Essential Thrombocytosis and Hepatocellular Carcinoma: A Mechanistic Challenge

Introduction/Objective Thrombocytosis has been reported in 8.2% of patients with Hepatocellular carcinoma [HCC] and has been attributed to increased Thrombopoietin [TPO] production by the tumor. Methods/Case Report We present a case of a 68-year-old male with a history of Hepatitis C and ethanol abuse presented with complaints of headache and chills. Imaging studies revealed a large mass in the liver [right lobe] suggesting an HCC of fibrolamellar type. His platelet count [PC] was 965 K/mcL. The serum AFP [Alpha feto protein] concentration was >51,800 ng/ml. There was no evidence of metastatic disease and the patient was started on Capecitabine and radiation therapy targeting the liver mass. Lung metastasis became evident and Sorafenib was added. His PCs and AFP concentrations however continued decreasing to 306 K/mcL and 460.7 ng/ml respectively. His PC then increased to a peak of 1.32 million/mcL 10 months later. The molecular workup done at our reference lab was positive for CALR [Calreticulin] mutation. The patient was started on Hydroxyurea and his PC decreased to 323 K/mcL at the time of writing. His AFP has remained stable. This case illuminates the complicated relationship between HCC and thrombocytosis. The therapy directed against the HCC did decrease PC and AFP concentration in our case. The second wave of thrombocytosis led to the discovery of CALR mutation, and the patient was diagnosed with Essential Thrombocytosis [ET]. Treatment with Hydroxyurea again decreased the PC while AFP concentration was increased but stable. Thrombopoietin levels have remained stable for the patient after treatment with Hydroxyurea. Results (if a Case Study enter NA) NA Conclusion Although the question of which mechanism(or possibly both) was at work in this case cannot be resolved definitely, two points are worth noting. The first is that- the assumption that the thrombocytosis was related to the HCC delayed testing for mutations associated with Essential Thrombocytosis. The second is that the TPO assay that may have yielded useful information in sorting out the alternatives was not ordered early enough before starting hydroxyurea.

Identification and Quantification of Proteins from Preparative Partition Chromatography and Peptides from Organic Extraction of Fetal Versus Adult Bovine Serum using Nano-Spray-LC-ESI-MS/MS

Blood proteins communicate with many different cells, tissue and organs; perform key functions in the immune system and may be of particular biological complexity. One of the most widely used blood products in the laboratory is fetal bovine serum for cell culture. There are ethical and practical concerns regarding the use of fetal serum from animals and alternative serum-free replacements have been attempted using platelet lysates. Previous biochemical experiments have shown that FBS apparently contained factors such as alpha-feto protein (AFP) and insulin-like growth factors that may support the indefinite cell growth and division of certain cell lines. It is presumed that a set of as yet undefined growth factors transform cells growth resulting in rapid proliferation. Cultured Raw cells 264.7 in adult bovine serum multiplied slowly and differentiated into elongated cells with a dendritic shape, which died after the first few generations. On the contrary, in fetal bovine serum, cultured cells multiplied rapidly and formed many smaller cells with a rounded shape through many cell passages. Three independent batches of fetal bovine serum were tested on Raw cells 264.7 macrophages to confirm that they supported cell growth in culture compared to three independent batches of adult bovine serum. The intact proteins of each serum sample were separated by partition chromatography into 16 fractions with an increasing step gradient of salts over quaternary amine resin (proteomics). The endogenous peptides were precipitated with 90% of acetonitrile and extracted into 10 fractions with a decreasing step gradient of acetonitrile in water (peptidomics). Trypsin digested intact proteins and endogenous peptides were then analyzed on a fresh C18 nano-HPLC column with random and independent sampling by LC-ESI-MS/MS. The fractionated mass spectra were identified with SEQUEST and X!TANDEM algorithms. Redundant use of MS/MS spectra were flirted out with the SQL Server system and the R statistical analysis system was used to perform Chi Square (X2) analysis of frequency counts and ANOVA of the log10 precursor intensity results. Alpha-feto protein, fetal albumin, insulin, insulin like growth factors, platelet derived growth factors and proteins associated with HRAS/AKT growth pathway at the level of ligand, receptors, receptor associated enzyme and nucleic acid binding proteins including transcription factors were observed to be specifically enriched in fetal serum.

Identification and Quantification of Proteins from Preparative Partition Chromatography and Peptides from Organic Extraction of Fetal Versus Adult Bovine Serum using Nano-Spray-LC-ESI-MS/MS

Blood proteins communicate with many different cells, tissue and organs; perform key functions in the immune system and may be of particular biological complexity. One of the most widely used blood products in the laboratory is fetal bovine serum for cell culture. There are ethical and practical concerns regarding the use of fetal serum from animals and alternative serum-free replacements have been attempted using platelet lysates. Previous biochemical experiments have shown that FBS apparently contained factors such as alpha-feto protein (AFP) and insulin-like growth factors that may support the indefinite cell growth and division of certain cell lines. It is presumed that a set of as yet undefined growth factors transform cells growth resulting in rapid proliferation. Cultured Raw cells 264.7 in adult bovine serum multiplied slowly and differentiated into elongated cells with a dendritic shape, which died after the first few generations. On the contrary, in fetal bovine serum, cultured cells multiplied rapidly and formed many smaller cells with a rounded shape through many cell passages. Three independent batches of fetal bovine serum were tested on Raw cells 264.7 macrophages to confirm that they supported cell growth in culture compared to three independent batches of adult bovine serum. The intact proteins of each serum sample were separated by partition chromatography into 16 fractions with an increasing step gradient of salts over quaternary amine resin (proteomics). The endogenous peptides were precipitated with 90% of acetonitrile and extracted into 10 fractions with a decreasing step gradient of acetonitrile in water (peptidomics). Trypsin digested intact proteins and endogenous peptides were then analyzed on a fresh C18 nano-HPLC column with random and independent sampling by LC-ESI-MS/MS. The fractionated mass spectra were identified with SEQUEST and X!TANDEM algorithms. Redundant use of MS/MS spectra were flirted out with the SQL Server system and the R statistical analysis system was used to perform Chi Square (X2) analysis of frequency counts and ANOVA of the log10 precursor intensity results. Alpha-feto protein, fetal albumin, insulin, insulin like growth factors, platelet derived growth factors and proteins associated with HRAS/AKT growth pathway at the level of ligand, receptors, receptor associated enzyme and nucleic acid binding proteins including transcription factors were observed to be specifically enriched in fetal serum.

Estimation of Levels of Glutathione Peroxidase (Gpx), Malondialdehyde (Mda), Tumor Necrosis Factor Alpha (Tnf Alpha) and Alpha Feto Protein (Afp) In Saliva of Potentially Malignant Disorders and Oral Squamous Cell Carcinoma.

The aim of the study was to estimate the levels of Glutathione peroxidase (GPx), malondialdehyde (MDA), Tumor necrosis factor alpha (TNF alpha) and Alpha feto protein (AFP) in saliva of potentially malignant disorders and oral squamous cell carcinoma, use them as an effective biomarkers in screening and diagnosis of oral squamous cell carcinoma as it is less invasive and more economical. 30 newly diagnosed patients with oral sub mucous fibrosis, oral leukoplakia, oral squamous cell carcinoma, who were not previously treated for the disease was selected for the study. 5ml of unstimulated saliva was collected from the patient for one minute in a sterile URICOL container and stored in sub-zero temperature before processing of the samples. Glutathione, malondialdehyde, alpha feto protein and TNF alpha was biochemically estimated and tabulated. There was increase mean concentration of glutathione, malondialdehyde, alpha feto protein and TNF alpha in carcinoma group and PMD group when compared to control group (p<0.05). Glutathione, Malondialdehyde, TNF alpha and alpha Feto protein are found to increase gradually from potentially malignant disorder to malignant condition. These factors can be used as potential biomarkers to indicate the prognosis of the disease and can be used as diagnostic tool for screening and early detection.