Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine.Hepatocellular carcinoma (HCC) is a global health concern with an estimated 750,000 new cases per year (1). In more than 80% of cases, HCC arises in a setting of liver cirrhosis mainly of alcoholic or viral origin (2). The prognosis for HCC patients is poor, with less than 30% qualifying for curative treatments such as tumor resection or liver transplantation (2). Median survival time of patients that cannot be treated surgically is less than 1 y. Sorafenib is the only approved targeted therapy for HCC, prolonging median patient survival by ∼3 mo (3). Sorafenib is a multikinase inhibitor of Raf (B and C), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) (4), which presumably inhibits not only tumor cells but also endothelial cells responsible for tumor vascularization.Resistance to a targeted cancer drug can be intrinsic or adaptive (5). Sorafenib is largely cytostatic (6), suggesting that intrinsic resistance is more common in tumors, although some reports describe tumor shrinkage upon sorafenib treatment (7). Studies involving HCC cell lines or immunohistochemical staining of tumor sections revealed that sorafenib resistance correlates with the up-regulation of several signaling pathways, including the mammalian target of rapamycin (mTOR) pathway as assayed by S6 S235/236 (8) and Akt S473 phosphorylation (9). Other potential resistance mechanisms involve epithelial-to-mesenchymal transition (EMT) and autophagy (10, 11). However, the molecular mechanisms of sorafenib resistance in patients are largely unknown. Understanding the pathways that confer intrinsic or adaptive resistance would allow precision medicine and increase treatment efficacy.Proteomic analysis allows the identification of drug targets for cancer treatment and biomarkers for cancer classification or recurrence. In particular, MS is a powerful tool for resolving the complexity of cancer signaling pathways. With regard to HCC, qualitative proteomics has been performed on resected tumor material (12), laser-capture microdissected material from tissue sections (13, 14), and primary hepatocytes or serum derived from patients (15, 16). These studies (17, 18) identified HCC biomarkers such as glutamine synthetase and heat shock protein 70 (Hsp70) that are currently in use for diagnosis (19, 20). Quantitative proteomics has been performed on HCC resected tissue and serum (21, 22). Recently, proteomics has been performed on tumor biopsies of renal cell carcinoma patients (23). Several studies also have described phosphoproteomic analyses of resected HCC or other cancer material (24–26), in some cases quantifying up to 8,000 phosphorylated sites (hereafter referred to as “phosphosites”) starting with 2 mg of protein (18, 27–30). However, to our knowledge, quantitative proteomics and phosphoproteomics, hereafter collectively referred to as “(phospho)proteomics,” have yet to be performed on tumor biopsies, possibly because biopsy material is nonrenewable and typically provides only a very small amount of protein. Importantly, quantitative (phospho)proteomics on serial biopsies taken before and during treatment has not been described. We note that although a biopsy procedure generates less material than a resection, it has the important advantage of capturing normally dynamic properties of a tumor, such as the phosphorylation status of signaling pathways. Biopsies are immediately snap-frozen upon removal from the patient and, unlike resected tissue, are obtained without causing ischemia or hypoglycemia in the collected tissue. Needle biopsies are taken routinely to diagnose and stage the disease. Another important consideration is a method to perform quantitative (phospho)proteomics, such as super-SILAC (“SILAC” is an acronym for “stable isotope labeling of amino acids in cell culture”), that allows direct comparison of biopsies obtained at different times or from different patients (31).We describe quantitative (phospho)proteomic analyses of needle biopsies of HCC and matched nontumor tissue from a human patient. These analyses provide a global snapshot of signaling pathways in the biopsy material. Analyzing serial biopsies taken from a patient before and during therapy, we measured differences in signaling pathways between tumor and matched nontumor control tissue and the changes in these signaling pathways upon sorafenib treatment. Our findings provide insight into mechanisms of tumor progression and resistance to cancer therapy.     

Clinical and haematological improvement induced by etidronate in a patient with idiopathic myelofibrosis and osteosclerosis

Summary We report a patient with agnogenic myeloid metaplasia associated with debilitating bone pain due to increased bone turnover and osteosclerosis. Treatment with etidronate at a dose of 6 mg/kg per day on alternate months resulted in a complete recovery of bone symptoms and normalization of metabolic parameters of bone turnover; unexpectedly, a sustained haematological improvement was also observed after several months of therapy, suggesting that bone marrow microenvironment improvement was able to restore a nearly normal haemopoiesis. We suggest that diphosphonate therapy may be of value in patients with AMM and increased bone turnover.     

Juvenile patients with the homozygous MTHFR C677T genotype develop ischemic stroke 5 years earlier than wild type

Journal of Thrombosis and Thrombolysis - To compare age at 1st ischaemic stroke (IS) in a cohort of juvenile (<?46 years of age) IS patients evaluated for the rs1801133...     

Stress hyperglycemia is predictive of worse outcome in patients with acute ischemic stroke undergoing intravenous thrombolysis

Journal of Thrombosis and Thrombolysis - No study investigated the possible detrimental effect of stress hyperglycemia on patients affected acute ischemic stroke (AIS) undergoing intravenous...