Therapeutic applications of germline testing for cancer predisposition genes in Asia in the real world.

BACKGROUND: Germline genetic testing is traditionally carried out in patients suspected with hereditary cancer syndrome for enhanced cancer surveillance and/or preventive strategies, but is increasingly carried out for therapeutic indications. MATERIALS AND METHODS: We conducted a retrospective review of patients who underwent germline genetic testing at our centre to determine the prevalence of actionable pathogenic germline variants (PGV) and their clinical utility. RESULTS: From 2000 to 2022, 1154 cancer patients underwent germline testing, with the majority (945/1154) tested with multi-gene panels. Four hundred and eleven (35.6%) patients harboured a PGV and 334 (81%) were clinically actionable. BRCA1/2 accounted for 62.3% of actionable mutations, followed by mismatch repair (18%), and other homologous recombination repair (HRR) genes (19.7%). One hundred and fifty-two germline-positive patients have advanced cancers, and 79 received germline-directed therapies (poly ADP ribose polymerase inhibitors = 75; immunotherapy = 4). Median duration of immunotherapy and poly ADP ribose polymerase were 20.5 months (range 5-40 months) and 8 months (range 1-76 months), respectively. Among BRCA/HRR mutation carriers who received platinum-based chemotherapy, pathological complete response rate in the neoadjuvant setting was 53% (n = 17 breast cancers) and objective response rate was >80% in the advanced setting (n = 71). CONCLUSIONS: One-third of cancer patients tested carried a PGV and ∼80% were clinically actionable. Three-quarters of germline-positive advanced cancer patients received germline-directed therapies in the real world, underscoring the practical utility of germline testing to guide cancer therapeutics.

Prophylactic lamivudine prevents hepatitis B reactivation in chemotherapy patients.

BACKGROUND: Chronic hepatitis B virus carriers receiving chemotherapy develop a high hepatitis B virus reactivation rate (38-53%) with a high mortality (37-60%). Few studies have characterized the efficacy of lamivudine in the treatment of chemotherapy-induced hepatitis B virus reactivation. AIM: To determine whether lamivudine prophylaxis reduces chemotherapy-induced hepatitis B virus reactivation and mortality. METHODS: The medical records of all hepatitis B surface antigen-positive patients with malignancy treated with chemotherapy since 1995 at the National University Hospital of Singapore were identified, and divided into those who received lamivudine prophylaxis before chemotherapy (P) and those who did not (NP). The parameters examined included gender, age, malignancy type, steroid usage, number of chemotherapy courses and regimens, follow-up duration and hepatitis B virus status. The outcome measures were hepatitis B virus reactivation (abrupt rise of serum alanine aminotransferase to > 200 IU/L) and reactivation death. Patients with primary hepatoma or liver metastasis were excluded. RESULTS: Thirty-five patients were identified: 16 in the P group and 19 in the NP group. The baseline characteristics of the two groups were similar. Seven of the 19 patients in the NP group and none of the 16 patients in the P group developed reactivation (36.8% vs. 0%, P=0.009). Six of the seven patients in the NP group who developed reactivation received lamivudine at that time, but five died (mortality, 71.4%), whilst no patient in the P group died from reactivation (P=0.064). CONCLUSIONS: Prophylactic lamivudine appears to prevent hepatitis B virus reactivation and its associated mortality in patients treated with chemotherapy. This should be confirmed with prospective studies.

Simulation of the delivery of doxorubicin to hepatoma.

PURPOSE: To develop a two-dimensional simulation platform for the transport of doxorubicin to the hepatoma. To examine the temporal and spatial variation of doxorubicin concentration and its penetration into the tumor and the surrounding normal tissues. METHODS: Simulations are carried out with Fluent/UNS using the finite volume method to obtain the interstitial fluid pressure, velocity, and concentration profiles. RESULTS: Interstitial fluid pressure in the tumor and core reaches a steady state value in about 800 s, corresponding well with the assumed time scale for interstitial matrix fluid percolation (-1,000 s). There is a strong correlation between the drug concentration in the interstitial space of tumor and blood plasma for time >> 1 h. Concentration of doxorubicin is highest in the viable zone of the tumor at early times and in the necrotic core at later times, and lowest in the surrounding normal tissues. Diffusion is the dominant form of transport for doxorubicin. CONCLUSIONS: Varying the volume of solution injected, while keeping the dosage the same, does not cause significant changes in the amount and distribution of drug in the tumor. A higher vascular exchange area leads to higher concentrations of drug in the tumor. Lymphatic drainage in the tumor causes negligible reductions in the mean concentrations in all three different zones. Cellular metabolism and DNA binding kinetics decrease the mean concentrations of drug by about 15 to 40%, as compared to the baseline case.

Phase II trial of docetaxel in Asian patients with inoperable stage III non-small cell lung cancer.

Docetaxel has a response rate of greater than 30% in first-line treatment of Western patients with advanced non-small cell lung cancer (NSCLC). The goal of this open-label. phase II study was to evaluate the activity and safety profile of docetaxel in Asian patients with inoperable untreated stage III NSCLC. Docetaxel was given at 100 mg/m2 as a 1-h infusion every 3 weeks. Prophylactic dexamethasone was given to reduce hypersensitivity reactions and edema. Thirty-five patients were enrolled in the study. The response rate was 34% (95% CI, 19%-50%) according to intent-to-treat analysis. No complete response was observed. Twenty-four patients (69%) had grade 3 or 4 neutropenia in cycle 1, and febrile neutropenia was seen in 12 patients. Six patients (17%) experienced mild fluid retention. Docetaxel is an active agent in first-line treatment of Asian patients with locally advanced NSCLC, with the main toxicity being neutropenia. Fluid retention was a minor problem in this study.

Persistent, antigen-specific, therapeutic antitumor immunity by dendritic cells genetically modified with an adenoviral vector to express a model tumor antigen.

Dendritic cells (DC) are potent antigen-presenting cells that play a critical role in the initiation of cellular immune responses. Using a BALB/c syngeneic colon carcinoma cell line expressing a model tumor antigen beta-galactosidase (betagal), we previously reported (Song et al, J Exp Med 1997; 186: 1247-1256) that immunization of mice with a single injection of DCs genetically modified with an adenovirus vector expressing betagal confers potent protection against a lethal intravenous tumor challenge, as well as suppression of pre-established lung tumors, resulting in a significant survival advantage. In the present study, we have addressed the question: how long does the memory of tumor antigen- specific immunity persists after DC priming in vivo using this genetically modified DC-based cancer vaccination strategy? To accomplish this, two groups of mice were evaluated: (1) mice surviving >400 days following protection from an initial intravenous tumor challenge after immunization with DC genetically modified to express betagal; and (2) mice surviving >300 days that had previously demonstrated regression of pre-established lung tumors after treatment with DC immunization. By analyzing the antigen-specific cytotoxic T lymphocyte response and challenging these long-term survival mice with a second subcutaneous tumor administration, the data demonstrate that a single administration of DC genetically modified to express a model antigen induces long-lasting, antigen-specific antitumor immunity in both naive and tumor-bearing hosts, observations that have important implications in the development of genetically modified DC-based antitumor vaccination strategies. Gene Therapy (2000) 7, 2080-2086.

Cancer gene therapy--fantasy or foresight?

Gene therapy is an exciting new method of treatment that may have far-reaching implications on the way we manage diseases in the future. Cancer has become the principal focus of this futuristic research. The breathtaking pace of gene discovery in the last two decades, coupled with the birth of recombinant DNA technology, gave rise to the concept that genes may be manipulated and used as drugs. Genetic modification of cells can be carried out in petri dishes (ex vivo) or within the living system (in vivo). In order for the therapeutic genes to exert their effect, they have to be transported into the cell nucleus where transcription takes place. Liposomes and genetically modified viruses have been extensively used as gene vectors. The ideal vector remains elusive. It would be one that can achieve tumour-specific, sustained, and regulatable gene expression without host toxicity. As a result of the past decade of intense gene therapy research, we have learned that it is a rational scientific concept that works remarkably well in petri dishes and in laboratory animals. However, early clinical gene therapy experimentations paled in comparison. This apparent disparity between dramatic preclinical successes and the very modest clinical results of gene therapy does not in anyway nullify the concept of gene therapy. Instead, it exposes the folly of underestimating the technical complexity of gene manipulation in human diseases. Fortunately, technical hurdles such as those confronting gene therapy today are not insurmountable; they need, however, much ingenuity, resolution and time to be overcome. It is reassuring that recent advances in gene therapy provide abundant evidence that the premature infant, born of unrealistic pressure, is indeed healthy and thriving. With proper nurturing and patience, there is no doubt that, in time, it will bear fruit.

Murine metastatic tumour models for cancer gene therapy research.

The appropriate use of animal tumour models has a pivotal role in the evaluation of any new anti-cancer therapy. Indeed, animal models of human diseases have been emphasised in the early development and evaluation of gene therapy. Given that most cancer treatment failures and cancer-related mortality are the direct results of metastatic cancers, the increasing use of clinically-relevant metastatic tumour models in the study of cancer therapeutics is both logical and necessary. Murine metastatic tumour models may be established either "experimentally" or "spontaneously". The yield and reproducibility of spontaneous metastasis models can be enhanced through manoeuvres such as using highly-metastatic sublines for primary tumour implantation and orthotropic transplantation. The use of immunodeficient rodents, although popular, suffers from the absence of T-cell responses in the host which may impact on therapeutic efficacy. While many gene therapy strategies today are capable of regressing primary tumours in experimental animals, only a limited number of approaches (viz. genetic immunotherapy and gene-mediated anti-angiogenesis) are designed to address the challenges posed by metastatic tumours. In extrapolating the results of gene therapy in animal models to humans, it is important to appreciate the heterogeneity of the latter populations, and anticipate greater variability in the treatment outcome.

Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor.

Vascular endothelial growth factor (VEGF), a potent angiogenic mediator, is overexpressed in most solid tumors. On the basis of the knowledge that solid tumor growth beyond a small volume is critically dependent on angiogenesis, and that adenovirus (Ad) vectors can mediate efficient in vivo gene transfer and expression, we hypothesized that Ad-mediated transfer of a secreted form of the extracellular domain of the flt-1 VEGF receptor (Adsflt) would suppress tumor growth on a regional basis. To evaluate this concept, three tumor models were examined using a murine colon carcinoma cell line and syngeneic BALB/c mice. First, mice with preestablished splenic CT26.CL25 tumors and liver metastases were given Adsflt on AdNull intravenously and, after 15 days, spleens and livers were harvested to quantify tumor burden. Adslft-treated animals had minimal residual splenic tumors and liver metastases; in contrast, control animals had bulky splenic tumors and extensive liver metastases (p < 0.003). Second, mice with preestablished lung metastases showed a significant reduction in pulmonary metastases with regionally administered Adslft (intratracheal, p < 0.02) but not when the vector was systemically administered (intravenous, p > 0.9). Finally, mice with primary subcutaneous tumors treated with intratumoral administration of Adslft showed significant tumor suppression (p < 0.05) not observed in AdNull-treated mice or mice given Adslft intravenously (p > 0.3). We conclude that Ad-mediated in vivo regional delivery of a secreted form of the extracellular domain of the flt-1 VEGF receptor can effectively inhibit regional tumor growth, a strategy that may provide a means to control tumor growth within the treated organ without the risk of systemic antiangiogenesis.

Gene therapy strategies for tumor antiangiogenesis.

Based on the concept that solid tumors cannot grow without the generation of new blood vessels, there is growing interest in the use of antiangiogenesis agents to inhibit tumor growth. This review summarizes the concepts of using gene transfer vectors to provide high concentration of antiangiogenic proteins within an organ. While there are many challenges that must be met before antiangiogenesis can be used to effectively treat human tumors, gene transfer strategies have the potential to provide sustained, high, local concentrations of antiangiogenic mediators specifically targeted to organs containing tumors, minimizing systemic toxicity. Antiangiogenesis gene therapy strategies will most likely be effective in a state of low tumor burden, where this "genetic tourniquet" can provide trans (i.e., acting in the extracellular milieu as opposed to within tumor cells) suppression of the growth of endothelial cells in the milieu of micrometastases.

Construction of an adenovirus type 7a E1A- vector.

A strategy for constructing replication-defective adenovirus vectors from non-subgroup C viruses has been successfully demonstrated with adenovirus type 7 strain a (Ad7a) as the prototype. An E1A-deleted Ad7a reporter virus expressing the chloramphenicol acetyltransferase (CAT) gene from the cytomegalovirus promoter enhancer was constructed with DNA fragments isolated from Ad7a, an Ad7a recombination reporter plasmid, and the 293 cell line. The Ad7a-CAT virus particle transduces A549 cells as efficiently as Ad5-based vectors. Intravenous infections in a murine model indicate that the Ad7a-CAT virus infects a variety of tissues, with maximal levels of CAT gene expression found in the liver. The duration of Ad7a-CAT transgene expression in the liver was maximally maintained 2 weeks postinfection, with a decline to baseline activity by the week 4 postinfection. Ad7a-CAT represents the first example of a non-subgroup C E1A- adenovirus gene transfer vector.

Dendritic cells genetically modified with an adenovirus vector encoding the cDNA for a model antigen induce protective and therapeutic antitumor immunity.

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the initiation of antitumor immune responses. In this study, we show that genetic modifications of a murine epidermis-derived DC line and primary bone marrow-derived DCs to express a model antigen beta-galactosidase (betagal) can be achieved through the use of a replication-deficient, recombinant adenovirus vector, and that the modified DCs are capable of eliciting antigen-specific, MHC-restricted CTL responses. Importantly, using a murine metastatic lung tumor model with syngeneic colon carcinoma cells expressing betagal, we show that immunization of mice with the genetically modified DC line or bone marrow DCs confers potent protection against a lethal tumor challenge, as well as suppression of preestablished tumors, resulting in a significant survival advantage. We conclude that genetic modification of DCs to express antigens that are also expressed in tumors can lead to antigen-specific, antitumor killer cells, with a concomitant resistance to tumor challenge and a decrease in the size of existing tumors.

Cytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors.

Although replication-deficient adenovirus (Ad) vectors are efficient vehicles for in vivo gene transfer, persistence of expression of the Ad genome is limited in immunocompetent hosts by cellular immunity directed against the gene product of the vector. While most attention has been focused on cytotoxic T lymphocytes (CTL) directed against the low-level early and late Ad gene expression in the Ad vector-infected target cells, significant cellular immunity is likely also directed against the product of heterologous transgenes. To evaluate this concept, in vivo generation of CTL was evaluated in C57B1/6 and BALB/c mice with Ad vectors expressing a variety of heterologous transgenes, including Escherichia coli chloramphenicol acetyl transferase (CAT), beta-galactosidase (beta-Gal), cytosine deaminase, and human thrombopoietin (hTPO), with an Ad vector expressing no transgene ("null") as a control. Following intravenous administration of Ad vectors, spleen cells were harvested 2 weeks later, stimulated for 5 days with syngeneic cells infected with various Ad vectors, and then evaluated for CTL activity using 51Cr-release from syngeneic Ad vector-infected targets. In all cases, CTL directed against the heterologous transgene products was observed, although there were differences in the amounts of transgene-specific CTL. CTL directed against the transgene were also observed with other routes of administration, including intratracheal, subcutaneous, and intraperitoneal administration. These observations suggest that inclusion of a heterologous transgene in Ad vectors enhances the elimination of vector-infected cells, a circumstance that will be partially circumvented using autologous genes. For some applications, specific immune responses to products of transgenes delivered by Ad vectors might be exploited for therapeutic purposes.

Wilson's disease presenting as haemolytic anaemia and its successful treatment with penicillamine and zinc.

Haemolysis is an uncommon first manifestation of Wilson's disease. We describe a young woman who presented with episodic haemolysis and abnormal liver functions; the diagnosis of Wilson's disease was not made until nine months later. She responded well to a combination of penicillamine and zinc. This report underscores the importance of considering Wilson's disease as a cause in a patient with haemolysis of uncertain aetiology, since the disease can be successfully treated in the early stages. the mechanism of oxidative damage to erythrocytes by the excessive copper and the present role of zinc therapy are also discussed.

Bone changes in tuberous sclerosis mimicking metastases.

Sclerotic and lytic bone changes of tuberous sclerosis (TS) can mimic bone metastases. We report a case of bone metastases from bronchogenic carcinoma in a patient with TS bone changes. Bone scintigraphy and magnetic resonance imaging are useful in distinguishing the TS bone lesions from bone metastases.

Medical treatment of Cushing's syndrome with aminoglutethimide and ketoconazole.

We report a 54-year-old Chinese man with Cushing's syndrome from bilateral adrenal adenomata in whom surgery was contraindicated because of his intercurrent medical conditions. Instead, he was successfully treated with aminoglutethimide 0.75 gm/day (which reduced his 24-hour urinary free cortisol from 628 nmol/day to 177 nmol/day in 4 weeks), followed by ketoconazole 0.6 gm/day which continued to suppress the urinary free cortisol level. We conclude that aminoglutethimide or ketoconazole offers a viable non-surgical alternative to the treatment of Cushing's syndrome.

Plasmid-mediated biodegradative fate of monohalogenated biphenyls in facultatively anaerobic sediments.

The results of these studies have demonstrated that model PCB substrates can be mineralized by indigenous microbial population in contaminated sediments. This catabolic function can be rate limited at the microenvironmental level by physical-chemical processes such as physical partitioning and accumulation. At the biochemical level, this catabolic function is determined by the existence of plasmid borne genes that, under laboratory conditions, can be maintained and expressed in pure or mixed culture. Numerous limitations are encountered in establishing the significance of these biodegradative bacteria and the catabolic plasmids at the environmental level. Relatively little information is available concerning frequencies and stability of the bacteria or the plasmid encoded genes within the community. There is no information on the incompatibility grouping of the isolated plasmid relative to other plasmids maintained within the populations. Such factors will influence the development of gene screening techniques to monitor gene frequency distributions in the sediment community. Although mineralization of 4CBP was observed under moderately reducing conditions, it remains suspect that transient or trace levels of dissolved oxygen may have permitted conventional aerobic metabolism of the substrate. If this is true, demonstrating anaerobic metabolism of environmental contaminants will require strict and tedious cultivation under highly reduced conditions (approximately-300 mV). Large deletions of cryptic DNA observed under laboratory conditions may affect bacterial survival and gene maintenance and transfer under environmental conditions. Little information exists on regulation of catabolic activity of selective pressures required to maintain the degradative genes under environmental conditions. Such limitation encountered in these studies are shared by virtually all attempts to utilize genetically manipulated bacteria or newly isolated strains and plasmids. Perhaps the fundamental question is whether the catabolic genes are maintained and expressed within the community rather than whether the host bacterium can survive in the environment.

Degradation and total mineralization of monohalogenated biphenyls in natural sediment and mixed bacterial culture.

Mixed bacterial cultures obtained from polychlorinated biphenyl-contaminated river sediments are capable of degrading monohalogenated biphenyls under simulated natural conditions. Culture conditions include river water as supportive medium and mixed bacterial cultures obtained from river sediments. Degradation occurs when the substrates are supplied as the sole carbon source or when added together with glucose. The degradation rates of 2-, 3-, and 4-chlorobiphenyl, at 30 micrograms ml-1, were 1.1, 1.6, and 2.0 micrograms ml-1 day-1, respectively. Monobrominated biphenyls, including 2-, 3-, and 4-bromobiphenyl, were degraded at rates of 2.3, 4.2, and 1.4 micrograms ml-1 day-1, respectively. Metabolites, including halogenated benzoates, were detected by high-performance liquid chromatography and mass spectrometry. By using chlorophenyl ring-labeled monochlorobiphenyls as substrates, total mineralization (defined as CO2 production from the chlorophenyl ring) was observed for 4-chlorobiphenyl but not for 2-chlorobiphenyl. Rates of total mineralization of 4-chlorobiphenyl (at 39 to 385 micrograms ml-1 levels) were dependent on substrate concentration, whereas variation of cell number in the range of 10(5) to 10(7) cells ml-1 had no significant effects. Simulated sunlight enhanced the rate of mineralization by ca. 400%.

Microcosm and experimental pond evaluation of microbial community response to synthetic oil contamination in freshwater sediments.

A multivariate approach was used to evaluate the significance of synthetic oil-induced perturbations in the functional activity of sediment microbial communities. Total viable cell densities, ATP-biomass, alkaline phosphatase and dehydrogenase activity, and mineralization rates of glucose, protein, oleic acid, starch, naphthalene, and phenanthrene were monitored on a periodic basis in microcosms and experimental ponds for 11 months, both before and after exposure to synthetic oil. All variables contributed to significant discrimination between sediment microbial responses in control communities and communities exposed to a gradient of synthetic oil contamination. At high synthetic oil concentrations (4,000 ml/12 m), a transient reduction in sediment ATP concentrations and increased rates of oleic acid mineralization were demonstrated within 1 week of exposure. These transient effects were followed within 1 month by a significant increase in rates of naphthalene and phenanthrene mineralization. After initial construction, both control and synthetic oil-exposed microbial communities demonstrated wide variability in community activity. All experimental microbial communities approached equilibrium and demonstrated good replication. However, synthetic oil perturbation was demonstrated by wide transient variability in community activity. This variability was primarily the result of the stimulation of polyaromatic hydrocarbon mineralization rates. In general, microcosms and pond communities demonstrated sufficient resiliency to recover from the effects of synthetic oil exposure within 3 months, although polyaromatic hydrocarbon mineralization rates remained significantly elevated.