The Histopathological Growth Pattern of Colorectal Liver Metastases Impacts Local Recurrence Risk and the Adequate Width of the Surgical Margin.

BACKGROUND: The histopathological growth pattern (HGP) of colorectal liver metastases (CLM) has been associated with prognosis. This study was designed to elucidate if the HGP is associated with local recurrence risk and impacts the adequate width of surgical margin. METHODS: All consecutive patients resected for CLM in 2018-2019 were considered. HGP was prospectively classified as follows: desmoplastic, pushing, and replacement. Surgical margin was classified as follows: R0 (margin ≥ 1 mm), R1vasc (0-mm margin, tumor detachment from intrahepatic vessels), and R1par (tumor exposure along transection plane). R0 resections were further distinguished in R0min (1-mm margin) and R0wide (> 1-mm margin). RESULTS: A total of 340 resection areas in 136 patients were analyzed (70 R0min, 143 R0wide, 31 R1vasc, 96 R1par). HGP was desmoplastic in 26 cases, pushing in 221, and replacement in 93. Thirty-six local recurrences occurred (11%, median follow-up 21 months): 1 after R0wide, 4 after R0min, 3 after R1vasc, and 28 after R1par resection. In R1par group, local recurrence rate was high independently of HGP (29%). In R1vasc and R0min groups, local recurrence risk was higher in the replacement group (R1vasc: 29% vs. 4% if pushing/desmoplastic; R0min: 11% vs. 4%). In R0wide group, local recurrence risk was low for all HGP ( < 1%). Independent predictors of local recurrence were replacement HGP (odds ratio = 1.654, P = 0.036), and R1par resection (odds ratio = 57.209, P < 0.001 vs. R0). CONCLUSIONS: Replacement HGP is associated with an increased risk of local recurrence. In these patients, a wide surgical margin should be pursued, because R1vasc and R0min resections could be insufficient. R1par resection is inadequate, independently of the HGP.

Catecolaminas vs. metanefrinas en el diagnóstico bioquímico de feocromocitoma y paraganglioma / Catecholamines vs. metanephrines in the biochemical diagnosis of pheochromocytoma and paraganglioma

RESUMEN Los tumores secretores de catecolaminas, feocromocitoma y paraganglioma son entidades poco frecuentes y potencialmente letales si no son diagnosticadas y tratadas a tiempo. El laboratorio cumple un rol fundamental en el diagnóstico y seguimiento de estos tumores a través de la evidencia bioquímica de un exceso de catecolaminas. Sin embargo, muchas veces suele ser dificultoso arribar a un diagnóstico temprano, debido a la baja incidencia de estos tumores y a la dificultad de hallar laboratorios con equipamiento especializado. El marcador bioquímico y las técnicas utilizadas para su medición han ido cambiando con el correr de los años. Tradicionalmente, la medición de catecolaminas en orina era la prueba bioquímica utilizada. Posteriores hallazgos de metabolitos aumentados en la orina de paciente llevaron al uso de ensayos colorimétricos para la detección de ácido vainillin mandélico y metanefrinas como marcadores diagnósticos adicionales de tumor. Las pruebas actuales para el diagnóstico bioquímico muestran una excelente precisión diagnóstica. La medición de metanefrinas libres de plasma utilizando cromatografía líquida de alta resolución con detección electroquímica o de espectrometría de masas en tándem proporciona la máxima precisión para el diagnóstico de estos tumores.

ABSTRACT Catecholamine-secreting neuroendocrine tumors called Pheochromocytoma and Paraganglioma are rare entities, but potentially lethal if diagnosis and treatment are not established early enough. Clinical Laboratory plays an important role in the diagnosis and follow-up of these tumors, through the biochemical evidence of a hyperproduction of catecholamines and its metabolites.

Prostate cancer diagnosis through electronic nose in the urine headspace setting: a pilot study.

BACKGROUND: To evaluate the efficacy of prostate cancer (PCa) detection by the electronic nose (EN) on human urine samples. METHODS: Urine samples were obtained from candidates of prostate biopsy (PB). Exclusion criteria were a history of urothelial carcinoma or other malignant disease, urine infection, fasting for <12 h before PB or ingestion of alcohol or foods that might alter the urine smell in the last 24 h. The initial part of the voided urine and the midstream were collected separately in two sterile containers. Both samples were analyzed by the EN immediately after the collection. All patients underwent a standard transperineal, transrectal-ultrasound-guided PB. The pathological results were compared with the outcomes of the EN. Sensitivity and specificity of EN were assessed. RESULTS: Forty-one men were included in the study. Fourteen out of the 41 patients were positive for PCa. Midstream urine did not correlate significantly neither with a positive nor with a negative PB. Instead, significantly different results on the initial part of the urine stream between positive and negative PBs were obtained. The EN correctly recognized 10 out of the 14 cases (that is, sensitivity 71.4% (confidence interval (CI) 42-92%)) of PCa while four were false negatives. Moreover, the device recognized as negative 25 out of the 27 (that is, specificity 92.6% (CI 76-99%)) samples of negative PBs, with only two false positives. CONCLUSIONS: We believe this is the first demonstration of an olfactory imprinting of the initial part of the urine stream in patients with PCa that was revealed by an EN, with high specificity.

Functional regulation of the apurinic/apyrimidinic endonuclease 1 by nucleophosmin: impact on tumor biology.

Nucleophosmin 1 (NPM1) is a nucleolar protein involved in ribosome biogenesis, stress responses and maintaining genome stability. One-third of acute myeloid leukemias (AMLs) are associated with aberrant localization of NPM1 to the cytoplasm (NPM1c+). This mutation is critical during leukemogenesis and constitutes a good prognostic factor for chemotherapy. At present, there is no clear molecular basis for the role of NPM1 in DNA repair and the tumorigenic process. We found that the nuclear apurinic/apyrimidinic endonuclease 1 (APE1), a core enzyme in base excision DNA repair (BER) of DNA lesions, specifically interacts with NPM1 within nucleoli and the nucleoplasm. Cytoplasmic accumulation of APE1 is associated with cancers including, as we show, NPM1c+ AML. Here we show that NPM1 stimulates APE1 BER activity in cells. We provide evidence that expression of the NPM1c+ variant causes cytoplasmic accumulation of APE1 in: (i) a heterologous cell system (HeLa cells); (ii) the myeloid cell line OCI/AML3 stably expressing NPM1c+; and (iii) primary lymphoblasts of NPM1c+ AML patients. Consistent with impaired APE1 localization, OCI/AML3 cells and blasts of AML patients have impaired BER activity. Cytoplasmic APE1 in NPM1c+ myeloid cells is truncated due to proteolysis. Thus, the good prognostic response of NPM1c+ AML to chemotherapy may result from the cytoplasmic relocalization of APE1 and the consequent BER deficiency. NPM1 thus has an indirect but significant role in BER in vivo that may also be important for NPM1c+ tumorigenesis.

EGFR expression is linked to osteopontin and Nf-κB signaling in clear cell renal cell carcinoma.

AIM AND BACKGROUND: Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in many important aspects of cell biology that are related to tumorigenesis. There are opposite evidences of the role of EGFR in renal cancer and the outcome of EGFR-targeted therapies, suggesting the complexity of EGFR signaling pathways. In vitro, osteopontin (OPN) and nuclear factor kappa B (NF-κB) are thought to be involved in specific ligand-independent EGFR activation that could have a role in resistance to EGFR mAb therapy. Aim of this study was to analyze the relationship between EGFR and OPN at the protein and mRNA level, as well as their relation to NF-κB in clear cell renal cell carcinoma (CCRCC). MATERIALS AND METHODS: Expression of EGFR, OPN, and p65 NF-κB protein was analyzed using immunohistochemistry and compared mutually in 88 CCRCC samples. Expression of EGFR and OPN mRNAs was analyzed using quantitative Real-time PCR in 22 CCRCC samples and compared mutually and with NF-κB protein expression. RESULTS: Epidermal growth factor receptor mRNA level was higher in CCRCC samples in comparison with normal renal tissue (p = 0.012) and was associated with high OPN mRNA level, and with NF-κB activation (p < 0.001 and p = 0.045, respectively). Immunohistochemical staining showed the inverse association; high EGFR protein expression was related with low OPN and NF-κB protein expression (p < 0.001 and p = 0.047, respectively). CONCLUSION: Epidermal growth factor receptor gene is upregulated in CRCC and associated with OPN gene expression and NF-kB signaling. The inverse relation between OPN and EGFR at the protein level could probably reflect dynamic changes that EGFR undergoes following activation.

Salvage hepatic resection after incomplete interstitial therapy for primary and secondary liver tumours.

BACKGROUND: When the response to percutaneous ablation therapy (PAT) of liver tumours is incomplete, surgery may be undertaken as a salvage therapy. To validate the safety and effectiveness of salvage hepatectomy, patients who had undergone PAT or no treatment before hepatectomy were compared. METHODS: Of 137 patients who had hepatectomy for primary and secondary tumours, 21 had undergone PAT and 116 had surgery as primary treatment. Tumour features and the incidence of liver cirrhosis were similar in the two groups. RESULTS: Peroperative mortality and major morbidity rates were zero and 5 per cent (one of 21) respectively among patients who had PAT before surgery, and 0.9 per cent (one of 116) and zero in those who did not. Duration of operation (mean 495 versus 336 min; P<0.001), clamping time (mean 81 versus 53 min; P<0.001), blood loss (mean 519 versus 286 ml; P=0.004), need for blood transfusion (six of 21 patients versus nine of 116; P=0.001), and rates of thoracophrenolaparotomy (eight of 21 versus 14 of 116; P<0.001) and resection of other tissues (six of 21 versus nine of 116; P<0.001) were significantly higher in the PAT group. CONCLUSION: Hepatectomy after incomplete PAT is safe and effective, but more extensive procedures are necessary. The effect of salvage hepatectomy on long-term outcome is still unclear.

Ultrasonographically guided surgical approach to liver tumours involving the hepatic veins close to the caval confluence.

BACKGROUND: Intraoperative ultrasonography (IOUS) may allow a more conservative procedure in patients with liver tumours involving a hepatic vein at the caval confluence. The aim of this study was to determine whether IOUS and colour Doppler IOUS might reduce the rate of major hepatectomy and vascular reconstruction in patients with such tumours. METHODS: Of 133 consecutive patients with a liver tumour who underwent hepatectomy, 22 had involvement of a hepatic vein at the caval confluence. The surgical strategy employed was determined by IOUS findings of the relationship between the tumour and hepatic vein, the presence of accessory veins, and portal flow as measured by colour Doppler IOUS following clamping of the hepatic vein to be resected. Mortality, morbidity, major resection, hepatic vein reconstruction and local recurrence rates were evaluated. RESULTS: There were no hospital deaths and only one patient suffered major morbidity. Although hepatic vein resection was performed in 15 patients, only two underwent major hepatectomy and none had vascular reconstruction. No patients had tumour recurrence at a mean follow-up of 23 months. CONCLUSION: IOUS allowed sparing of the liver parenchyma without tumour recurrence in most patients with a tumour involving a hepatic vein at the caval confluence, avoiding more extensive hepatectomy or vascular reconstruction.

Liver resection for primary hepatic neuroendocrine tumours: report of three cases and review of the literature.

Primary neuroendocrine tumours are rare especially in the liver, which is more often site of metastatic tumours. We report three cases of primary hepatic neuroendocrine tumours, which underwent hepatic resection. Review of the diagnostic and therapeutic approaches to these tumours are discussed.

Utility of the hooking technique for cases of major hepatectomy.

Currently, resective hepatic surgery should be considered an echoguided surgical procedure to guarantee conservative but radical resections. A simple and original technique guided by intraoperative ultrasonography, termed the "hooking technique," had been described previously. It enables the ligation sites of the intrahepatic vessels during systematic segmentectomy to be chosen precisely. This report describes a further application of this technique to allow safe ligation of portal vein main branches invaded by tumor thrombi during major hepatectomies.

Technical tricks for radical but conservative liver resection. The ultrasound guidance.

Rate of major resection is still high in most surgical institutions due to fear of incomplete tumor removal: this is in spite mortality and major morbidity of major hepatectomies, particularly in cirrhotic are still not negligible. Intraoperative ultrasonography (IOUS), when used not only for tumor staging but also for resection guidance, minimises the rate of major hepatectomies maintaining treatment radicality. Maintaining this policy, the rate of major resection in our experience is 15% if major hepatectomy is classified as removal of at least 1 sector or 2 adjacent segments, and 5% if we consider major resections only those which include at least 3 segments. This policy has allowed us a safe surgical approach with no mortality and minimal major morbidity and effective local treatment with no tumor relapses at the site of the resection after a mean follow-up of 18 months. Tricks for safe and radical IOUS-guided liver resections are here discussed.

Pyrrolopyrimidine c-Src inhibitors reduce growth, adhesion, motility and invasion of prostate cancer cells in vitro.

Two bona fide c-Src inhibitors, denominated CGP77675 and CGP76030, reduced in a time- and concentration-dependent manner (i) the proliferation of the PC3 prostate carcinoma cell line, as assessed by the [3H]-thymidine incorporation test, (ii) the capacity of PC3 cells to adhere and spread on Matrigel substrate, as determined by crystal violet staining, (iii) the ability of PC3 cells to migrate through a gelatine boundary and invade a Matrigel substrate. The latter effect was not due to a decrease of urokinase-type plasminogen activator (uPA), nor of metalloproteinase-2 (MMP-2) activities. The MMP-9 activity, along with the expression of the Tissue Inhibitor of Metalloproteinases (TIMP)-1 and TIMP-2, were reduced by the two inhibitors, consistent with the ability of c-Src to enhance MMP-9 and TIMP expression levels. Collectively, these data demonstrate that the pyrrolopyrimidine-derived c-Src inhibitors significantly reduced PC3 cell activities associated with their malignant phenotype.

Imatinib: a selective tyrosine kinase inhibitor.

The understanding of the pathophysiology of a large number of cancer types provides a strategy to target cancer cells with minimal effect on normal cells. Protein phosphorylation and dephosphorylation play a pivotal role in intracellular signaling; to regulate signal transduction pathways, there are approximately 700 protein kinases and 100 protein phosphatases encoded within the human genome. In cancer, as well as in other proliferative diseases, unregulated cell proliferation, differentiation and survival frequently results from abnormal protein phosphorylation. Although it is often possible to identify a single kinase that plays a pivotal role in a given disease, the development of drugs based upon protein kinase inhibition has been hampered by unacceptable side effects resulting from a lack of target selectivity. With the growing understanding of the molecular biology of protein tyrosine kinases and the use of structural information, the design of potential drugs directed towards the bind adenosine triphosphate (ATP)-binding site of a single target has become possible. These advances have transferred emphasis away from the identification of potent kinase inhibitors and more towards issues of target selectivity, cellular efficacy, therapeutic effectiveness and tolerability. In this paper, the relationship between molecular biology and drug discovery methods, as utilized for the identification of anticancer drugs, will be illustrated.

Effect of VEGF receptor inhibitor PTK787/ZK222584 [correction of ZK222548] combined with ionizing radiation on endothelial cells and tumour growth.

The vascular endothelial growth factor (VEGF) receptor is a major target for anti-angiogenesis-based cancer treatment. Here we report the treatment effect of ionizing radiation in combination with the novel orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on endothelial cell proliferation in vitro and with tumour xenografts in vivo. Combined treatment of human umbilical vein endothelial cells with increasing doses of PTK787/ZK222584 and ionizing radiation abrogated VEGF-dependent proliferation in a dose-dependent way, but inhibition of endothelial cell proliferation was not due to apoptosis induction. In vivo, a combined treatment regimen of PTK787/ZK222584 (4 x 100 mg/kg) during 4 consecutive days in combination with ionizing radiation (4 x 3 Gy) exerted a substantial tumour growth delay for radiation-resistant p53-dysfunctional tumour xenografts derived from SW480 colon adenocarcinoma cells while each treatment modality alone had only a minimal effect on tumour size and neovascularization. SW480 tumours from animals that received a combined treatment regimen, displayed not only an extended tumour growth delay but also a significant decrease in the number of microvessels in the tumour xenograft. These results support the model of a cooperative anti-tumoral effect of angiogenesis inhibitor and irradiation and show that the orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 is suitable for combination therapy with irradiation.

The phosphatidylinositide 3'-kinase/Akt survival pathway is a target for the anticancer and radiosensitizing agent PKC412, an inhibitor of protein kinase C.

Activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt survival pathway protects against apoptotic stress stimuli. Therefore, compounds that down-regulate this pathway are of clinical interest for single and combined anticancer treatment modalities. Here we demonstrate that the cytotoxic effect of the protein kinase C (PKC)-inhibitor N-benzoylated staurosporine (PKC412) is mediated via the PI3K/Akt pathway. Dose-dependent down-regulation of the proliferative activity, activation of the apoptotic machinery, and cell killing by PKC412 (0-1 microM) in Rat1a-fibroblasts and H-ras-oncogene-transformed fibroblasts correlated with a decrease of Akt phosphorylation and a reduced phosphorylation of the endogenous Akt-substrate GSK3-alpha. Expression of the dominant-active myristoylated form of Akt abrogated this cytotoxic effect of PKC412. Experiments with Apaf-1-deficient cells revealed that PKC412-induced cytotoxicity depends on an intact apoptosome but that the decrease of Akt phosphorylation is not attributable to apoptosis execution. Comparative experiments indicate that PKC412 and the parent-compound staurosporine down-regulate this survival pathway upstream or at the level of Akt but by a different mechanism than the PI3K-inhibitor LY294002. Furthermore, inhibition of this pathway by PKC412 is relevant for sensitization to ionizing radiation. These results demonstrate the specific role of this signaling pathway for the PKC412-mediated down-regulation of an apoptotic threshold and its cytotoxicity.

Ultrasound-guided reduction of an incarcerated Spigelian hernia.

Spigelian hernia is a rare abdominal hernia. We report a case in which its diagnosis proved ultrasonography to be an effective tool, not only to diagnose an incarcerated Spigelian hernia but, moreover, to reduce it by echo-probe palpation. Ultrasound (US) is an aid for therapy of various diseases. In our experience, US-guidance prevented possible damage related to forced and wrongly applied compression during the hernia reduction, and allowed us to perform surgical repair on an elective basis. In conclusion, if an incarcerated Spigelian hernia is suspected, a US examination should be done on an emergency basis to confirm the diagnosis and to attempt US-guided reduction.

Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase.

Full activation of protein kinase B (PKB, also called Akt) requires phosphorylation on two regulatory sites, Thr-308 in the activation loop and Ser-473 in the hydrophobic C-terminal regulatory domain (numbering for PKB alpha/Akt-1). Although 3'-phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of the Ser-473 phosphorylation remains controversial. As a step to further characterize the Ser-473 kinase, we examined the effects of a range of protein kinase inhibitors on the activation and phosphorylation of PKB. We found that staurosporine, a broad-specificity kinase inhibitor and inducer of cell apoptosis, attenuated PKB activation exclusively through the inhibition of Thr-308 phosphorylation, with Ser-473 phosphorylation unaffected. The increase in Thr-308 phosphorylation because of overexpression of PDK1 was also inhibited by staurosporine. We further show that staurosporine (CGP 39360) potently inhibited PDK1 activity in vitro with an IC(50) of approximately 0.22 microm. These data indicate that agonist-induced phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB activity and occurs through a distinct Ser-473 kinase that is not inhibited by staurosporine. Moreover, our results suggest that inhibition of PKB signaling is involved in the proapoptotic action of staurosporine.

Selective in vivo and in vitro effects of a small molecule inhibitor of cyclin-dependent kinase 4.

BACKGROUND: Cyclin-dependent kinase 4 (Cdk4) represents a prime target for the treatment of cancer because most human cancers are characterized by overexpression of its activating partner cyclin D1, loss of the natural Cdk4-specific inhibitor p16, or mutation(s) in Cdk4's catalytic subunit. All of these can cause deregulated cell growth, resulting in tumor formation. We sought to identify a small molecule that could inhibit the kinase activity of Cdk4 in vitro and to then ascertain the effects of that inhibitor on cell growth and tumor volume in vivo. METHODS: A triaminopyrimidine derivative, CINK4 (a chemical inhibitor of Cdk4), was identified by screening for compounds that could inhibit Cdk4 enzyme activity in vitro. Kinase assays were performed on diverse human Cdks and on other kinases that were expressed in and purified from insect cells to determine the specificity of CINK4. Cell cycle effects of CINK4 on tumor and normal cells were studied by flow cytometry, and changes in phosphorylation of the retinoblastoma protein (pRb), a substrate of Cdk4, were determined by western blotting. The effect of the inhibitor on tumor growth in vivo was studied by use of tumors established through xenografts of HCT116 colon carcinoma cells in mice. Statistical tests were two-sided. RESULTS: CINK4 specifically inhibited Cdk4/cyclin D1 in vitro. It caused growth arrest in tumor cells and in normal cells and prevented pRb phosphorylation. CINK4 treatment resulted in statistically significantly (P: =.031) smaller mean tumor volumes in a mouse xenograft model. CONCLUSIONS: Like p16, the natural inhibitor of Cdk4, CINK4 inhibits Cdk4 activity in vitro and slows tumor growth in vivo. The specificity of CINK4 for Cdk4 raises the possibility that this small molecule or one with a similar structure could have therapeutic value.

Differential p53-dependent mechanism of radiosensitization in vitro and in vivo by the protein kinase C-specific inhibitor PKC412.

The cellular response to ionizing radiation is governed by the DNA-damage recognition process but is also modulated by cytoplasmic signal transduction cascades that are part of the cellular stress response. Growth-promoting protein kinase C activity antagonizes irradiation-induced cell death, and, therefore, protein kinase C inhibitors might be potent radiosensitizers. The antiproliferative and radiosensitizing effect of the novel N-benzoylated staurosporine analogue PKC412 was tested in vitro against genetically defined p53-wild type (+/+) and p53-deficient (-/-) murine fibrosarcoma cells and in vivo against radioresistant p53-/- murine fibrosarcoma and human colon adenocarcinoma tumor xenograft (SW480, p53-mutated). PKC412 sensitized both p53+/+ and p53-/- tumor cells in vitro and in vivo for treatment with ionizing radiation but with a different mechanism of radiosensitization depending on the p53 status. In p53+/+, cells combined treatment with PKC412 and ionizing radiation drastically induced apoptotic cell death, whereas no apoptosis induction could be observed in p53-deficient cells in vitro and in histological tumor sections. Combined treatment resulted in an increased G2 cell cycle distribution in p53-/- cells at PKC412 concentrations that did not alter cell cycle distribution when applied alone. In vivo, a minimal treatment regimen during 4 consecutive days of PKC412 (4 x 100 mg/kg) in combination with ionizing radiation (4 x 3 Gy) exerted a substantial tumor growth delay for both p53-disfunctional tumor xenografts and showed that the clinically relevant protein kinase C inhibitor PKC412 is a promising new radiosensitizer with a potentially broad therapeutic window.