Identifying Clonal Origin of Multifocal Hepatocellular Carcinoma and Its Clinical Implications.

Hepatocellular carcinoma (HCC) is characterized by high prevalence of multifocality. Multifocal HCC can arise synchronously or metachronously either from intrahepatic metastasis (IM) or multicentric occurrence (MO). To date, there have been no established criteria to accurately distinguish whether multifocal HCC originates from IM or MO. Histopathological features remain the most convenient strategy but with subjectivity and limited accuracy. Various molecular biological techniques involving assessment of TP53 mutation status, hepatitis B virus integration sites, and chromosomal alterations have been applied to determine the clonal origin. The introduction of next-generation sequencing facilitates a more comprehensive annotation of intertumor heterogeneity, resulting in more sensitive and accurate clonal discrimination. Generally, MO-HCC has better overall survival than IM-HCC after curative resection. Adjuvant antiviral treatment has been proved to decrease post-treatment recurrence probably by reducing MO-HCC recurrence, whereas adjuvant sorafenib treatment targeting prior micrometastasis failed to reduce IM-HCC recurrence. Recent studies recommended transcatheter arterial chemoembolization (TACE) and traditional Chinese medicine Huaier granule as effective adjuvant treatments probably by preventing IM and both types of recurrences respectively. Immunotherapy that inhibits immune checkpoint interaction may be an optimal choice for both MO- and IM-HCC. In the future, effective personalized therapy against multifocal HCC may be achieved.

A 'complex solution' to a 'complex' problem: tackling the complexity of cancer with botanicals.

Several biological and physical factors confer complexity on cancer, which may be responsible for its associated morbidity. The successful management of this hyperproliferative disease is beyond the realm of therapeutics that may include the use of a single or at times a combination of treatment modalities. It is apparent that prevention with complex agents such as botanicals could serve as a possible potential solution. The present review focuses on the aspects of cancer that contribute toward making it an extremely complex disease and that may be tackled effectively with the use of 'multicomponent' botanicals.

Cutaneous T cell lymphoma: translating immunobiology into therapeutic opportunities.

Cutaneous T cell lymphoma (CTCL) has always served as a proving ground where conceptual advances in immunology can be tested and the results translated into clinical practice. From the earliest studies that used sheep red blood cells to identify the malignant cell as a T lymphocyte to molecular demonstration of the clonalilty of the disease, basic science techniques have provided sign posts that allow us to understand the clinical features seen in the patients. We continue to apply this paradigm to develop new insights into the role of the immune system in CTCL with the goal of using this knowledge to enhance the therapeutic options available to the patient. This article will review the studies that have led to our current understanding of the immunobiology of CTCL and the new therapeutic approaches that are being tested in this disease.

Seasonal changes in antigen-specific T-helper clone sizes in patients with Japanese cedar pollinosis: a 2-year study.

BACKGROUND: Allergic rhinitis (AR) is a typical type I allergic disease that occurs through the induction of allergen-specific effector T cells. Once established, new effector T cells derive mostly from memory T cells that are capable of surviving for extended periods, although the mechanisms by which these memory functions are maintained have not yet been clarified. In particular, the exact life-span of memory T cells is still not well understood. OBJECTIVE: Pollinosis patients seemed to be suitable subjects to investigate because such patients are exposed to antigens strongly for only a limited period once a year. We compared the seasonal changes in memory T-helper type 2 (Th2) between pollinosis and perennial allergic subjects. METHODS: The clone sizes of the Japanese cedar pollen-specific memory Th cells were measured by an ELISPOT assay using specific peptides from the patients with cedar pollinosis, and the seasonal changes were noted. This study was performed for 2 years. The cedar-specific IgE levels in the peripheral blood were also studied. Mite allergy patients were also enrolled in the study. RESULTS: The Japanese cedar-specific IL-4-producing Th2 cells were detected in all patients examined, although the number of cells was low. These Th memory cells increased during the pollen season and decreased during the off-season. However, more than 60% of the cedar-specific memory Th2 cells survived up to 8 months after the pollen season. The cedar-specific IgE levels exhibited changes similar to the cedar-specific Th cells. On the other hand, there was no drifting of Th memory clone size with the mite allergics, and the IgE levels also did not change. CONCLUSIONS: While pollen-specific Th cells decreased after pollen exposure, their memory functions continued. Memory clone size maintenance therefore requires repetitive antigen irritation.

Late complications following treatment for severe aplastic anemia (SAA) with high-dose cyclophosphamide (Cy): follow-up of a randomized trial.

High-dose cyclophosphamide (Cy) has been promoted as curative therapy for severe aplastic anemia (SAA). However, our randomized trial comparing antithymocyte globulin (ATG) and Cy was terminated early because of excess morbidity/early mortality in the Cy arm. We now report analysis of secondary endpoints at a median of 38 months. Relapse occurred in 6 (46%) of 13 responders in the ATG arm versus 2 (25%) of 8 in the Cy arm (P =.38). Five (31%) of 16 patients in the ATG arm and 4 (27%) of 15 patients in the Cy arm had evidence of paroxysmal nocturnal hemoglobinuria (PNH) at diagnosis, with no substantial change in the overall percentage of glycophosphatidyl inositol (GPI)-anchored protein-deficient neutrophils over extended follow-up in individual patients in either arm. Bone marrow cytogenetic abnormalities have been observed among surviving patients in both arms (2 of 14 ATG versus 1 of 12 Cy, P =.70). High-dose Cy does not prevent relapse or clonal evolution in SAA.

Decreased susceptibility of leukemic cells with PIG-A mutation to natural killer cells in vitro.

The cloning of the PIG-A gene has facilitated the unraveling of the complex pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH). Of current major concern is the mechanism by which a PNH clone expands. Many reports have suggested that an immune mechanism operates to cause bone marrow failure in some patients with PNH, aplastic anemia, and myelodysplastic syndromes. Because blood cells of PNH phenotype are often found in patients with these marrow diseases, one hypothesis is that the PNH clone escapes immune attack, producing a survival advantage by immunoselection. To test this hypothesis, we examined the sensitivity of blood cells, with or without PIG-A mutations, to killing by natural killer (NK) cells, using 51Cr-release assay in vitro. To both peripheral blood and cultured NK cells, PIG-A mutant cells prepared from myeloid and lymphoid leukemic cell lines were less susceptible than their control counterparts (reverted from the mutant cells by transfection with a PIG-A cDNA). NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was perforin-dependent. There were no differences in major histocompatibility (MHC) class I expression or sensitivity to either purified perforin or to interleukin-2-activated NK cells between PIG-A mutant and control cells. From these results, we infer that PIG-A mutant cells lack molecules needed for NK activation or to trigger perforin-mediated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the setting of marrow injury by cytotoxic lymphocytes.

Gamma-glutamyl transferase expression in higher-grade astrocytic glioma.

Increased expression of gamma-glutamyltransferase (GGT) has been detected in a range of human malignancies and is thought to be involved in neoplastic proliferation and treatment resistance. Since GGT expression and its role in malignant glioma biology remain largely unknown, we investigated this phenomenon by immunostaining 26 higher-grade human astrocytic gliomas (WHO grades III and IV) with a monoclonal anti-GGT-antibody (138H11). Further, human pancreatic GGT cDNA was used for liposome-mediated transfection of 9L gliosarcoma cells. GGT-expressing and control 9L cells were cultured in media containing different amounts of essential amino acids and/or cytotoxic agents. Cell viability was evaluated by microplate MTT assay. Immunohistochemical staining of tumor specimens demonstrated that GGT expression is a frequent feature of higher-grade human astrocytic gliomas, but not of normal brain tissue. Human tumors were strongly GGT-positive in 6 of 7 cases of grade III astrocytoma, and in 12 of 19 grade IV astrocytoma (glioblastoma multiforme, GBM) cases. In the cell culture model, 9L-GGT cells had a growth advantage over control cells in cysteine-deficient medium. but not in standard or glutamine-free medium. No significant difference in numbers of viable cells of either clone was found in media containing the alkylating drug BCNU (5-200 microg/ml). In conclusion, GGT is expressed in a high percentage of human WHO grade III astrocytomas and GBM, but not in normal brain tissue. This molecule seems to give neoplastic cells a moderate growth advantage under in vivo conditions.

Adenoviral p53 gene therapy promotes heat-induced apoptosis in a nasopharyngeal carcinoma cell line.

BACKGROUND: It has previously been demonstrated that Ad5CMV-p53 gene transfer, either used alone or delivered concomitantly with ionizing radiation, resulted in cytotoxicity mediated by apoptosis in nasopharyngeal carcinoma (NPC) cell lines. In this study, a novel approach was evaluated of combining Ad5CMV-p53 gene therapy with hyperthermia (HT), in the CNE-1 NPC cell line, which harbours a mutation in codon 249 of the p53 gene. MATERIALS AND METHODS: CNE-1 cells were infected using either Ad5CMV-p53 or Ad5CMV-B-gal, followed, 24 h later, by HT (43 degrees C x 0-2 h). Protein was extracted for Western blot analysis, and apoptosis was evaluated using acridine-orange ethidium bromide staining, followed immediately by fluorescent microscopy examination for the proportion of cells displaying morphologic features of apoptosis. RESULTS: Ad5CMV-p53 gene therapy combined with HT resulted in a dose-dependent cytotoxicity with less than 1% clonogenic survival when 10 pfu/cell of Ad5CMV-p53 was combined with 2 h heating at 43 degrees C. Western blotting demonstrated that treatment with Ad5CMV-p53 resulted in the rapid expression of p53, which was minimally affected by HT. The inducible form of hsp70 was maximally expressed at 48 h post-HT, with minimal effect when cells were additionally treated with Ad5CMV-p53. Clonogenic cytotoxicity was associated with the development of apoptosis, with up to 70% of CNE-1 cells displaying morphologic features of apoptosis after the combination treatments. CONCLUSION: Based on the shapes of the clonogenic survival curves, Ad5CMV-p53 gene therapy and HT appear to interact in an additive manner, suggesting the therapeutic potential of this combined treatment approach for patients with NPC.

Prognostic significance of a polymerase chain reaction-detectable dominant T-lymphocyte clone in cutaneous lesions of patients with mycosis fungoides.

Although mycosis fungoides (MF) is considered to be an indolent lymphoma, survival is highly influenced by TNM stage. At diagnosis, most MF patients present with early stage disease and a high probability of long-term survival. Treatment is generally directed towards skin lesions, and achievement and duration of complete responses are variable. A dominant T-cell clone is detectable in the cutaneous lesions of 60% of patients. The aim of this study was to determine whether the presence of a T-cell clonal population influences the clinical course of the disease after topical therapy. Cutaneous biopsies from 68 patients were histologically diagnosed as MF and T-cell clonality was analyzed by in vitro amplification of TCR-gamma chain gene rearrangements (polymerase chain reaction gamma [PCRgamma]). After a median follow-up of 48 months, response to treatment was clinically assessed. Age, sex, duration of symptoms before diagnosis, type of cutaneous lesions (T stage), TNM stage, and PCRgamma were evaluated as predictive factors of response to treatment in univariate and multivariate analyses. Univariate analysis demonstrated that T1 cutaneous lesions (P = .05) and PCRgamma negativity (P = .007) were associated with a higher complete remission rate. Using multivariate analysis, T stage (relative risk, 3.13; P = .06) and PCRgamma (relative risk, 4.4; P = .01) remained independent significant predictive parameters of response. In conclusion, T stage and cutaneous PCRgamma at diagnosis are the two predictive parameters of treatment response for MF. Therefore, the cutaneous PCRgamma findings should be considered in the analysis of future therapeutic trials.

Analysis of immunoglobulin VH gene repertoire by an anchored PCR-ELISA.

We developed a novel technique to analyze the relative concentration of the expressed immunoglobulin (Ig) VH genes using an enzyme-linked immunosorbent assay (ELISA). Expressed Ig cDNA are amplified via anchored PCR and then subjected to a "nested PCR" reaction that attaches biotin to the 5' end of the antisense strand. This allows us to tether the antisense strand of PCR products onto avidin-coated ELISA plates. Digoxigenin-labeled oligonucleotides specific for the leader sequence sense strand of each major Ig VH gene subgroup are used to probe the plate-tethered, alkaline-denatured, and single-stranded antisense cDNA. Bound probes then are detected with alkaline-phosphatase-conjugated anti-digoxigenin antibodies. Using this method, we assessed the distribution of Ig VH genes used by IgM-expressing blood B cells of normal adults. We found the predominant subgroup is VH3, representing approximately half (range 41-59%) of the expressed IgM repertoire. The next largest subgroups used are VH4 (19-23%), VH1 (15-17%), and VH5 (7-11%). The VH2, VH6, and VH7 subgroups each constitute less than 3% of the expressed IgM repertoire. These results agree with those obtained using traditional and more laborious methods that analyze the distribution of Ig clones in cDNA libraries. In addition, we find that this method compares favorably in sensitivity and specificity to more conventional techniques for assessing the clonality of blood or tissue B-cell populations. This rapid and nonradioactive method should have utility for assessing the Ig repertoires expressed by normal, autoimmune, or neoplastic B-cell populations.

Influence of molecular characteristics on clinical outcome in human immunodeficiency virus-associated non-Hodgkin's lymphoma: identification of a subgroup with favorable clinical outcome.

The relationship between clinical and molecular characteristics of 45 treated individuals with histologically-documented human immunodeficiency virus (HIV)-associated B-cell non-Hodgkin's lymphoma was examined to determine whether differences in molecular features of lymphoma were associated with differences in clinical outcome. Tissue specimens from these tumors were evaluated for evidence of Ig heavy-chain gene rearrangements using both Southern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Lymphomas were also evaluated for the presence of Epstein-Barr virus (EBV) DNA sequences and c-myc gene rearrangements. Twenty-five lymphomas were characterized as polyclonal and 20 as monoclonal. PCR amplification of expressed Ig variable (V)-region genes confirmed polyclonality in three extensively studied polyclonal lymphomas. The median CD4 count was significantly higher in the group with polyclonal disease (277/microL) than in the group with monoclonal disease (123/microL), P = .04. The complete response rate to therapy was significantly higher in patients with polyclonal disease (78%) and CD4 greater than 200/microL (81%) than in those with monoclonal disease (31%) and CD4 less than 200/microL (33%). CD4 count, clonality, and presence of EBV DNA sequences were the most important predictors of survival. Both Kaplan-Meier and Cox proportional hazards analyses showed a markedly prolonged survival in those patients with both CD4 > or = 200/microL and polyclonal disease. Histologically the polyclonal lymphomas were high grade in appearance and contained prominent macrophages. All seven surviving patients were in this group. Median survival for those individuals whose tumors contained EBV sequences was only 3.2 months (range, 0.4 to 19.5), whereas those with EBV- tumors survived for a median of 9.0 months (range, 0.7 to 65.2), P = .0007. These data indicate that molecular features of HIV-associated lymphomas may be important predictors of clinical outcome. These characteristics define a distinct subset of patients with polyclonal EBV- tumors and CD4 counts greater than 200/microL that appear to have a less aggressive clinical course.

Characterization of selected strongly and weakly invasive sublines of a primary human melanoma cell line and isolation of subtractive cDNA clones.

Invasion of basement membranes is a key step in systemic spread of tumour cells. To analyze genetic mechanisms involved in this process, we have selected strongly and weakly invasive sublines with stable phenotypes from a primary human melanoma cell line by repeated passage through a reconstituted basement membrane in vitro. The sublines differed approximately 5-fold in their invasive potential. Invasiveness correlated with better attachment and overexpression of the integrin alpha v/beta 3 (vitronectin/laminin-receptor). Treatment with retinoic acid inhibited proliferation in both sublines and invasion in the weakly invasive cells but stimulated invasion in the strongly invasive subline. Northern-blot analyses revealed equal levels of mRNA expression regarding collagenase type-IV and retinoic-acid receptors but enhanced expression of TIMP-2 mRNA in weakly invasive cells. The 2 sublines differed significantly in their respective DNA ploidy when compared to the wild-type Mel Im cell line, suggesting that they represent heterogeneous clones present in the primary tumour. We have started to exploit this in vitro system for tumour heterogeneity to clone genes involved in invasion. By a subtractive cDNA cloning strategy, 12 partial cDNA clones were obtained that are specifically overexpressed in the strongly or weakly invasive subline. These results illustrate that stable genetic alterations lead to heterogeneous subpopulations within primary melanomas which differ in their ability to invade basement membranes and interact with components of the extracellular matrix.

[Growth-stimulating, cytostatic and cytotoxic activity of the secretory products of the RPMI-6410t human B-cell lymphoblastoid line]. / Roststimuliruiushchaia, tsitostaticheskaia i tsitotoksicheskaia aktivnost' sekretornykh produktov limfoblastnoi linii B-kletok cheloveka RPMI-6410t.

The cells of human lymphoblastoid line RPMI-6410t were shown to synthesize constitutively a factor(s) with different types of biological activity. The factor(s) stimulated the growth of both B cells 6410t, obtained from the blood of a patient with acute myeloblastic leukemia, and the human embryonic diploid fibroblasts. With B cell lines Raji and P3HR-1.G5, obtained from the patients with Burkitt's lymphoma. The growth factor(s) displayed cytotoxic and cytostatic effects, respectively. Growth-stimulating and cytotoxic activating of the factor were destroyed by a 15 hour exposure to low or high pH. The activity was stable within pH values of 6-8. With regard to heat stability, the activity destroyed at 70 degrees C within 1 hour but remained stable at 56 degrees C during 1 hour. The above factor(s) displayed biological activities similar to those of the previously known tumor necrosis factor (TNF).

Effects of type beta transforming growth factor in combination with retinoic acid or tumor necrosis factor on proliferation of a human glioblastoma cell line and clonogenic cells from freshly resected human brain tumors.

Type beta transforming growth factor (beta-TGF) is a potent regulator of cell growth and differentiation. The human glioblastoma cell line, T-MGI, was growth inhibited by beta-TGF under anchorage independent conditions. The antiproliferative effect of beta-TGF was potentiated to nearly total arrest by low doses of retinoic acid (RA) or tumor necrosis factor (TNF), while epidermal growth factor, platelet-derived growth factor, interleukin-2, and gamma interferon did not have this potentiating effect. The potentiation of the beta-TGF effect by RA and TNF could not be explained by modulation of the epidermal growth factor receptor, the beta-TGF receptor, or the TNF receptor. beta-TGF alone and in combination with RA or TNF were further tested on primary cultures from freshly resected human glioma biopsies (n = 13). There was great individual variation in sensitivity to beta-TGF, RA, or TNF. The astrocytoma and oligodendroglioma cells were inhibited to various degrees by beta-TGF or TNF, while most of the glioblastomas were not sensitive to these agents. Most of the biopsies were stimulated by RA. RA or TNF did not potentiate the growth inhibitory effect of beta-TGF on biopsy cells. We therefore think it unlikely that beta-TGF in combination with RA or TNF will be effective agents in the treatment of gliomas.

[Clonogenic cells in human tumors and the sensitivity to chemotherapy]. / Klonogennye kletki v opukholiakh cheloveka i chuvstvitel'nost' k khimioterapii.

The current literature data on the cloning of human tumour cells, the use of clonal assays of tumour growth for studying biology, cell kinetics and cytogenetic analysis of human tumour cells, drug sensitivity and related clinical and preclinical studies are reviewed.

Pathogenesis of metastatic disease: implications for current therapy and for the development of new therapeutic strategies.

Different tumor cell subpopulations coexisting within the same tumor exhibit varied susceptibilities to antineoplastic agents. Tumor cell heterogeneity is now recognized as the principal cause of treatment failure in cancer, and is a formidable obstacle to effective therapy and to the development of drug delivery systems for selective targeting of antineoplastic agents to tumor cells. Recent insights into the genesis of tumor cell heterogeneity during progressive tumor growth reveal new complexities that raise challenging questions about the adequacy of certain approaches to the current therapy of metastatic disease and impose challenging criteria for the development of improved therapeutic strategies. Many of the experimental approaches used in the search for new antineoplastic agents and targeted drug delivery systems ignore the pathogenesis of metastasis and the problem of tumor cell heterogeneity. The adoption of more relevant assay systems is an urgent priority. These include the greater use of metastatic tumor models and the increased use of human tumor cells to replace rodent cell systems which have been of limited predictive value in identifying effective anticancer agents. In contrast to current strategies for the development of new antineoplastic drugs which seek to identify agents with activity against a broad range of histologically diverse tumors, greater success may be achieved by seeking agents active only against specific cell lineages. Many established human tumor cell lines may not be suitable for this purpose because of extensive phenotypic change produced by prolonged passage ex vivo. Development of histiotype-specific human tumor cell screens will require an extensive research effort to identify target cells that display demonstrable phenotypic relatedness to tumor cells in neoplastic lesions. Major advances in the therapy of metastatic disease are considered unlikely in the next few years, and progress will stem from improved use of existing agents in refined combination therapy protocols in which greater attention is given to the duration, frequency, and sequence of therapy with different agents to limit emergence of tumor cell variants resistant to one or more antineoplastic agents. Advances in molecular biology offer exciting prospects for the identification of new therapeutic targets in human tumor cells, for the induction of alterations in tumor cells that could serve as therapeutic targets, and for the elucidation of the mechanisms responsible for the rapid phenotypic diversification of tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of human tumor colony growth in soft agar by serum.

The factors controlling the growth of human tumor cells in soft agar are poorly understood. However, it has been demonstrated that serum provides factors which promote anchorage-independent growth. We tested 58 tumor specimens, which were obtained from patients with adenocarcinoma of the lung, colon, ovary or squamous cell carcinoma, for their ability to form colonies in soft agar in serum-free or serum-supplemented media. The cells were unable to replicate, and none of the hormones or growth factors tested: insulin (I), transferrin (T), selenium (S), estradiol (E), hydrocortisone (H) or epidermal growth factor (EGF) could substitute for serum. Examination of the serum dose-response curves indicated that growth factors reduced the serum concentrations needed to support anchorage-independent growth. The addition of the supplements and the lowering of serum concentrations increased cloning efficiencies (C.E.) in 38/51 trials, when cells were able to grow initially. The addition of ITS increased C.E. in 18/21 cases, HITES in 15/17 cases and EGF in 12/18 cases as compared to controls. ITS and HITES increased the number of colonies only when serum was the limiting factor. EGF, however, increased the number of colonies even when serum was not the limiting factor. The ability of the supplements to enhance growth could not be correlated to tumor type or initial cloning efficiencies. However, in only 1/25 cases were cells that were unable to form colonies under standard conditions induced to form colonies in the presence of the growth factors. Normal and tumor-derived human fibroblasts did not form colonies in soft agar in the presence of these growth factors. The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.

Cell kinetic analysis of human tumor stem cells.

The application of the tumor stem cell assay to the study of human tumor cell kinetics has the potential for major advances in our knowledge of proliferative characteristics of clonogenic human tumor cells. From simultaneous evaluation of in vitro drug sensitivity, in vitro doubling time, and the thymidine suicide index, plus flow cytometry, cytogenetic analysis, and assessment of differentiation markers, substantial insights into the basic biology of tumor cell growth may be attained. As discussed in other chapters, using modifications of the two-layer system, one can assess both local cell-mediated and humoral factors that might influence in vitro kinetics and drug sensitivity. The next few years should see the acquisition and integration of valuable new information that should allow more rational approaches to the treatment of tumors by taking full advantage of information on both kinetics and drug sensitivity of clonogenic human tumor cells.