Dynamic Contrast Enhanced MRI and Intravoxel Incoherent Motion to Identify Molecular Subtypes of Breast Cancer with Different Vascular Normalization Gene Expression.

OBJECTIVE: To assess the expression of vascular normalization genes in different molecular subtypes of breast cancer and to determine whether molecular subtypes with a higher vascular normalization gene expression can be identified using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) and intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI). MATERIALS AND METHODS: This prospective study evaluated 306 female (mean age ± standard deviation, 50 ± 10 years), recruited between January 2014 and August 2017, who had de novo breast cancer larger than 1 cm in diameter (308 tumors). DCE MRI followed by IVIM DWI studies using 11 different b-values (0 to 1200 s/mm²) were performed on a 1.5T MRI system. The Tofts model and segmented biexponential IVIM analysis were used. For each tumor, the molecular subtype (according to six [I-VI] subtypes and PAM50 subtypes), expression profile of genes for vascular normalization, pericytes, and normal vascular signatures were determined using freshly frozen tissue. Statistical associations between imaging parameters and molecular subtypes were examined using logistic regression or linear regression with a significance level of p = 0.05. RESULTS: Breast cancer subtypes III and VI and PAM50 subtypes luminal A and normal-like exhibited a higher expression of genes for vascular normalization, pericyte markers, and normal vessel function signature (p < 0.001 for all) compared to other subtypes. Subtypes III and VI and PAM50 subtypes luminal A and normal-like, versus the remaining subtypes, showed significant associations with Ktrans, kep, vp, and IAUGCBN90 on DEC MRI, with relatively smaller values in the former. The subtype grouping was significantly associated with D, with relatively less restricted diffusion in subtypes III and VI and PAM50 subtypes luminal A and normal-like. CONCLUSION: DCE MRI and IVIM parameters may identify molecular subtypes of breast cancers with a different vascular normalization gene expression.

Why Cannot BCLC 0- or A-Stage Patients Receive Curative Treatment?

INTRODUCTION: Barcelona Clinic Liver Cancer (BCLC) staging has been an important clinical guideline for the management of hepatocellular carcinoma (HCC). BCLC 0 and A stages (BCLC 0/A) have been designated as the early-stage HCC, and the curative treatment is recommended as the primary therapeutic modality. However, a recent study indicated that a significant number of BCLC 0/A patients were not initially managed with the curative treatment without knowing why. METHODS: We, therefore, conducted a study on BCLC 0/A patients who had and had not received initial curative treatment cared at our cancer center from January 2011 to December 2015 and analyzed causes contributing to not having the initial curative treatment. RESULTS: One hundred and sixty-nine BCLC 0/A patients were identified and included in the study. Seventy two patients (43%) received the initial curative treatment and 97 patients (57%) did not. After careful review of medical records, all 97 patients without the initial curative treatment had identifiable reasons for not having the initial curative treatment. Two main reasons for not having the initial curative treatment were "probable presence of additional HCC and requiring diagnostic angiography" (28%) and "difficult or complicating anatomical location of tumors" (17%). When the relevant clinical parameters were compared between the 2 groups of patients, it was found that patients without the initial curative treatment had more serious clinical conditions and worse overall and recurrence-free survival outcomes compared with those who had the initial curative treatment. DISCUSSION/CONCLUSION: Our finding indicates that a significant fraction of the BCLC 0/A HCC patients is unable to have initial curative treatment as recommended by BCLC guidelines. These early stages of HCC patients represent a distinctive subpopulation and are in need of further investigation to improve their survival outcomes.

Clinical Factors Predicting Better Survival Outcome for Pulmonary Metastasectomy of Hepatocellular Carcinoma.

BACKGROUND: In patients with lung metastasis of hepatocellular carcinoma (HCC), it remains uncertain how a better survival outcome can be predicted after metastasectomy. This study aims to identify clinical factors that may be used to guide patient selection for such a therapeutic modality. A total of 28 patients who received pulmonary metastasectomy for HCC between 1993 and 2012 were identified. All relevant clinical factors were extracted from medical records up to September 2015. Patients were classified into high- and low-risk groups according to survival outcome after metastasectomy. All pertinent clinical factors were analyzed for correlation with survival outcome. SUMMARY: The overall survival of 28 patients after pulmonary metastasectomy was studied first. The survival curve was biphasic and reached a plateau at 40 months after metastasectomy. The results indicate the presence of 2 groups of patients with a different survival outcome. Among all clinical parameters, remission status in the liver before pulmonary metastasectomy and distant metastasis-free interval between the last treatment of HCC and the occurrence of lung metastasis were found to be significantly associated with excellent survival outcome after pulmonary metastasectomy (p = 0.019 and 0.007 by Fisher exact test, and p = 0.002 and 0.0002 by Cox regression analysis).

B1 Field Correction of T1 Estimation Should Be Considered for Breast Dynamic Contrast-enhanced MR Imaging Even at 1.5 T.

Purpose To prospectively quantify the effect of T1 estimation in fat by B1 correction in breast magnetic resonance (MR) imaging at 1.5 T and to examine the subsequent quantitative dynamic contrast material-enhanced parameters in breast cancer with and without B1 correction. Materials and Methods This study had institutional review board approval, and informed consent was obtained from 72 patients with breast cancer before breast MR imaging studies were performed between January and July 2015. B1+ field and variable flip angle (FA) mapping were included in the dynamic contrast-enhanced breast MR imaging protocol with a 1.5-T MR imaging system. Precontrast T1 relaxation in fat and breast tumors was computed with and without B1 correction. The pharmacokinetic parameters of breast cancer were calculated by using the Tofts model with T1 values before and after B1 correction. The Mann-Whitney U test and linear regression model were used for statistical analysis. Results The FA was 19% higher in the left breast and 3% lower in the right breast than the prescribed value. This 22% average FA difference created a 43% T1 estimation bias in fat between the breasts. The T1 variation in fat was reduced to 0.96% after B1 correction. There was a 50% overestimation and a 7% underestimation of tumor T1 in the left breast and the right, respectively, associated with B1 error. Assuming T1 after B1 correction represents the true tumor T1, 41% underestimation in the left breast and 10% overestimation in the right without B1 correction were seen in the dynamic contrast-enhanced parameters (including the volume transfer constant, or Ktrans, fraction of extracellular extravascular space, or ve, and blood normalized initial area under the gadolinium concentration curve to 90 seconds, or IAUGCBN90). Conclusion B1 correction for more accurate T1 values should be considered for quantitative dynamic contrast-enhanced breast MR imaging, even at 1.5 T, to offset significant systemic error. © RSNA, 2016.

Plasmalemmal Vesicle Associated Protein (PLVAP) as a therapeutic target for treatment of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignancy with poor survival outcome. New treatment options for the disease are needed. In this study, we identified and evaluated tumor vascular PLVAP as a therapeutic target for treatment of HCC. METHODS: Genes showing extreme differential expression between paired human HCC and adjacent non-tumorous liver tissue were investigated. PLVAP was identified as one of such genes with potential to serve as a therapeutic target for treatment of HCC. A recombinant monoclonal anti-PLVAP Fab fragment co-expressing extracellular domain of human tissue factor (TF) was developed. The potential therapeutic effect and toxicity to treat HCC were studied using a Hep3B HCC xenograft model in SCID mice. RESULTS: PLVAP was identified as a gene specifically expressed in vascular endothelial cells of HCC but not in non-tumorous liver tissues. This finding was confirmed by RT-PCR analysis of micro-dissected cells and immunohistochemical staining of tissue sections. Infusion of recombinant monoclonal anti-PLVAP Fab-TF into the main tumor feeding artery induced tumor vascular thrombosis and extensive tumor necrosis at doses between 2.5 µg and 12 µg. Tumor growth was suppressed for 40 days after a single treatment. Systemic administration did not induce tumor necrosis. Little systemic toxicity was noted for this therapeutic agent. CONCLUSIONS: The results of this study suggest that anti-PLVAP Fab-TF may be used to treat HCC cases for which transcatheter arterial chemoembolization (TACE) is currently used and potentially avoid the drawback of high viscosity of chemoembolic emulsion for TACE to improve therapeutic outcome. Anti-PLVAP Fab-TF may become a viable therapeutic agent in patients with advanced disease and compromised liver function.

Inhibition of Hec1 as a novel approach for treatment of primary liver cancer.

PURPOSE: Highly expressed in cancer protein 1 (Hec1) is an oncogene and a promising molecular target for novel anticancer drugs. The purpose of this study was to evaluate the potential of a Hec1 inhibitor, TAI-95, as a treatment for primary liver cancer. METHODS: In vitro and in vivo methods were used to test the activity of TAI-95. Gene expression analysis was used to evaluate clinical correlation of the target. RESULTS: In vitro growth inhibition results showed that TAI-95 has excellent potency on a wide range of primary liver cancer cell lines (hepatoblastoma or hepatocellular carcinoma) (GI(50) 30-70 nM), which was superior to sorafenib and other cytotoxic agents. TAI-95 was relatively inactive in non-cancerous cell lines (GI(50) > 10 µM). TAI-95 disrupts the interaction between Hec1 and Nek2 and leads to degradation of Nek2, chromosomal misalignment, and apoptotic cell death. TAI-95 showed synergistic activity in selected cancer cell lines with doxorubicin, paclitaxel, and topotecan, but not with sorafenib. TAI-95 shows excellent potency in a Huh-7 xenograft mouse model when administered orally. Gene expression analysis of clinical samples demonstrated increased expression of Hec1/NDC80 and associated genes (Nek2, SMC1A, and SMC2) in 27 % of patients, highlighting the potential for using this therapeutic approach to target patients with high Hec1 expression. CONCLUSION: Inhibition of Hec1 using small molecule approach may represent a promising novel approach for the treatment of primary liver cancers.

Activity of a novel Hec1-targeted anticancer compound against breast cancer cell lines in vitro and in vivo.

Current cytotoxic chemotherapy produces clinical benefit in patients with breast cancer but the survival impact is modest. To explore novel cytotoxic agents for the treatment of advanced disease, we have characterized a new and pharmacokinetically improved Hec1-targeted compound, TAI-95. Nine of 11 breast cancer cell lines tested were sensitive to nanomolar levels of TAI-95 (GI(50) = 14.29-73.65 nmol/L), and more importantly, TAI-95 was active on a number of cell lines that were resistant (GI(50) > 10 µmol/L) to other established cytotoxic agents. TAI-95 demonstrates strong inhibition of in vivo tumor growth of breast cancer model when administered orally, without inducing weight loss or other obvious toxicity. Mechanistically, TAI-95 acts by disrupting the interaction between Hec1 and Nek2, leading to apoptotic cell death in breast cancer cells. Furthermore, TAI-95 is active on multidrug-resistant (MDR) cell lines and led to downregulation of the expression of P-glycoprotein (Pgp), an MDR gene. In addition, TAI-95 increased the potency of cytotoxic Pgp substrates, including doxorubicin and topotecan. Certain clinical subtypes of breast cancer more likely to respond to Hec1-targeted therapy were identified and these subtypes are the ones associated with poor prognosis. This study highlights the potential of the novel anticancer compound TAI-95 in difficult-to-treat breast cancers.

Characterization of the biological activity of a potent small molecule Hec1 inhibitor TAI-1.

BACKGROUND: Hec1 (NDC80) is an integral part of the kinetochore and is overexpressed in a variety of human cancers, making it an attractive molecular target for the design of novel anticancer therapeutics. A highly potent first-in-class compound targeting Hec1, TAI-1, was identified and is characterized in this study to determine its potential as an anticancer agent for clinical utility. METHODS: The in vitro potency, cancer cell specificity, synergy activity, and markers for response of TAI-1 were evaluated with cell lines. Mechanism of action was confirmed with western blotting and immunofluorescent staining. The in vivo potency of TAI-1 was evaluated in three xenograft models in mice. Preliminary toxicity was evaluated in mice. Specificity to the target was tested with a kinase panel. Cardiac safety was evaluated with hERG assay. Clinical correlation was performed with human gene database. RESULTS: TAI-1 showed strong potency across a broad spectrum of tumor cells. TAI-1 disrupted Hec1-Nek2 protein interaction, led to Nek2 degradation, induced significant chromosomal misalignment in metaphase, and induced apoptotic cell death. TAI-1 was effective orally in in vivo animal models of triple negative breast cancer, colon cancer and liver cancer. Preliminary toxicity shows no effect on the body weights, organ weights, and blood indices at efficacious doses. TAI-1 shows high specificity to cancer cells and to target and had no effect on the cardiac channel hERG. TAI-1 is synergistic with doxorubicin, topotecan and paclitaxel in leukemia, breast and liver cancer cells. Sensitivity to TAI-1 was associated with the status of RB and P53 gene. Knockdown of RB and P53 in cancer cells increased sensitivity to TAI-1. Hec1-overexpressing molecular subtypes of human lung cancer were identified. CONCLUSIONS: The excellent potency, safety and synergistic profiles of this potent first-in-class Hec1-targeted small molecule TAI-1 show its potential for clinically utility in anti-cancer treatment regimens.

Serum midkine as a prognostic biomarker for patients with hepatocellular carcinoma.

Gene expression profiles of paired hepatocellular carcinoma (HCC) and adjacent noncancerous liver tissue samples revealed preferential expression of midkine in HCC. This finding suggested the clinical usefulness of midkine measurement in serum for monitoring HCC treatment response, recurrence, and progression. A prospective study in 285 patients, 144 in complete remission and 141 at risk for developing de novo HCC, was conducted. The changes in serum midkine level were in parallel with disease activity in about 81% of patients with HCC. The study also revealed that rapidly rising serum midkine levels occurred in patients in the terminal stage of HCC. The rising rate of serum midkine levels was inversely correlated with remaining survival days. However, serum midkine measurement did not detect emergence of new HCC in most patients in complete remission and in high-risk people without a history of HCC. Serum midkine levels can be useful to monitor HCC progression, and a sharp rise signals the approach of end of life in patients with HCC.

Correlation of microarray-based breast cancer molecular subtypes and clinical outcomes: implications for treatment optimization.

BACKGROUND: Optimizing treatment through microarray-based molecular subtyping is a promising method to address the problem of heterogeneity in breast cancer; however, current application is restricted to prediction of distant recurrence risk. This study investigated whether breast cancer molecular subtyping according to its global intrinsic biology could be used for treatment customization. METHODS: Gene expression profiling was conducted on fresh frozen breast cancer tissue collected from 327 patients in conjunction with thoroughly documented clinical data. A method of molecular subtyping based on 783 probe-sets was established and validated. Statistical analysis was performed to correlate molecular subtypes with survival outcome and adjuvant chemotherapy regimens. Heterogeneity of molecular subtypes within groups sharing the same distant recurrence risk predicted by genes of the Oncotype and MammaPrint predictors was studied. RESULTS: We identified six molecular subtypes of breast cancer demonstrating distinctive molecular and clinical characteristics. These six subtypes showed similarities and significant differences from the Perou-Sørlie intrinsic types. Subtype I breast cancer was in concordance with chemosensitive basal-like intrinsic type. Adjuvant chemotherapy of lower intensity with CMF yielded survival outcome similar to those of CAF in this subtype. Subtype IV breast cancer was positive for ER with a full-range expression of HER2, responding poorly to CMF; however, this subtype showed excellent survival when treated with CAF. Reduced expression of a gene associated with methotrexate sensitivity in subtype IV was the likely reason for poor response to methotrexate. All subtype V breast cancer was positive for ER and had excellent long-term survival with hormonal therapy alone following surgery and/or radiation therapy. Adjuvant chemotherapy did not provide any survival benefit in early stages of subtype V patients. Subtype V was consistent with a unique subset of luminal A intrinsic type. When molecular subtypes were correlated with recurrence risk predicted by genes of Oncotype and MammaPrint predictors, a significant degree of heterogeneity within the same risk group was noted. This heterogeneity was distributed over several subtypes, suggesting that patients in the same risk groups require different treatment approaches. CONCLUSIONS: Our results indicate that the molecular subtypes established in this study can be utilized for customization of breast cancer treatment.

Persistence of lymphocytotoxic antibodies in patients in the trial to reduce alloimmunization to platelets: implications for using modified blood products.

Patients with acute myelogenous leukemia undergoing induction chemotherapy have significant decreases in alloimmune platelet refractoriness if they receive filter-leukoreduced or UV-B-irradiated vs standard platelet transfusions (3%-5% vs 13%, respectively; P ≤ .03) with no differences among the treated platelet arms (Trial to Reduce Alloimmunization to Platelets). Therefore, measuring antibody persistence might identify the best platelets for transfusion. Lymphocytotoxic (LCT) antibody duration was evaluated for association with patient age, sex, prior transfusion and pregnancy history, study-assigned platelet transfusions, and percentage LCT panel reactive antibodies. During the Trial to Reduce Alloimmunization to Platelets, 145 patients became antibody positive; and 81 (56%) of them subsequently became antibody negative. Using Kaplan-Meier estimates, projected antibody loss was 73% at 1 year. Major factors associated with antibody persistence were prior pregnancy and percentage panel reactive antibody positivity, whereas neither the assigned type of platelets transfused during the 8 weeks of the trial nor prior transfusion history was predictive. After 5 to 8 weeks, the number and type of blood products transfused had no effect on either antibody development or loss. A majority of patients with acute myelogenous leukemia who develop LCT antibodies during induction chemotherapy will lose their antibodies within 4 months regardless of the type or number of blood products they receive.

Factors affecting posttransfusion platelet increments, platelet refractoriness, and platelet transfusion intervals in thrombocytopenic patients.

A variety of patient and product-related factors influenced the outcome of 6379 transfusions given to 533 patients in the Trial to Reduce Alloimmunization to Platelets (TRAP). Responses measured were platelet increments, interval between platelet transfusions, and platelet refractoriness. Patient factors that improved platelet responses were splenectomy and increasing patient age. In contrast, at least 2 prior pregnancies, male gender, splenomegaly, bleeding, fever, infection, disseminated intravascular coagulation, increasing height and weight, lymphocytotoxic antibody positivity, an increasing number of platelet transfusions, or receiving heparin or amphotericin were associated with decreased posttransfusion platelet responses. Platelet factors that were associated with improved platelet responses were giving ABO-compatible platelets, platelets stored for 48 hours or less, and giving large doses of platelets while ultraviolet B (UV-B) or gamma irradiation decreased platelet responses. However, in alloimmunized lymphocytoxic antibody-positive patients, the immediate increment to UV-B-irradiated platelets was well maintained, whereas all other products showed substantial reductions. Refractoriness to platelet transfusions developed in 27% of the patients. Platelet refractoriness was associated with lymphocytotoxic antibody positivity, heparin administration, fever, bleeding, increasing number of platelet transfusions, increasing weight, at least 2 pregnancies, and male gender. The only factors that reduced platelet refractoriness rates were increasing the dose of platelets transfused or transfusing filtered apheresis platelets.

Effect of CXC chemokine platelet factor 4 on differentiation and function of monocyte-derived dendritic cells.

Platelet factor 4 (PF4) is a CXC chemokine secreted by activated platelets. PF4 has been shown to promote monocyte survival and induce the differentiation of monocytes into macrophages. However, the effect of PF4 on differentiation of monocytes into dendritic cells (DC) has yet to be determined. As reported previously, monocytes cultured in RPMI medium containing FCS, granulocyte macrophage colony stimulating factor and IL-4 differentiated into CD1a+ DC. When PF4 was added, the expression of CD1a on DC was inhibited. This inhibitory effect was not observed with the other platelet-derived CXC chemokine, beta-thromboglobulin. The relative number of CD1a- DC increased from 17 to 92% when the PF4 concentration was increased from 0 to 10 micro g/ml. The inhibitory effect of PF4 on CD1a expression was reversed by 50 U/ml heparin. DC developed in the PF4-containing media appeared more adhesive to plastic culture wells and had higher light side scatter by flow cytometry. Immunophenotypically, monocyte-derived DC in the presence of increasing concentrations of PF4 proportionally expressed higher CD86 and lower HLA-DR. The levels of CD11c, CD40 and CD80 remained unchanged with or without PF4. Both CD1a+ DC and CD1a- DC were negative for CD14, CD68 and CD83. Functionally, DC developed in the presence of PF4 had their secretion of tumor necrosis factor-alpha and IL-12 reduced by 75 +/- 10 and 79 +/- 13% respectively when they were stimulated by 100 ng/ml lipopolysaccharide and 50 ng/ml IFN-gamma. CD1a- DC developed in the presence of PF4 were not as active as the control CD1a+ DC in stimulating allogeneic T cells to proliferate. In addition, CD1a- DC were less potent in priming naive CD4+ T cells to secrete both type 1 and 2 cytokines. These results indicate that PF4 can influence differentiation and function of monocyte-derived DC.

Molecular cloning and characterization of a novel gene family of four ancient conserved domain proteins (ACDP).

We have recently cloned four novel human genes that encode the ancient conserved domain proteins (ACDP). The full-length cDNA sequence of ACDP1 consists of 5898 bp and encodes a predicted protein of 951 amino acids (AA). The transcript for ACDP2 has 4058 bp of cDNA sequence, encoding a protein of 875 AA. ACDP3 contains 3113 bp of cDNA sequence and encodes a putative protein of 707 AA. ACDP4 contains 4765 bp of cDNA sequence and encodes a protein of 775 AA. The ACDP genes belong to a highly conserved new gene family. The conserved region showed 62.8% of nucleotide sequence identity, and 65.5% of AA identity with 92% of AA homologies among ACDP members. The conserved domain is also found in genes from evolutionarily divergent species from bacteria, yeast, Caenorhabditis elegans, and Drosophila melanogaster to mammals. All ACDP genes except ACDP1 have a ubiquitous expression pattern while ACDP1 expression is restricted to the brain and testis. Immunofluorescence staining of premeablized HeLa cells showed that ACDP proteins are predominantly localized in the nucleus. Sequence homology analyses revealed AA property and structural homologies between the ACD domain and cyclin molecules.

Effects of X-ray radiation on the rheologic properties of platelets and lymphocytes.

BACKGROUND: Blood is often irradiated before transfusion for severely immunocompromised patients to prevent a potentially fatal complication of transfusion-associated GVHD. STUDY DESIGN AND METHODS: This study evaluates the effects of X-ray radiation on platelet and lymphocyte rheology because the ability of these blood cells to deform is vital to their flow throughout the microvascular system. Micropipette aspiration experiments were conducted on platelets and lymphocytes exposed to X-ray radiation doses of 0 (control), 25, and 50 Gy. RESULTS: A significant increase in the Young modulus of elasticity was observed between control platelets and irradiated platelets at 25 Gy (p = 0.02) and 50 Gy (p = 0.03). Percent cell activation increased significantly in 25 Gy-irradiated platelets (p = 0.008). In addition, lymphocytes irradiated at 25 Gy have a higher viscosity than controls (p < 0.02). A significantly larger number of activated cells is found in the 50 Gy-irradiated lymphocyte population (p < 0.04). CONCLUSION: The changes in the deformability and activation of irradiated platelets and lymphocytes may reduce local blood flow and lead to intermittent blockage, which may cause a change of blood flow in microvasculatures.

Heparin induces differentiation of CD1a+ dendritic cells from monocytes: phenotypic and functional characterization.

Dendritic cells (DCs) play important roles in initiation and regulation of immune responses. DCs derived from human monocytes can be classified according to presence of CD1a molecules. Although CD1a+ DCs can be prepared from monocytes in media containing GM-CSF, IL-4, and FCS, it has been reported that CD1a+ DCs could not be easily obtained from monocytes using media containing human serum or plasma. In this study, we demonstrate for the first time that heparin can reliably induce differentiation of CD1a+ DCs from monocytes with or without autologous serum or plasma. The development of CD1a+ DCs is heparin concentration dependent (0-50 U/ml). Comparing with CD1a- DCs developed without heparin, CD1a+ DCs express higher CD40 and CD80 and lower CD86. Both CD1a+ and CD1a- DCs express similar levels of HLA-DR. CD80, CD86, HLA-DR, and CD40 are proportionally up-regulated when both types of DCs are stimulated with LPS or LPS plus IFN-gamma. The effect of heparin is neutralized by heparin-binding proteins, such as protamine sulfate, platelet factor-4, and beta-thromboglobulin. Functionally, heparin-treated DCs respond to LPS or LPS plus IFN-gamma with higher IL-10 and less IL-12 production than heparin-untreated DCs. Heparin-treated DCs are more potent in priming allogeneic and autologous CD4+ T cells to proliferate and to produce both type 1 and type 2 cytokines. The results of our study show that CD1a+ DCs can be prepared from monocytes ex vivo without using xenogeneic serum and may be used for immunotherapy.