László Damjanovich

Intraoperative Tissue Identification Using Rapid Evaporative Ionization Mass Spectrometry

A mass spectrometric approach was developed for intraoperative identification of cancerous tissue, in near–real-time. Diagnosing the Masses One of the best options for curing cancer is surgery. Yet, surgeons can leave cancerous tissue behind by not seeing the “tumor margins”—or edges of the tumor—clearly. If a surgeon isn’t sure whether tissue is normal or cancerous, the tissue is sent to a pathologist for testing. During this time (20 to 30 min), the patient remains under anesthesia, and, quite often, additional samples are required. To ensure that all malignant tissue is removed in the operating room, Balog and colleagues developed a mass spectrometry–based approach that identifies cancer during surgery. After analyzing ex vivo samples of cancerous, healthy, and benign/inflammatory tissue with rapid evaporative ionization mass spectrometry (REIMS), the authors created a database of the nearly 3000 tissue-specific mass spectra. These spectra were unique for each cancer type, with lipids such as phosphatidylcholine and phosphotidylinositol showing different ratios. Using these ratios, Balog et al. were even able to identify the origin of metastatic tumors ex vivo. To adapt this technology for use in vivo, during surgery, the authors created the “intelligent knife” (iKnife), which samples surgical smoke for mass spectrometric analysis. More than 800 spectra were acquired with the iKnife from 81 patients. These spectra, when matched against the previously created database, confirmed the results of normal histology, with low rates of false-positive and false-negative readouts. This first-in-human demonstration shows that the iKnife technology is ready for widespread use in the operating room to improve the accuracy of surgical intervention in cancer. Rapid evaporative ionization mass spectrometry (REIMS) is an emerging technique that allows near–real-time characterization of human tissue in vivo by analysis of the aerosol (“smoke”) released during electrosurgical dissection. The coupling of REIMS technology with electrosurgery for tissue diagnostics is known as the intelligent knife (iKnife). This study aimed to validate the technique by applying it to the analysis of fresh human tissue samples ex vivo and to demonstrate the translation to real-time use in vivo in a surgical environment. A variety of tissue samples from 302 patients were analyzed in the laboratory, resulting in 1624 cancerous and 1309 noncancerous database entries. The technology was then transferred to the operating theater, where the device was coupled to existing electrosurgical equipment to collect data during a total of 81 resections. Mass spectrometric data were analyzed using multivariate statistical methods, including principal components analysis (PCA) and linear discriminant analysis (LDA), and a spectral identification algorithm using a similar approach was implemented. The REIMS approach differentiated accurately between distinct histological and histopathological tissue types, with malignant tissues yielding chemical characteristics specific to their histopathological subtypes. Tissue identification via intraoperative REIMS matched the postoperative histological diagnosis in 100% (all 81) of the cases studied. The mass spectra reflected lipidomic profiles that varied between distinct histological tumor types and also between primary and metastatic tumors. Thus, in addition to real-time diagnostic information, the spectra provided additional information on divergent tumor biochemistry that may have mechanistic importance in cancer.

Two germline alterations in mismatch repair genes found in a HNPCC patient with poor family history

The Bethesda guidelines may offer more useful criteria in patients’ selection for germline mismatch repair gene mutation analysis than guidelines merely based on family background. An early onset double primary colorectal cancer patient with poor family history with MSI-H status was investigated for MLH1 promoter methylation, expression of the MLH1 and MSH2 gene by immunohistochemistry and mutations in the MLH1 and MSH2 genes. The index patient carried two germline alterations, the p.Val716Met in MLH1 and the c.2210+lG>C in MSH2 genes, and both tumors failed to express MLH1 and MSH2 proteins. After subsequent analysis of the whole family of the index patient, the p.Val716Met variant can be defined as a rare polymorphism with the possible contribution of pathogenicity to tumor formation and c.2210+1G>C as a true pathogenic mutation causing an out-of-frame deletion of exon 13.

Crohn's disease alters MHC-rafts in CD4+ T-cells

Clusters of MHCI, ICAM‐1, CD44, CD59, IL‐2R, and IL‐15R molecules have been studied on the surface of CD4+ T‐cells from peripheral blood and lymph nodes of patients in Crohns disease and healthy individuals as controls by using a dual‐laser flow cytometric fluorescence resonance energy transfer (FRET) technique and fluorescently stained Fabs. When cells from patients in Crohns disease are compared to those of controls, the surface expression level for the MHCI reduced by ∼45%, for CD44 enhanced by ∼100%, and for IL‐2Rα, IL‐15Rα, and common γc enhanced by ∼50%, ∼70%, and ∼130%, respectively. Efficiencies of FRET monitoring homoassociation for the MHCI and CD44 reduced, that for IL‐2Rα enhanced. While efficiencies of FRET monitoring the association of γc and ICAM‐1 with the MHCI reduced, those monitoring association of IL‐2/15Rα, CD44, and CD59 with MHCI enhanced. Efficiencies of FRET measured between the MHCI and IL‐2Rα, IL‐15Rα differently enhanced to the advantage of IL‐15Rα, the one measured between γc and IL‐2Rα reduced, suggesting modulations in the strength of interaction of MHCI with IL‐2R, IL‐15R, and γc. The increases in density of surface bound cTx and in the associations of the receptors with the GM1‐ganglioside lipid molecules suggest stronger lipid raft interactions of the receptors. The observed alterations of MHC‐rafts in Crohns disease—summarized in models of receptor patterns of diseased and control cells—may have functional consequences regarding signaling by the raft components.

A new mutation in Muir-Torre syndrome associated with familiar transmission of different gastrointestinal adenocarcinomas

Hereditary Nonpolyposis Colorectal Carcinoma (HNPCC) is the most frequent inherited disease which can lead to the development of tumors in the colon and other locations. Its genetic basis is related to the germline mutation of the Mismatch Repair (MMR) genes. Muir-Torre syndrome is considered one of the subtypes of this disease, in which the HNPCC tumor spectrum is frequently associated with sebaceous carcinoma of the skin or keratoacanthoma. A 57 years old male patient is presented with a mucinous carcinoma of the caecum and an adenocarcinoma of the pancreas head. A malignant sebaceous carcinoma was removed from his left neck area. His family history was significant for two cases of colon carcinoma, two cases of stomach cancer and a case of metacron endometrial and skin tumor as well. Both the colon carcinoma and the skin tumor proved to be microsatellite unstable. An Arg>Pro switch missense mutation was found in codon 265 of the hMLH1 gene. This error was found in 4 other members of his family. The detected genetic alteration was considered pathogenic and was not published yet in English literature. The significance of this particular case is the rare tumor association in a patient with Muir-Torre syndrome (MTS). In cases of sebaceous skin lesions, evaluation of family history is of utmost importance in the early detection of HNPCC and in the follow up care of family members with the particular mutation.

Difficulties in recognizing families with Hereditary Non-polyposis Colorectal Carcinoma. Presentation of 4 families with proven mutation

INTRODUCTION Hereditary Non-polyposis Colorectal Carcinoma is the most frequent genetic disease leading to colon and other malignancies. Recognizing the condition requires extensive family history going back several generations focusing particularly on the types of tumors occurring in the family at different age groups. METHODS In families who met the Amsterdam and Bethesda Criteria, the removed tumor tissue was first examined by immunohistochemistry and microsatellite instability analysis. Subsequently DNA sequencing was performed to detect an underlying Mismatch Repair Gene mutation and multiple ligation dependent probe amplification was applied for recognizing large deletions in Mismatch Repair Genes. RESULTS In the investigated families 3 pathogen mutations, 1 large deletion and 2 cases of polymorphism were found. There is considerable difference between the families in terms of the types of malignancies and the age in which those appeared. CONCLUSION Recognizing families with Hereditary Non-polyposis Colorectal Carcinoma presents great difficulties because of the variety of phenotypes in presentation. Special attention should be paid to small families and those who present with cancer of other than colon origin. Practicing physicians should be made aware of the fact that this disease may have atypical presentations. Follow up of families who have already been screened may be difficult for social, economical or religious reasons.

Primary angiosarcoma of the pancreas mimicking severe acute pancreatitis – Case report

Primary angiosarcoma of the pancreas is an extremely rare neoplasm that often mimicks severe acute pancreatitis. A 58-year-old man was admitted with clinical and laboratory signs of severe acute pancreatitis. Contrast enhanced CT scan demonstrated haemorrhagic necrotizing inflammation of the pancreas involving the pancreatic tail, splenic hilum and small bowels with multiple peripancreatic and free abdominal fluid collection. Percutaneous drainage was performed. After 13 days, laparotomy was indicated because of persistent intra-abdominal bleeding, fever and a palpable, rapidly growing mass in the left upper quadrant of the abdomen. During the operation a necrotic, haemorrhagic mass was found in the pancreatic tail; a frozen section showed malignancy, although the tumour was unresectable. Despite all conservative and surgical therapeutic attempts, the patient died within four weeks after diagnosis. Final histology justified primary angiosarcoma of the pancreas. If a patient with signs of severe acute pancreatitis has fever without elevated PCT, the presence of a malignant tumour of the pancreas should be considered.

Intensity correlation-based calibration of FRET.

Dual-laser flow cytometric resonance energy transfer (FCET) is a statistically efficient and accurate way of determining proximity relationships for molecules of cells even under living conditions. In the framework of this algorithm, absolute fluorescence resonance energy transfer (FRET) efficiency is determined by the simultaneous measurement of donor-quenching and sensitized emission. A crucial point is the determination of the scaling factor α responsible for balancing the different sensitivities of the donor and acceptor signal channels. The determination of α is not simple, requiring preparation of special samples that are generally different from a double-labeled FRET sample, or by the use of sophisticated statistical estimation (least-squares) procedures. We present an alternative, free-from-spectral-constants approach for the determination of α and the absolute FRET efficiency, by an extension of the presented framework of the FCET algorithm with an analysis of the second moments (variances and covariances) of the detected intensity distributions. A quadratic equation for α is formulated with the intensity fluctuations, which is proved sufficiently robust to give accurate α-values on a cell-by-cell basis in a wide system of conditions using the same double-labeled sample from which the FRET efficiency itself is determined. This seemingly new approach is illustrated by FRET measurements between epitopes of the MHCI receptor on the cell surface of two cell lines, FT and LS174T. The figures show that whereas the common way of α determination fails at large dye-per-protein labeling ratios of mAbs, this presented-as-new approach has sufficient ability to give accurate results. Although introduced in a flow cytometer, the new approach can also be straightforwardly used with fluorescence microscopes.

Single-laser polarization FRET (polFRET) on the cell surface

A new method for the simultaneous detection of rotational mobility and proximity of cell surface receptors is presented based on cell-by-cell basis measurement of polarized fluorescence intensity components of the donor and acceptor of a FRET system. In addition to the FRET efficiency and the donor and acceptor concentrations, the method makes also possible the determination of the rotational characteristics and the associated fraction of the donors (FRET-fraction). The method is illustrated with flow cytometric and rFLIM measurements on donor-acceptor systems comprising fluorescently labeled whole antibodies and their Fab fragments against epitopes of the MHCI and MHCII cell surface receptors on human lymphoblast cells. Fluorescence anisotropy of donor and acceptor and FRET efficiency were measured for samples of different acceptor-to-donor concentration ratios. Acceptor anisotropy proved to be more sensitive than the donor anisotropy for sensing FRET. After determining the rotational constants of the donor-conjugated antibodies by measurements of FRET in the steady state, and by rFLIM as a reference, the associated fractions of the MHCI and MHCII molecules in their clusters were determined. Besides the flow cytometer and the wide-field rFLIM used in this study, the method can be applied also in other devices capable of dual-anisotropy detection.

Role of core needle biopsy in the treatment of radial scar.

Invasive tumor or ductal carcinoma in situ occur in radial sclerosing lesions in one third of the cases therefore, surgical excision is mandatory. Forty-five patients with radial scar morphology were examined. Ultrasound guided fine-needle aspiration biopsy (FNAB) and core biopsy (CB) were performed in all cases. The postoperative pathological findings were compared to the results of preoperative biopsies. Sensitivity of preoperative percutaneous biopsies (FNAB and CB) was 17.6% and 70.6%, false-negative rate was 82.4% with FNAB and 29.4% with CB. The negative predictive value was 48.1% and 84.8% respectively. Had we done preoperative cytology only, we would have had to perform a two-step procedure (sentinel lymph node biopsy) in 7 patients (15.6%), while with preoperative core biopsy it has decreased to 2 patients (4.4%). Preoperative CB in small radial stellate lesions is recommended to achieve accurate diagnosis in order to avoid a two-step surgical procedures.

[Analysis of clinical course of severe acute biliary and non biliary pancreatitis: a comparative study].

UNLABELLED The acute pancreatitis is a relative common disease with incidence of 5-80 per 100000 people of the population. The number of new cases has steadily increased in recent years. The two main etiological factors are alcohol and cholelithiasis. The incidence of alcoholic pancreatitis is higher in male, and the incidence of gallstone pancreatitis is higher in female. AIM To summarize the difference between the clinical course of biliary and not biliary type of severe acute pancreatitis by analyzing the data of these patients. METHODS 139 patients treated with severe acute pancreatitis were divided in two groups: biliary (A group) and non biliary (B group) of origin. The two groups were compared on the basis of sex and age, mortality, morbidity, number of surgery and hospital stay. chi 2 probe was used for the statistical analysis. RESULTS The complications in biliary group were more serious. The average mortality rate was 15.1%, 17.8% in group A and 13.8% in group B. The mortality rate of female patients in group A was significantly higher. CONCLUSION Female patients suffering from severe biliary acute pancreatitis have higher morbidity and mortality rate. Therefore an elective cholecystectomy is suggested in old female patients with serious co-morbidity and gallstones, before any complications.