In vitro expansion of tumour cells derived from blood and tumour tissue is useful to redefine personalized treatment in non-small cell lung cancer patients.

The clinical development of locally and advanced non-small cell lung cancer (NSCLC) suffers from a lack of biomarkers as a guide in the selection of optimal prognostic prediction. Circulating Tumour Cells (CTCs) are correlated to prognosis and show efficacy in cancer monitoring in patients. However, their enumeration alone might be inadequate; it might also be critical to understand the viability, the apoptotic state and the kinetics of these cells. Here, we report what we believe to be a new and selective approach to visually detect tumour specific CTCs. Firstly, using labelled human lung cancer cells, we detected a specific density interval in which NSCL-CTCs were concentrated. Secondly, to better characterize CTCs in respect to their heterogeneous composition and tumour reference, blood and tumour biopsy were performed on specimens taken from the same patient. The approach consisted in comparing phenotype profile of CTCs, and their progenitor Tumour Stem Cells, (TSCs). Moreover, NSCL-CTCs were cultivated in short-time human cultures to provide response to drug sensitivity. Our bimodal approach allowed to reveal two items. Firstly, that one part of a tumour, proximal to the bronchial structure, displays a predominance of CD133+. Secondly, specific NSCL-CTCs Epithelial Cell Adhesion Molecule (EpCAM)+CD29+ can be used as a negative prognostic factor as well the high expression of CTCs EpCAM+. These data were confirmed by drug-sensitivity tests, in vitro, and by the survival curves, in vivo.

Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay.

Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of ß-galactosides and galectin-3--a protein that is correlated to the progress of tumor and metastasis--is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction's strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine.

Experimental modified orthotopic piggy-back liver autotransplantation.

UNLABELLED: The classical orthotopic liver autotransplantation is a very challenging and time wasting technique; it includes the division of major hepatic vessels and choledocus, and subsequent reconnection by end to end anastomoses. The caval end to end anastomoses are the most difficult to be performed and the interposition of a prosthesis can be required. We adopted the classical orthotopic liver autotransplantation technique in 2 patients affected with diffused liver metastases from colorectal cancer, for extracorporeal neutron capture therapy (BNCT). The procedure required very long operating times and the extracorporeal circulation (ECC) set up; furthermore the vena cava reconstruction was performed by the interposition of a goretex-prosthesis. We propose a "modified orthotopic piggy-back technique" to simplify liver reconnection and shorten the operating time. MATERIALS AND METHODS: The technique was developed in the swine (25 kg body weight), under general anaesthesia. We performed the resection of the retro-hepatic vena cava with preservation of the caval flow during the anhepatic phase, by interposing a goretex-prosthesis. The reconstruction of the vena cava was then performed by a side-to-side cava-prosthesis anastomosis with lateral clamping of the prosthesis. The procedure was then completed according to the classical technique of liver transplantation. RESULTS: The mean time for VC reconstruction was 56 (+/-10)min. and the mean time for side-to-side VC-prosthesis anastomosis was 13(+/-4)min. CONCLUSIONS: The "modified orthotopic piggy-back technique" can simplify the reimplant of the liver during autotransplantation and shorten the operating time. Furthermore also the time of total extracorporeal circulation is reduced, as during the anhepatic phase and during the side-to-side cava-prosthesis anastomosis the flow in the inferior vena cava is uninterrupted.

Extra-corporeal liver BNCT for the treatment of diffuse metastases: what was learned and what is still to be learned.

Almost eight years ago, in December 2001, we performed for the first time in the world thermal neutron irradiation on an isolated liver of a patient. The organ was affected by diffuse metastases of a colon carcinoma and had been previously loaded with a (10)B compound. In July 2003, the same procedure was applied again on a patient for the treatment of unresectable and incurable hepatic metastases of a carcinoma of the rectum. Both patients are dead at present. Now we can analyze in depth the clinical history of these patients and evaluate the effectiveness of this therapy. From this exciting experience we learned much, and we also found out about complications till then unknown, which need to be studied and addressed experimentally. Unfortunately we can base our conclusions just on the experience we had with these two patients. We could have been much more detailed and firm in our statements if the number of clinical cases was larger. The BNCT Pavia project has been suspended, but it is more than likely to resume in a short time. Good findings were many. The procedure is feasible; the original concept of complete immersion of the diseased liver in a homogeneous neutron field proved effective and winning. The tumor masses resulted completely necrotic and unknown metastases too appeared radically treated; healthy hepatic tissue was preserved from both morphological and functional points of view; no symptoms of cirrhosis appeared even four years after treatment. For the long term surviving patient, quality of life was excellent. Other findings require to be tackled in depth. The "post-irradiation syndrome" we observed in both patients, with identical symptoms and biochemical derangements, creates a dramatic--even though totally reversible--clinical condition, that is the probable cause of death for our second patient, suffering from cardiomyopathy, 33 days after treatment. For the first patient, recurrences were a late yet fatal complication, for which even a further surgical revision was ineffective. We offer some hypotheses about their origin and possible prevention.

Neutron autoradiography imaging of selective boron uptake in human metastatic tumours.

The ability to selectively hit the tumour cells is an essential characteristic of an anti-tumour therapy. In boron neutron capture therapy (BNCT) this characteristic is based on the selective uptake of (10)B in the tumour cells with respect to normal tissues. An important step in the BNCT planning is the measurement of the boron concentration in the tissue samples, both tumour and healthy. When the tumour is spread through the healthy tissue, as in the case of metastases, the knowledge of the different kinds of tissues in the sample being analysed is crucial. If the percentage of tumour and normal tissues cannot be evaluated, the obtained concentration is a mean value depending on the composition of the different samples being measured. In this case an imaging method that could give information both on the morphology and on the spatial distribution of boron concentration in the sample would be a fundamental support. In this paper, the results of the boron uptake analysis in the tumour and in the healthy samples taken from human livers after boron phenylalanine (BPA) infusion are shown; boron imaging was performed using neutron autoradiography.

Surface properties and implantation site affect the capsular fibrotic overgrowth.

Transplantation of encapsulated pancreatic islets is a promising approach for the treatment of type 1 diabetes. Large-scale application of this technique, however, is hampered by insufficient biocompatibility of the capsules. In this study, we have evaluated the biocompatibility of a new synthetic material with six different chemical groups on their surface (amino, carboxy-sulfate, carboxylate, hydroxylate, sulfate, and PMMA) used for the fabrication of the microcapsules. Eight Lewis rats were inoculated with a suspension of empty capsules made for each candidate material in the retroperitoneal ileopsoas muscle and renal subcapsular space. Four weeks later kidney and muscle containing the capsules were explanted, paraffin embedded, sectioned and stained with Sirius Red and Masson's Trichrome for histological analysis. The amount of fibrosis was also ultrastructurally evaluated with scanning electron microscopy. The samples were then subjected to digitalized quantitative analysis using specific software to determine the degree of fibrotic overgrowth. The quantification of collagen deposition, calculated in proximity of the microcapsules, was expressed as a percentage of the total area and can be considered a good index for the biocompatibility, an essential prerequisite for functional pancreatic islet transplantation. The results show that subcapsular renal space is the best implantation site and the positive surface charge induces a more intense collagen synthesis.

How to study boron biodistribution in liver metastases from colorectal cancer.

The purpose of this study was to evaluate boron distribution for a safe and effective BNCT (Boron Neutron Capture Therapy) of liver metastases. Samples both from healthy and tumour liver parenchyma were analysed, after i.v. boron administration, by: alpha particles counting under neutron irradiation; morphological analysis by standard haematoxylin-eosin staining; neutron autoradiography. Our method was unaffected by the cytological heterogeneity inside tumour nodules; it demonstrated selective boron distribution in tumour tissue and predicted estimated mean therapeutic doses in tumour and safety doses in healthy tissue. The time interval for efficient BNCT was 2 to 4 hours after i.v. boron administration.

Boron-loaded liposomes in the treatment of hepatic metastases: preliminary investigation by autoradiography analysis.

Boronophenylalanine (BPA)-loaded conventional and stabilized liposomes were prepared by the reversed phase evaporation method to treat liver metastases by boron neutron capture therapy. Conventional vesicles were composed of phosphatidylcholine and cholesterol, molar ratio 1:1. To obtain stealth liposomes, GM1 or PEG were included in the lipidic bilayer at a concentration of 6.67 or 5 mol%, respectively. Large unilamellar vesicles were formulated encapsulating BPA in the liposome aqueous compartment as a complex with fructose; BPA free base also was embedded into the lipidic bilayer. In vivo experiments were carried out after intravenous injection of liposome suspensions in BD-IX strain rats in which liver metastases had been induced. Alpha particle spectroscopy associated with histological analysis was performed to visualize boron spatial distribution in liver. Simultaneously, tissue boron concentrations were determined using inductively coupled plasma-mass spectroscopy. Results showed that PEG-modified liposomes accumulated boron in therapeutic concentrations (> 30 micrograms boron/g tissue) in metastatic tissue. The PEG-liposomes could be further explored in enhancing boron delivery to tumor cells.

Feasibility and safety of a new technique of extracorporeal photochemotherapy: experience of 240 procedures.

BACKGROUND AND OBJECTIVE: Extracorporeal photochemotherapy (ECP) is a therapeutic approach based on the biological effects of ultraviolet light (UV) - A and psoralens on mononuclear cells collected by apheresis. Recently, ECP has been under investigation as an alternative treatment for various immune and autoimmune diseases. The aim of this study was to evaluate the safety and feasibility of a new three-step ECP technique, in terms of reproducibility, acceptance, tolerability, and short and long term side effects. DESIGN AND METHODS: Seventeen patients affected by acute or chronic graft-versus-host disease (GvHD), pemphigus vulgaris, or interferon-resistant chronic hepatitis C and one patient being treated for prevention of heart transplant rejection underwent 240 ECP procedures. MNC collection and processing parameters were recorded, biological effects of UV-A/8 methoxy-psoralen (8-MOP) were evaluated, and short and long term side effects were monitored. RESULTS: At a mean follow up of 7 months (range 2-19) 240 ECP had been completed, a mean of 7,136 mL (range 1,998-10,591) of whole blood having beenprocessed per procedure. The mean of total nucleated cells collected per procedure was 6.5x109 (range 0.65-23.8), with a mean MNC percentage of 85% (41. 4-98%) in a mean final volume of 115.5 mL (37-160). No severe side effects were documented and no infectious episodes occurred throughout the course of the treatment. INTERPRETATION AND CONCLUSIONS: The new ECP technique was highly reproducible as regards the collection and each processing step. Short and long term side effects were mild. No increase in infectious episodes was recorded. All patients willingly underwent ECP, demonstrating an excellent tolerability for the procedure even after several courses.

Radioimmunoassisted surgery for lung metastases from colorectal cancer: results and perspectives.

The high incidence of resectable lung metastases from colorectal cancer and the very poor prognosis of untreated patients (less than 24-month survival) has promoted the surgical approach to treatment. Since the main aims of this kind of surgery are the complete resection of the tumor, the preservation of tumor-free parenchyma, and a minimal surgical trauma, innovative surgical techniques have been developed. We report on our experience in the radioimmunoassisted pulmonary metastasectomy by the use of a hand-held gamma-detecting probe (GDP) and describe the application of the intraoperative radioimmunolocalization of tumor to video-assisted minimally invasive surgery.

Potentials of liposomes in diagnosis and treatment of pulmonary metastases: an experimental study in the rat.

OBJECTIVE: The ultimate goal of the therapy of lung metastases is to destroy all malignant cells while sparing normal ones. Liposomes represent a novel approach for the selective transport of tracers and therapeutic agents to cancer cells because of their flexibility, low toxicity, wide range of possible variants, simplicity to make, and because agents can be entrapped in them in their native states in large amounts. We have studied the biodistribution of "Stealth" liposomes in the experimental model of lung metastases in the rat. METHODS: The secondaries were induced by i.v. injection 20. 10(6) cancer cells (DHD/K12/TRb line) in BD-IX rats. The study of the liposome biodistribution in the rat was carried out by the use of unilamellar liposomes with homogeneous size distribution (0.1 microns), the liposomes were labeled with Cholesteryl-Bodipy. The rats were sacrificed at scheduled times after the injection; blood, urine, metastatic and healthy lung, colon, liver and spleen were analysed by a microcytofluorimetric examination. RESULTS: Liposomes prolonged the circulation time of Cholesteryl-Bodipy. Only spleen and lung metastases exhibited an accretion of fluorescent liposomes. CONCLUSIONS: The biodistribution of such formulation of liposomes in rats with lung metastases, may be of considerable importance in diagnosis and therapy of the secondaries, for increasing the concentration of tracers and therapeutic agents in tumor tissue while minimizing the likelihood of aspecific distribution and toxicity to non target tissue.

Radioimmunoassisted follow-up and surgery vs traditional examinations and surgery after radical excision of colorectal cancer.

The authors report their experience in the radioimmunodetection of recurrent colorectal cancer in comparison with traditional examinations. 485 patients were studied after radical surgery for colorectal cancer: 168 (Group 1) were studied with a radioimmunoassisted follow-up plan including: Immunoscintigraphy (IS), serum markers assays, Radioimmunoguided Endoscopy (RIGE), Intraoperative Radioimmuno-localization (IORIL) in case of reoperation; 317 (Group 2) underwent a protocol with traditional examinations. In 24 patients of Group 1 IS was performed even at the time of their admission for primary cancer. 7 patients underwent RIGE for primary cancer and 16 in the follow-up. IORIL was performed in 12 patients with primary cancer and in 16 in the course of reoperation. The radioimmunodiagnostic methods were performed after a single administration of the radiolabeled MAb (111In F(ab')2 a-CEA and 111In B72.3). The radioimmunoassisted follow-up plan detected a greater number of recurrences than traditional examinations (27% vs 13%). The rates of radical reoperation in Group 1 and 2 were 61% and 37% respectively. 33% of the patients of Group 2 were alive 24 months after radical reoperation vs 62.5% of the patients of Group 1. Immunoscintigraphy demonstrated a good sensitivity and specificity, mainly in the detection of pelvic recurrences (sens. 92%, spec. 84%). The radioimmunoassisted follow-up plan was well accepted by the patients. RIGE led to the detection of 3 periluminal recurrences of rectal cancer that traditional investigations failed to demonstrate and in 5 cases influenced the patients management. IORIL detected minimal tumor foci (2 mm.) where pre and intraoperative study were negative, while the histopathologic examination gave evidence of tumor. Considering that the costs of the radioimmunodiagnostic methods and of traditional examinations are very similar, we can conclude that the radioimmunoassisted follow-up plan has a favorable cost/benefit rate and a remarkable impact on the treatment of patients with colorectal cancer.

[Tumors of the breast in old age]. / I tumori della mammella nell'età geriatrica.

After reviewing the literature on this topic the authors studied all patients admitted to the Institute of Surgical Pathology I at the University of Pavia for breast cancer between 1-1-1974. An analysis of the findings revealed that: (a) Out of 993 patients with breast cancer, 204 (20.54%) belonged to the geriatric age group (> 65 years old) and of the latter 135 (66.17%) belonged to the 3rd age group and 69 (33.82%) belonged to the 4th age group (> 75 years old). (b) In the 3rd age group 25 (18.51%) were benign tumours and 110 (81.48%) were malignant, whereas in the 4th age group 4 (5.79%) were benign and 65 (94.20%) were malignant. (c) Familial patterns were also examined and were found to be positive in 47 (23.03%) cases in the 3rd age group and in 30 (14.71%) in the 4th age group. (d) In the physiological anamnesis it is interesting to note that there was 1 menarche before 12 (0.9%) in the 3rd age group; delayed menopause (after 50) in 66 cases (60%) in the 3 rd age group and in 29 cases (44.61%) in the 4 th age group. (e) In the pathological anamnesis of the patients in question it was observed that the most frequent site of previous tumours was the breast (72 cases, equivalent to 10.9% in the 3rd age group; 9 cases, 13.8% in the 4th age group). (f) In the next pathological anamnesis and at EOL it was observed that the tumours was most often found in the supero-external quadrant, in the right breast in the 3rd age group (66 cases, 48.89%) and in the left one in the 4th age group (42 cases, 60.87%). (g) Test included breast cancer scan, mammography, aspirated needle and biopsy where required. (h) Surgery in the form of Halsted's mastectomy was the preferred therapy and as early as possible. Some Authors use conservative therapy. (i) Histological tests were performed in all patients and revealed that the most frequent malignant tumour was ductal carcinoma. (j) Postoperative complications took the form of wound suppuration. (k) Only one tumour (carcinoma) was observed in male patient aged 83 years old, without metastasis. From the above findings it can be concluded that the most evident risk factor for breast cancer is the influence of the estrogen hormone.

Radioimmuno-guided endoscopy (RIGE) in the detection of primary and recurrent rectal tumor.

The usefulness of radioimmunoguided endoscopy in the detection of primary and recurrent rectal cancer was investigated. Of the 15 patients included in our study, 4 with suspected primary rectal cancer were examined preoperatively, while the remaining 11 were studied after radical resection of rectal carcinoma with the aim of detecting local recurrence. Radioimmunoguided endoscopy was performed employing a hand-held gamma-detecting probe (mod. 2 Oris, France), after the administration of a 111In labeled monoclonal antibody to CEA. Radioimmuno-guided endoscopy results detected the presence of primary or recurrent periluminal cancer in seven cases. In four it modified the preoperative stage based on the findings of conventional investigation and it influenced the surgical decision in five cases. No toxicity was noted and none of the patients developed HAMAs.

Radioimmunolocalization of pelvic recurrences from rectosigmoid cancer employing 111In anti-CEA F(ab')2.

Thirty-two immunoscintigraphic studies have been performed in 26 patients previously operated upon for rectosigmoid cancer, for an early detection of pelvic recurrence, employing 111In anti-CEA, F(ab')2. The sensitivity was 1, specificity 0.76 and accuracy 0.84, leading to a positive predictive value of 0.69 and a negative value of 1. The combined use of a non-imaging surgical probe in 6 of the 10 patients who underwent second look surgery, detecting the 111In radioactivity used for RIS, improved diagnostic accuracy, ruling out the presence of tumors in 3 patients with false positive RIS.

[Radioimmunoscintigraphy with monoclonal antibodies in recurrences and metastases of colorectal tumors]. / Radioimmunoscintigrafia con anticorpi monoclonali delle recidive e delle metastasi dei tumori colorettali.

An experimental follow-up plan based on immunoscintigraphy was evaluated in 48 patients, who had undergone resection for colorectal cancer. The results were compared with those concerning other 317 patients submitted to a traditional follow-up (physical examination, blood analysis, endoscopy, ultrasonography, CT scan, barium enema, etc.). Immunoscintigraphy was performed with Iodine 131--or Indium 111--labeled anti-CEA and-19.9 monoclonal antibody, using a gamma-detecting probe (GDP) enabling to perform radioimmunodetections. In addition, radioimmunoguided surgery was performed in two cases of highly suspected recurrence, and radioimmunoguided endoscopy was made in two cases of suspected recurrence after anterior resection for tumor of the rectum. Our gamma-detecting probe differs from the other ones because of its collimator which enables to detect all the radioisotopes that are used for external scanning and because of its particular shape that enables to perform endoscopic radioimmuno detections. The experimental follow-up plan showed cancer recurrence in 27% of the patients vs. 13% of extensive instrumental investigations. Radioimmunoguided surgery appears to provide a higher level of radicality. The same GDP used for intraoperative immunoscintigraphies seems to be effective in the endoscopic study of the pelvis after anterior resection, a clinical occurrence which can hardly be investigated by means of other instrumental diagnostic procedures.

High-resolution protein patterns of human expressed prostatic secretion: a new tool for the diagnosis of prostatitis.

Expressed prostatic secretions (EPS) obtained from 62 infertile and urological patients have been analyzed for their protein content by sodium dodecylsulfate electrophoresis and isoelectrofocusing. Independent cytological and bacteriological screening on the same EPS samples was carried out to detect prostatic inflammation. EPS from both urological and infertile patients without inflammation had simple and characteristic protein patterns. Important qualitative and quantitative differences in the protein composition of EPS from patients with severe dysspermia or prostatic inflammation were found. The comparison of EPS protein patterns with those of seminal plasma obtained from the same patients led to the detection of a characteristic group of low-molecular-weight bands that were not of prostatic origin.