Glycoproteins or glycosylated proteins are carbohydrates (oligosaccharide chains or glycan’s) linked proteins and execute important functions in the biological systems, such as embryonic development, cell-to-cell recognition, adhesion, pathogen identification and immune functions. It is evident that the alteration of glycoproteins in cells are associated with a number of human diseases, including cancer, rheumatoid arthritis, inflammatory diseases as well as immunodeficiency diseases. Recent advances in modern technologies in cancer treatment are promising. However, researchers and clinicians are still searching for appropriate biomarkers for the early detection and management of patients with cancer. Altered glycoprotein levels are associated with critical events in cancer pathogenesis and progression. Also, abnormal glycosylation of protein is a common regulatory event in carcinogenesis, therefore, aberrant glycosylation could act as a promising resource in identifying a cancer biomarker for diagnosis and monitoring of the progression of patients with cancers. This chapter summarizes the major clinically approved glycoproteins utilized for screening, diagnosis, and monitoring of the treatment response of patients with cancers.

Supplementary Table S4: Dose reduction indices for BKM120

Table S2: Source and dilution factors of antibodies used

Supplementary Table S3: IC50 values for BKM120 and Erlotinib at 72 h

This file contains supplementary information for publication

Supplementary Table S6: Pair-wise p values for animal studies

Supp lementary Table S5: Dose reduction indices for erlotinib

BACKGROUND:Nucleophosmin-anaplastic lymphoma kinase-expressing (NPM-ALK+) T cell lymphoma is an aggressive neoplasm. NPM-ALK, an oncogenic tyrosine kinase, plays a critical role in this lymphoma. Recently, selective ALK inhibitors have emerged as a first-line therapy for this neoplasm. Unfortunately, ALK inhibitors were hindered by emergence of resistance and relapse. We have previously demonstrated that type I insulin-like growth factor receptor (IGF-IR) is commonly expressed and activated in this lymphoma. In addition, IGF-IR and NPM-ALK are physically associated and reciprocally enhance their phosphorylation/activation. Herein, we tested the hypothesis that combined inhibition of IGF-IR and NPM-ALK could significantly improve the effects of inhibiting each kinase alone.METHODS:We used clinically utilized inhibitors of IGF-IR (picropodophyllin; PPP) and ALK (ASP3026) to assess the in vitro cellular effects of combined treatment versus treatment using a single agent. Moreover, we used a systemic NPM-ALK+ T cell lymphoma mouse model to analyze the in vivo effects of PPP and ASP3026 alone or in combination.RESULTS:Our data show that combined treatment with PPP and ASP3026 decreased the viability, proliferation, and anchorage-independent colony formation, and increased apoptosis of NPM-ALK+ T cell lymphoma cells in vitro. The in vitro effects of combined treatment were synergistic and significantly more pronounced than the effects of PPP or ASP3026 alone. Biochemically, simultaneous antagonism of IGF-IR and ALK induced more pronounced decrease in pIGF-IRY1135/1136, pNPM-ALKY646, and pSTAT3Y705 levels than antagonizing IGF-IR or ALK alone. Moreover, combined targeting of IGF-IR and NPM-ALK decreased significantly systemic lymphoma tumor growth and improved mice survival in vivo. Consistent with the in vitro results, the in vivo effects of the combined therapy were more pronounced than the effects of targeting IGF-IR or ALK alone.CONCLUSIONS:Combined targeting of IGF-IR and ALK is more effective than targeting IGF-IR or ALK alone in NPM-ALK+ T cell lymphoma. This strategy might also limit emergence of resistance to high doses of ALK inhibitors. Therefore, it could represent a successful therapeutic approach to eradicate this aggressive lymphoma. Importantly, combined inhibition is feasible because of the clinical availability of IGF-IR and ALK inhibitors. Our findings are applicable to other types of cancer where IGF-IR and ALK are simultaneously expressed.

Supplementary Table S1: Characteristics of Cell Lines Used

Introduction: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancers. It is an aggressive neoplasm with dismal outcome because most of the patients present with an advanced-stage disease, which precludes curative surgical options. Therefore, these patients require systemic therapies that typically induce small improvements in overall survival. Hence, it is crucial to identify new and promising therapeutic targets for HCC to improve the current outcome. The liver is a key organ in the signaling cascade triggered by the growth hormone receptor (GHR). Previous studies have shown that GHR signaling stimulates the proliferation and regeneration of liver cells and tissues; however, a definitive role of GHR signaling in HCC pathogenesis has not been identified. Methods: In this study, we used a direct and specific approach to analyze the role of GHR in HCC development. This approach encompasses mice with global (Ghr-/-) or liver-specific (LiGhr-/-) disruption of GHR expression, and the injection of diethylnitrosamine (DEN) to develop HCC in these mice. Results: Our data show that DEN induced HCC in a substantial majority of the Ghr+/+ (93.5%) and Ghr+/- (87.1%) mice but not in the Ghr-/- (5.6%) mice (P < 0.0001). Although 57.7% of LiGhr-/- mice developed HCC after injection of DEN, these mice had significantly fewer tumors than LiGhr+/+ (P < 0.001), which implies that the expression of GHR in the liver cells might increase tumor burden. Notably, the pathologic, histologic, and biochemical characteristics of DEN-induced HCC in mice resembled to a great extent human HCC, despite the fact that etiologically this model does not mimic this cancer in humans. Our data also show that the effects of DEN on mice livers were primarily related to its carcinogenic effects and ability to induce HCC, with minimal effects related to toxic effects. Conclusion: Collectively, our data support an important role of GHR in HCC development, and suggest that exploiting GHR signaling may represent a promising approach to treat HCC.

Hepatocellular carcinoma (HCC) is an aggressive neoplasm with poor clinical outcome because most patients present at an advanced stage, at which point curative surgical options, such as tumor excision or liver transplantation, are not feasible. Therefore, the majority of HCC patients require systemic therapy. Nonetheless, the currently approved systemic therapies have limited effects, particularly in patients with advanced and resistant disease. Hence, there is a critical need to identify new molecular targets and effective systemic therapies to improve HCC outcome. The liver is a major target of the growth hormone receptor (GHR) signaling, and accumulating evidence suggests that GHR signaling plays an important role in HCC pathogenesis. We tested the hypothesis that GHR could represent a potential therapeutic target in this aggressive neoplasm. We measured GH levels in 767 HCC patients and 200 healthy controls, and then carried out clinicopathological correlation analyses. Moreover, specific inhibition of GHR was performed in vitro using siRNA and pegvisomant (a small peptide that blocks GHR signaling and is currently approved by the FDA to treat acromegaly) and in vivo, also using pegvisomant. GH was significantly elevated in 49.5% of HCC patients, and these patients had a more aggressive disease and poorer clinical outcome (P<0.0001). Blockade of GHR signaling with siRNA or pegvisomant induced substantial inhibitory cellular effects in vitro. In addition, pegvisomant potentiated the effects of sorafenib (P<0.01) and overcame sorafenib resistance (P<0.0001) in vivo. Mechanistically, pegvisomant decreased the phosphorylation of GHR downstream survival proteins including JAK2, STAT3, STAT5, IRS-1, AKT, ERK, and IGF-IR. In two patients with advanced-stage HCC and high GH who developed sorafenib resistance, pegvisomant caused tumor stability. Our data show that GHR signaling represents a novel “druggable” target, and pegvisomant may function as an effective systemic therapy in HCC. Our findings could also lead to testing GHR inhibition in other aggressive cancers.

The introduction of targeted therapy has revolutionized cancer treatment. Nonetheless, for this approach to succeed, it is crucial to identify the targets, particularly when activated, in tumor tissues. Phosphorylation is a posttranslational modification that causes activation of numerous oncogenic protein kinases and transcription regulators. Hence, phosphoproteins is a class of biomarkers that has therapeutic and prognostic implications directly relevant to cancer patients’ management. Despite the progress in histopathology methodology, analysis of the expression of phosphoproteins in tumor tissues still represents a challenge owing to preanalytical and analytical factors that include antigen retrieval strategies. In this study, we tested the hypothesis that optimizing antigen retrieval methods will improve phosphoproteins unmasking and enhance their immunohistochemical staining signal. We screened 4 antigen retrieval methods by using antibodies specific for 3 oncogenic phosphoproteins to stain human lymphoma tumors that were developed in severe combined immunodeficiency mice and subsequently fixed in formalin for 2 years. Then, we used antibodies specific for 15 survival phosphoproteins to compare the most effective method identified from our screening experiment to the antigen retrieval method that is most commonly utilized. Using the antigen retrieval buffer Tris-EDTA at pH 9.0 and heating for 45 minutes at 97°C unmasked and significantly enhanced the staining of 9 of the 15 phosphoproteins (P<0.0001). Our antigen retrieval approach is cost effective and feasible for clinical and research settings. We anticipate that combining this approach with the newly proposed methods to improve tissue fixation will further improve unmasking of phosphoproteins in human and animal tissues.

Background and Aims Therapeutic, clinical trial entry and stratification decisions for hepatocellular carcinoma (HCC) are made based on prognostic assessments, using clinical staging systems based on small numbers of empirically selected variables that insufficiently account for differences in biological characteristics of individual patients’ disease. Approach and Results We propose an approach for constructing risk scores from circulating biomarkers that produce a global biological characterization of individual patient’s disease. Plasma samples were collected prospectively from 767 patients with HCC and 200 controls, and 317 proteins were quantified in a Clinical Laboratory Improvement Amendments–certified biomarker testing laboratory. We constructed a circulating biomarker aberration score for each patient, a score between 0 and 1 that measures the degree of aberration of his or her biomarker panel relative to normal, which we call HepatoScore. We used log‐rank tests to assess its ability to substratify patients within existing staging systems/prognostic factors. To enhance clinical application, we constructed a single‐sample score, HepatoScore‐14, which requires only a subset of 14 representative proteins encompassing the global biological effects. Patients with HCC were split into three distinct groups (low, medium, and high HepatoScore) with vastly different prognoses (medial overall survival 38.2/18.3/7.1 months; P < 0.0001). Furthermore, HepatoScore accurately substratified patients within levels of existing prognostic factors and staging systems (P < 0.0001 for nearly all), providing substantial and sometimes dramatic refinement of expected patient outcomes with strong therapeutic implications. These results were recapitulated by HepatoScore‐14, rigorously validated in repeated training/test splits, concordant across Myriad RBM (Austin, TX) and enzyme‐linked immunosorbent assay kits, and established as an independent prognostic factor. Conclusions HepatoScore‐14 augments existing HCC staging systems, dramatically refining patient prognostic assessments and therapeutic decision making and enrollment in clinical trials. The underlying strategy provides a global biological characterization of disease, and can be applied broadly to other disease settings and biological media.

Cancer is the second deadliest disease worldwide. Although recent advances applying precision treatments with targeted (molecular and immune) agents are promising, the histological and molecular heterogeneity of cancer cells and huge mutational burdens (intrinsic or acquired after therapy) leading to drug resistance and treatment failure are posing continuous challenges. These recent advances do not negate the need for alternative approaches such as chemoprevention, the pharmacological approach to reverse, suppress or prevent the initial phases of carcinogenesis or the progression of premalignant cells to invasive disease by using non-toxic agents. Although data are limited, the success of several clinical trials in preventing cancer in high-risk populations suggests that chemoprevention is a rational, appealing and viable strategy to prevent carcinogenesis. Particularly among higher-risk groups, the use of safe, non-toxic agents is the utmost consideration because these individuals have not yet developed invasive disease. Natural dietary compounds present in fruits, vegetables and spices are especially attractive for chemoprevention and treatment because of their easy availability, high margin of safety, relatively low cost and widespread human consumption. Hundreds of such compounds have been widely investigated for chemoprevention and treatment in the last few decades. Previously, we reviewed the most widely studied natural compounds and their molecular mechanisms, which were highly exploited by the cancer research community. In the time since our initial review, many promising new compounds have been identified. In this review, we critically review these promising new natural compounds, their molecular targets and mechanisms of anticancer activity that may create novel opportunities for further design and conduct of preclinical and clinical studies.

Background: Sorafenib was the first systemic therapy approved for the treatment of Child-Turcotte-Pugh (CTP) class A patients with advanced hepatocellular carcinoma (HCC). However, there are no biomarkers to predict survival and treatment outcomes and guide HCC systemic therapy. Type 1 insulin-like growth factor (IGF-1)/CTP composite score has emerged as a potential hepatic reserve assessment tool. Our study investigated the association of the IGF/CTP score with overall survival (OS) and progression-free survival (PFS) of HCC patients treated with sorafenib. Materials and Methods: In this prospective study, patients with HCC were treated with sorafenib and followed up until progression/death. We calculated the IGF/CTP score and used the Kaplan-Meier method and log-rank test to estimate and compare the time-to-event outcomes between patient subgroups. Results: 171 patients were included, 116 of whom were CTP class A. Median PFS for IGF/CTP score AA and AB patients were 6.88 and 4.28 months, respectively (p = 0.1359). Median OS for IGF/CTP score AA and AB patients were 14.54 and 7.60 months, respectively (p = 0.1378). The PFS and OS was superior in AA patients, but the difference was not significant, likely due to the sample size. However, there was a significant difference in early OS and PFS curves between AA and AB (p = 0.0383 and p = 0.0099), respectively. Conclusions: In CTP class A patients, IGF/CTP score B was associated with shorter PFS and OS, however, study was underpowered to reach statistical significance. If validated in larger cohorts, IGF/CTP score may serve as stratification tool in clinical trials, a hepatic reserve assessment tool for HCC outcomes prediction and to assist in therapy decisions.

Objective: Chronic kidney disease (CKD) is a major public health concern worldwide. High costs of late-stage diagnosis and insufficient testing facilities can contribute to high morbidity and mortality rates in CKD patients, particularly in less developed countries. Thus, early diagnosis aided by vital parameter analytics using affordable computer-aided diagnosis could not only reduce diagnosis costs but improve patient management and outcomes. Methods: In this study, we developed machine learning models using selective key pathological categories to identify clinical test attributes that will aid in accurate early diagnosis of CKD. Such an approach will save time and costs for diagnostic screening. We have also evaluated the performance of several classifiers with k-fold cross-validation on optimized datasets derived using these selected clinical test attributes. Results: Our results suggest that the optimized datasets with important attributes perform well in diagnosis of CKD using our proposed machine learning models. Furthermore, we evaluated clinical test attributes based on urine and blood tests along with clinical parameters that have low costs of acquisition. The predictive models with the optimized and pathologically categorized attributes set yielded high levels of CKD diagnosis accuracy with random forest (RF) classifier being the best performing. Conclusions: Our machine learning approach has yielded effective predictive analytics for CKD screening which can be developed as a resource to facilitate improved CKD screening for enhanced and timely treatment plans.

Abstract Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. However, intrinsic and acquired resistance to targeted agents poses challenges to the success of such treatments. The purpose of the current study was to investigate the intrinsic resistance of head and neck cancer (HNC) cell lines to apoptosis induced by the combination of EGFR inhibitor erlotinib and PI3K inhibitor BKM120. Methods: In a panel of 10 malignant and 1 premalignant HNC cell lines, we evaluated cell growth inhibition (by SRB assay), IC50, combination index, dose reduction index (by CalcuSyn Software), and apoptosis (by Annexin-V staining). Protein expression levels were measured by Western blotting. Small molecule inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: We have reported previously that single targeting of EGFR with erlotinib or PI3K with BKM120 (pan-PI3K inhibitor) suppressed cellular growth without inducing significant apoptosis. Co-targeting EGFR and PI3K demonstrated in vitro synergy in all except the JHU022 cell line and more effectively inhibited HNC xenograft growth in vivo. The combination of erlotinib and BKM120 induced variable apoptosis: some cell lines were very sensitive (MDA686TU, MDA686LN, 93-VU-147T), some moderately (Fadu, SqCCy1, MDA1483, UPCI:SCC090) and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. We found that the resistant cell line JHU022 expressed very high levels of p-Met (Y1234/1235). Inhibition of Met with small molecule inhibitor crizotinib or siRNA rendered this cell line highly sensitive to apoptosis induced by the combination of erlotinib and BKM120. Although inhibition of hepatocyte growth factor (HGF), the only known ligand for Met, with soluble neutralization antibody had no effect on Met phosphorylation, inhibition of Src family kinases (SFK) with dasatinib abolished Met phosphorylation at Y1234/1235. Dasatinib also made this cell line sensitive to apoptosis. Ablation of c-Src with siRNA had similar effects on p-Met (Y1234/1235) and apoptosis. Interestingly, inhibition of Met had no effects on AKT and ERK phosphorylation, two critical downstream pathways of receptor tyrosine kinases including Met. Conclusions: Our results strongly suggest that co-targeting of EGFR and PI3K has synergistic activity, is effective in vivo, and induces apoptosis of some HNC cell lines. Some resistant cell lines express a high level of p-Met as a downstream target of SFK, which confers resistance of these cells to EGFR and PI3K co-targeting. Finally, Met confers apoptosis resistance independently of AKT and ERK pathway activation. Citation Format: Abu S. Anisuzzaman, Abedul Haque, Zhuo G. Chen, Dong M. Shin, A.R.M. Ruhul Amin. Src-Met signaling confers apoptosis resistance to EGFR and PI3K co-targeting independently of AKT and ERK pathway activation in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1907.

Background: Liver reserve affects survival in hepatocellular carcinoma (HCC). Model for End-Stage Liver Disease (MELD) score is used to predict overall survival (OS) and to prioritize HCC patients on the transplantation waiting list, but more accurate models are needed. We hypothesized that integrating insulin-like growth factor 1 (IGF-1) levels into MELD score (MELD-IGF-1) improves OS prediction as compared to MELD. Methods: We measured plasma IGF-1 levels in training (n = 310) and validation (n = 155) HCC cohorts and created MELD-IGF-1 score. Cox models were used to determine the association of MELD and MELD-IGF-1 with OS. Harrell’s c-index was used to compare the predictive capacity. Results: IGF-1 was significantly associated with OS in both cohorts. Patients with an IGF-1 level of ≤26 ng/mL in the training cohort and in the validation cohorts had significantly higher hazard ratios than patients with the same MELD but IGF-1 >26 ng/mL. In both cohorts, MELD-IGF-1 scores had higher c-indices (0.60 and 0.66) than MELD scores (0.58 and 0.60) (p < 0.001 in both cohorts). Overall, 26% of training and 52.9% of validation cohort patients were reclassified into different risk groups by MELD-IGF-1 (p < 0.001). Conclusions: After independent validation, the MELD-IGF-1 could be used to risk-stratify patients in clinical trials and for priority assignment for patients on liver transplantation waiting list.