon los volúmenes derivados con los GTV generados manualmente, utilizando RM (GTVMR). Resultados. El valor medio de GTV2SD, GTV40% y GTV50% fue de 85,3 cc; 16,2 cc y 24,1 cc, respectivamente. Se halló una buena concordancia entre GTV2SD y GTVMR (rho=0,88). GTV40% y GTV50% mostraron una menor correlación con GTVMR (rho=0,68 y rho=0,71, respectivamente). Conclusiones. Este estudio prueba de modo preliminar que la delimitación del volumen tumoral metabólico es posible utilizando las mediciones generadas informáticamente en las imágenes de 18F-FDG PET. La generación de los volúmenes tumorales basados en PET se ve afectada por la elección del nivel de umbral utilizado. El grueso del tumor metabólico calculado utilizando el píxel con el valor medio más 2 desviaciones de la intensidad del hígado (GTV2SD) guarda una mejor correlación con los volúmenes tumorales derivados de RM. El método constituye un enfoque simple y clínicamente aplicable para generar el GTV derivado del PET, para la planificación de la terapia de radiación del cáncer cervical(AU)

Objective. To evaluate a semi-automated PET-image tumor segmentation algorithm for gross tumor volume (GTV) delineation in patients with locally advanced cervical cancer. Material and methods. Thirty-two patients with locally advanced cervical cancer were retrospectively evaluated. Semi-automated PET-image-based GTV delineation was applied using a previous established algorithm (GTV2SD) and 2 fixed threshold-based methods (GTV40% and GTV50%). GTV2SD was determined as the pixel with the mean value plus 2-standard deviation of the liver intensity, and GTV40% and GTV50% with 40% and 50% of the maximum tumor intensity (Tmax), respectively. The derived volumes were then compared with the GTVs generated manually using MR (GTVMR). Results. The mean value of GTV2SD, GTV40% and GTV50% was 85.3cc, 16.2cc and 24.1cc, respectively. Good agreement was noticed between GTV2SD and GTVMR (rho=0.88). GTV40% and GTV50% showed weaker correlation with GTVMR (rho=0.68 and rho=0.71, respectively). Conclusions. This study provides preliminary evidence that metabolic tumor volume delineation is feasible using computer-generated measurements in 18F-FDG PET images. Generation of PET-based tumor volumes is affected by the choice of threshold level used. Metabolic tumor bulk calculated using the pixel with the mean value plus 2-standard deviations of the liver intensity (GTV2SD) correlates better with the MR-derived tumor volumes. The method is a simple and clinically applicable approach to generate PET-derived GTV for radiation therapy planning of cervical cancer(AU)

In vitro inhibition of canine distemper virus by flavonoids and phenolic acids: implications of structural differences for antiviral design.

Infection caused by canine distemper virus (CDV) is a highly contagious disease with high incidence and lethality in the canine population. Antiviral activity of flavonoids quercetin, morin, rutin and hesperidin, and phenolic cinnamic, trans-cinnamic and ferulic acids were evaluated in vitro against the CDV using the time of addition assay to determine which step of the viral replicative cycle was affected. All flavonoids displayed great viral inhibition when they were added at the times 0 (adsorption) and 1h (penetration) of the viral replicative cycle. Both quercetin and hesperidin presented antiviral activity at the time 2h (intracellular). In the other hand, cinnamic acid showed antiviral activity at the times 0 and 2h while trans-cinnamic acid showed antiviral effect at the times -1h (pre-treatment) and 0 h. Ferulic acid inhibited CDV replicative cycle at the times 0 and 1h. Our study revealed promising candidates to be considered in the treatment of CDV. Structural differences among compounds and correlation to their antiviral activity were also explored. Our analysis suggest that these compounds could be useful in order to design new antiviral drugs against CDV as well as other viruses of great meaning in veterinary medicine.

18F-FDG-PET-based tumor delineation in cervical cancer: threshold contouring and lesion volumes.

OBJECTIVE: To evaluate a semi-automated PET-image tumor segmentation algorithm for gross tumor volume (GTV) delineation in patients with locally advanced cervical cancer. MATERIAL AND METHODS: Thirty-two patients with locally advanced cervical cancer were retrospectively evaluated. Semi-automated PET-image-based GTV delineation was applied using a previous established algorithm (GTV2SD) and 2 fixed threshold-based methods (GTV40% and GTV50%). GTV2SD was determined as the pixel with the mean value plus 2-standard deviation of the liver intensity, and GTV40% and GTV50% with 40% and 50% of the maximum tumor intensity (Tmax), respectively. The derived volumes were then compared with the GTVs generated manually using MR (GTVMR). RESULTS: The mean value of GTV2SD, GTV40% and GTV50% was 85.3cc, 16.2cc and 24.1cc, respectively. Good agreement was noticed between GTV2SD and GTVMR (ρ=0.88). GTV40% and GTV50% showed weaker correlation with GTVMR (ρ=0.68 and ρ=0.71, respectively). CONCLUSIONS: This study provides preliminary evidence that metabolic tumor volume delineation is feasible using computer-generated measurements in (18)F-FDG PET images. Generation of PET-based tumor volumes is affected by the choice of threshold level used. Metabolic tumor bulk calculated using the pixel with the mean value plus 2-standard deviations of the liver intensity (GTV2SD) correlates better with the MR-derived tumor volumes. The method is a simple and clinically applicable approach to generate PET-derived GTV for radiation therapy planning of cervical cancer.

Identical sequence patterns in the ends of exons and introns of human protein-coding genes

Intron splicing is one of the most important steps involved in the maturation process of a pre-mRNA. Although the sequence profiles around the splice sites have been studied extensively, the levels of sequence identity between the exonic sequences preceding the donor sites and the intronic sequences preceding the acceptor sites has not been examined as thoroughly. In this study we investigated identity patterns between the last 15 nucleotides of the exonic sequence preceding the 5' splice site and the intronic sequence preceding the 3' splice site in a set of human protein-coding genes that do not exhibit intron retention. We found that almost 60% of consecutive exons and introns in human protein-coding genes share at least two identical nucleotides at their 3' ends and, on average, the sequence identity length is 2.47 nucleotides. Based on our findings we conclude that the 3' ends of exons and introns tend to have longer identical sequences within a gene than when being taken from different genes. Our results hold even if the pairs are non-consecutive in the transcription order.

Modified Spitz procedure using a Collis gastroplasty for the repair of long-gap esophageal atresia.

OBJECTIVE: We report here on our experience with a modified Spitz procedure using a Collis gastroplasty for the repair of long-gap esophageal atresia (EA). PATIENTS AND METHODS: The authors reviewed 5 cases of children with long-gap (5-6 vertebral bodies) EA. The repair was carried out after 3 months of permanent monitoring, enteral nutrition through a gastrostomy tube and permanent aspiration of the proximal esophagus. The Collis gastroplasty was performed to create a gastric tube along the gastric lesser curvature using an Endo-GIA linear stapler. The gastric tube can be easily mobilized into the thorax after ligation of the left gastric vessels and anastomosed to the proximal esophageal segment. RESULTS: There were no intraoperative complications. The median postoperative hospital stay was 39 days (range: 30-60) with a median follow-up of 20 months (range: 16-29). During the early follow-up period, a digestive occlusion occurred in one patient, and several months later, she suffered from hiatal herniation. Other postoperative complications included anastomotic stricture (n = 2) treated by dilatations, gastroesophageal reflux (GER) (n = 3), and weight delay (n = 3) requiring additional enteral nutrition in 2 cases. No mortality was seen in our series. CONCLUSIONS: We propose a modified Spitz procedure using a Collis gastroplasty in the primary repair of long-gap EA as an alternative option to esophageal replacement or elongation techniques. The complications noted in our series are common complications of EA surgical repair, such as GER, anastomotic stricture and ponderal stagnation.

Combination of cetuximab with chemoradiation, trastuzumab or MAPK inhibitors: mechanisms of sensitisation of cervical cancer cells.

BACKGROUND: Cervical cancer (CC) annually kills 288,000 women worldwide. Unfortunately, responses to chemoradiation are partial and are of short duration. As anti-EGFR monoclonal antibodies sensitise tumours, we investigated cetuximab's toxicity plus chemoradiation on CC cells, which express different EGFR levels. METHODS: EGFR, HER2, AKT and MAPK expression and phosphorylation status were determined by western blotting. Cytotoxicity was assessed by MTT or clonogenic assays (CA) in cell lines treated with cetuximab alone or in combinations. RESULTS: Cetuximab with cisplatin and radiation achieved maximum cytotoxic effects for A431, Caski and C33A cells (high, intermediate and low EGFR expression, respectively) in CA. Cetuximab efficiently decreased MAPK and AKT phosphorylation in A431 cells but slightly less in Caski and C33A cells. To check whether further EGFR, HER2 or MAPK inhibition would improve cetuximab's cytotoxicity, we combined it with an EGFR tyrosine kinase inhibitor (TKI), trastuzumab or a MEK1/2 inhibitor (PD98059). In Caski, but not in C33A cells, cetuximab cooperated with the TKI, reducing cell survival and AKT and MAPK phosphorylation. However, cetuximab with trastuzumab or PD98059 reduced survival and MAPK phosphorylation of both cell lines. CONCLUSION: Our data suggest that cetuximab combined with chemoradiation, trastuzumab or MAPK inhibitors has useful applications for CC treatment, independently of EGFR expression.

Changes in the status of p53 affect drug sensitivity to thymidylate synthase (TS) inhibitors by altering TS levels.

Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of several genes, it may modulate TS activity, and changes in the status of p53 might be responsible for chemoresistance. Therefore, this study was aimed to investigate TS levels and sensitivity to TS inhibitors in wild-type (wt) and mutant (mt) p53 CRC cells, Lovo and WiDr, respectively, transfected with mt and wt p53. Lovo 175X2 cells (transfected with mt p53) were more resistant to 5-fluorouracil (5-FU; 2-fold), nolatrexed (3-fold), raltitrexed (3-fold) and pemetrexed (10-fold) in comparison with the wt p53 parental cells Lovo 92. Resistance was associated with an increase in TS protein expression and catalytic activity, which might be caused by the loss of the inhibitory effect on the activity of TS promoter or by the lack of TS mRNA degradation, as suggested by the reversal of TS expression to the levels of Lovo 92 cells by adding actinomycin. In contrast, Lovo li cells, characterized by functionally inactive p53, were 3-13-fold more sensitive to nolatrexed, raltitrexed and pemetrexed, and had a lower TS mRNA, protein expression and catalytic activity than Lovo 92. However, MDM-2 expression was significantly higher in Lovo li, while no significant differences were observed in Lovo 175X2 cells with respect to Lovo 92. Finally, mt p53 WiDr transfected with wt p53 were not significantly different from mt p53 WiDr cells with respect to sensitivity to TS inhibitors or TS levels. Altogether, these results indicate that changes in the status of p53, can differently alter sensitivity to TS inhibitors by affecting TS levels, depending on activity or cell line, and might explain the lack of clear correlation between mutations in p53 and clinical outcome after chemotherapy with TS inhibitors.

Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines.

Thymidylate synthase (TS) is an important target for chemotherapy and increased levels are associated with resistance to colorectal cancer chemotherapy. TS can be inhibited by 5-fluorouracil (5-FU) and antifolates, ultimately resulting in apoptosis. We aimed to clarify whether activation of caspases and Fas signalling are crucial for the onset of apoptosis after specific inhibition of TS and whether p53 plays a role in activation of these downstream processes. For this purpose, wild-type (wt) and mutant (mt) p53 colon cancer cell lines, Lovo and WiDr, respectively, transfected with mt- and wt-p53, were treated with the specific TS inhibitor, AG337. Treatment with 10xIC(50) values of AG337 for 48 h resulted in S phase arrest in all Lovo and WiDr cells (up to 50% of cells being in S phase), irrespective of their p53 status. After 72 h, the induction of apoptosis was most pronounced in the AG337-sensitive cells. Approximately 30% apoptosis was detected in all of the WiDr cells, 20% in Lovo li (non-functional p53), 12-14% in Lovo 92 and B2 (wt p53) and only 7% in Lovo 175x2 cells (mt p53 transfected). The induction of apoptosis in Lovo cells, as determined using the classical sub-G1 peak after propidium iodide (PI) staining, was associated with an increase in the expression of Fas receptor. In addition, synergistic increases in apoptosis from approximately 10 to 35% after 48 h could be detected after simultaneous treatment of AG337 and the Fas activator antibody, CH11. Only additive effects were measurable in WiDr cells, without an increase in Fas receptor expression. Surprisingly, the Fas inhibitor, ZB4, could not decrease the amount of cell death in both cell lines after AG337 treatment. In contrast, simultaneous exposure of Lovo and WiDr cells to AG337 and inhibitors of caspases 8, 9 and 3 caused a decrease in the number of apoptotic cells compared with AG337 exposure alone. Inhibition of apoptosis by approximately 10-80% in Lovo and approximately 70-80% in WiDr cells could be detected. In conclusion, these results indicate that apoptosis induced after specific inhibition of TS is mediated via the caspases, but without clear involvement of Fas signalling. The status of p53 did not affect the onset of apoptosis by these caspases.

Expression of X-linked inhibitor of apoptosis as a novel prognostic marker in radically resected non-small cell lung cancer patients.

PURPOSE: To assess the pattern of expression and the prognostic value of the inhibitor of apoptosis family member X-linked inhibitor of apoptosis (XIAP; MIHA/ILP-a) in radically resected non-small cell lung cancer patients. EXPERIMENTAL DESIGN: The expression of XIAP and its relationship with overall survival was analyzed by immunohistochemistry on tumors from 144 patients with early-stage non-small cell lung cancer. In addition, the apoptotic and mitotic index, Ki-67, p53, and bcl-2 levels were also assessed. RESULTS: XIAP expression was specific for tumor cells, and the pattern was cytoplasmic. The median expression of XIAP was 20%, and when this value was used as a cutoff point for statistical analyses, 63 of the samples were considered high XIAP-expressing and 81 low XIAP-expressing. Surprisingly, high XIAP-expressing patients had a longer overall survival than the group expressing lower levels (60 versus 24 months of median survival; log rank, P = 0.01). The positive impact of XIAP expression on survival was confirmed by multivariate analysis (P = 0.026). Although no correlation was observed between XIAP expression and the apoptotic index, a significant inverse correlation was observed between XIAP, Ki-67 (P = 0.006), and mitotic index (P = 0.04). CONCLUSIONS: The unexpected inverse correlation of XIAP with proliferation markers and the absence of correlation with apoptotic index, coupled with its role as an independent positive prognostic factor for survival in radically resected NSCLC patients imply a more complex role for XIAP in tumor biology than anticipated by in vitro data.