Daniel C. Chung

Gastroenteropancreatic neuroendocrine tumours

Gastroenteropancreatic (GEP) neuroendocrine tumours (NETs) are fairly rare neoplasms that present many clinical challenges. They secrete peptides and neuroamines that cause distinct clinical syndromes, including carcinoid syndrome. However, many are clinically silent until late presentation with mass effects. Investigation and management should be highly individualised for a patient, taking into consideration the likely natural history of the tumour and general health of the patient. Management strategies include surgery for cure (which is achieved rarely) or for cytoreduction, radiological intervention (by chemoembolisation and radiofrequency ablation), chemotherapy, and somatostatin analogues to control symptoms that result from release of peptides and neuroamines. New biological agents and somatostatin-tagged radionuclides are under investigation. The complexity, heterogeneity, and rarity of GEP NETs have contributed to a paucity of relevant randomised trials and little or no survival increase over the past 30 years. To improve outcome from GEP NETs, a better understanding of their biology is needed, with emphasis on molecular genetics and disease modeling. More-reliable serum markers, better tumour localisation and identification of small lesions, and histological grading systems and classifications with prognostic application are needed. Comparison between treatments is currently very difficult. Progress is unlikely to occur without development of centers of excellence, with dedicated combined clinical teams to coordinate multicentre studies, maintain clinical and tissue databases, and refine molecularly targeted therapeutics.

Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial

BACKGROUND Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Lebers congenital amaurosis. METHODS We assessed the retinal and visual function in 12 patients (aged 8-44 years) with RPE65-associated Lebers congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1.5 x 10(10) vector genomes), medium (4.8 x 10(10) vector genomes), or high dose (1.5 x 10(11) vector genomes) for up to 2 years. FINDINGS AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with ClinicalTrials.gov, number NCT00516477. INTERPRETATION The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain. FUNDING Center for Cellular and Molecular Therapeutics at the Childrens Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.

Efficacy, Safety, and Biomarkers of Neoadjuvant Bevacizumab, Radiation Therapy, and Fluorouracil in Rectal Cancer: A Multidisciplinary Phase II Study

PURPOSE To assess the safety and efficacy of neoadjuvant bevacizumab with standard chemoradiotherapy in locally advanced rectal cancer and explore biomarkers for response. PATIENTS AND METHODS In a phase I/II study, 32 patients received four cycles of therapy consisting of: bevacizumab infusion (5 or 10 mg/kg) on day 1 of each cycle; fluorouracil infusion (225 mg/m(2)/24 hours) during cycles 2 to 4; external-beam irradiation (50.4 Gy in 28 fractions over 5.5 weeks); and surgery 7 to 10 weeks after completion of all therapies. We measured molecular, cellular, and physiologic biomarkers before treatment, during bevacizumab monotherapy, and during and after combination therapy. RESULTS Tumors regressed from a mass with mean size of 5 cm (range, 3 to 12 cm) to an ulcer/scar with mean size of 2.4 cm (range, 0.7 to 6.0 cm) in all 32 patients. Histologic examination revealed either no cancer or varying numbers of scattered cancer cells in a bed of fibrosis at the primary site. This treatment resulted in an actuarial 5-year local control and overall survival of 100%. Actuarial 5-year disease-free survival was 75% and five patients developed metastases postsurgery. Bevacizumab with chemoradiotherapy showed acceptable toxicity. Bevacizumab decreased tumor interstitial fluid pressure and blood flow. Baseline plasma soluble vascular endothelial growth factor receptor 1 (sVEGFR1), plasma vascular endothelial growth factor (VEGF), placental-derived growth factor (PlGF), and interleukin 6 (IL-6) during treatment, and circulating endothelial cells (CECs) after treatment showed significant correlations with outcome. CONCLUSION Bevacizumab with chemoradiotherapy appears safe and active and yields promising survival results in locally advanced rectal cancer. Plasma VEGF, PlGF, sVEGFR1, and IL-6 and CECs should be further evaluated as candidate biomarkers of response for this regimen.

Induction of interleukin-8 preserves the angiogenic response in HIF-1alpha-deficient colon cancer cells.

Hypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis. It represents an attractive therapeutic target in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis. In HIF-1α knockdown DLD-1 colon cancer cells (DLD-1HIF-kd), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1α (DLD-1HIF-wt). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1HIF-kd but not DLD-1HIF-wt cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-κB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1HIF-kd but not DLD-1HIF-wt xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1α may be most effective when IL-8 is simultaneously targeted.

The Chromosomal Instability Pathway in Colon Cancer

The acquisition of genomic instability is a crucial feature in tumor development and there are at least 3 distinct pathways in colorectal cancer pathogenesis: the chromosomal instability (CIN), microsatellite instability, and CpG island methylator phenotype pathways. Most cases of colorectal cancer arise through the CIN pathway, which is characterized by widespread imbalances in chromosome number (aneuploidy) and loss of heterozygosity. It can result from defects in chromosomal segregation, telomere stability, and the DNA damage response, although the full complement of genes underlying CIN remains incompletely described. Coupled with the karyotypic abnormalities observed in CIN tumors are the accumulation of a characteristic set of mutations in specific tumor suppressor genes and oncogenes that activate pathways critical for colorectal cancer initiation and progression. Whether CIN creates the appropriate milieu for the accumulation of these mutations or vice versa remains a provocative and unanswered question. The goal of this review is to provide an updated perspective on the mechanisms that lead to CIN and the key mutations that are acquired in this pathway.

Hereditary diffuse gastric cancer: updated consensus guidelines for clinical management and directions for future research

25–30% of families fulfilling the criteria for hereditary diffuse gastric cancer have germline mutations of the CDH1 (E-cadherin) gene. In light of new data and advancement of technologies, a multidisciplinary workshop was convened to discuss genetic testing, surgery, endoscopy and pathology reporting. The updated recommendations include broadening of CDH1 testing criteria such that: histological confirmation of diffuse gastric criteria is only required for one family member; inclusion of individuals with diffuse gastric cancer before the age of 40 years without a family history; and inclusion of individuals and families with diagnoses of both diffuse gastric cancer (including one before the age of 50 years) and lobular breast cancer. Testing is considered appropriate from the age of consent following counselling and discussion with a multidisciplinary team. In addition to direct sequencing, large genomic rearrangements should be sought. Annual mammography and breast MRI from the age of 35 years is recommended for women due to the increased risk for lobular breast cancer. In mutation positive individuals prophylactic total gastrectomy at a centre of excellence should be strongly considered. Protocolised endoscopic surveillance in centres with endoscopists and pathologists experienced with these patients is recommended for: those opting not to have gastrectomy, those with mutations of undetermined significance, and in those families for whom no germline mutation is yet identified. The systematic histological study of prophylactic gastrectomies almost universally shows pre-invasive lesions including in situ signet ring carcinoma with pagetoid spread of signet ring cells. Expert histopathological confirmation of these early lesions is recommended.

The Hereditary Nonpolyposis Colorectal Cancer Syndrome: Genetics and Clinical Implications

In the United States, an individuals lifetime risk for developing some form of cancer is estimated to be as high as 40% (1). Cancer is fundamentally a genetic disorder, meaning that mutations in specific genes confer a selective growth advantage for tumor cells. Such mutations may develop sporadically or may be inherited through the germline, which results in a hereditary predisposition to multiple cases of early-onset cancer. Identification of these cancer-susceptibility genes has revealed the cellular processes that ordinarily guard against tumor formation. One particularly instructive example is the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. Defining the genetic basis for HNPCC has demonstrated a fascinating connection between the cellular machinery that regulates the replication and repair of DNA and cancer formation. Replication is the process by which DNA is copied in preparation for cell division. Errors of replication are inevitable, but specialized repair systems have evolved to prevent the accumulation of harmful mutations in the genome. The DNA mismatch repair system functions as a critical spell checker that identifies and then corrects mismatched base pairs of DNA. A germline mutation in a DNA mismatch repair gene results in the HNPCC syndrome, which is inherited as an autosomal dominant disorder. The 1990 Amsterdam criteria strictly define HNPCC as occurring when colon cancer is diagnosed in at least three family members, one a first-degree relative of the other two. The cases must span two generations, and one case must be diagnosed before 50 years of age (2). Less stringent criteria, including the Amsterdam II, modified Amsterdam, and Bethesda criteria, have also been developed. This review first highlights the key features of normal DNA replication and the mechanisms that ensure the fidelity of this process. The next section focuses on the genes involved in DNA mismatch repair and illustrates the biological consequences that result when mutations arise in these genes. Finally, we discuss the clinical implications of these genetic insights in the management of HNPCC families as well as persons with sporadic colon cancer. DNA Replication Replication of DNA takes place in preparation for mitosis. Consequently, each daughter cell carries an exact replica of the entire parental genome. Many nuclear proteins work in concert to execute the three phases of initiation, elongation, and termination. These mechanisms of DNA replication have been defined primarily from studies in bacteria and yeast, but the fundamental processes are relevant to higher eukaryotic cells. Replication is always initiated at specific points of origin in a chromosome when a cell enters the S (or DNA Synthesis) phase of the cell cycle (3). The helicase enzyme separates the double strands of DNA at these replication origin points. This unwinding of DNA results in an open loop structure. Each of the two parental strands can then serve as a template for the synthesis of a new complementary strand. Because each daughter chromosome contains one parental strand and one newly synthesized strand, the replication process is termed semi-conservative. The DNA polymerase enzyme carries out new DNA synthesis, but it cannot begin until a primer is available. Most commonly, a specific RNA polymerase called primase synthesizes a short RNA primer at the replication origin. Two replication forks are created as DNA is synthesized in a bi-directional manner away from the point of origin (4). A multicomponent complex containing DNA polymerase carries out the elongation process that adds each complementary nucleotide to the primer. Replication always proceeds in a 5 to 3 direction. This results in continuous DNA synthesis along one strand (the leading strand), but it is necessarily discontinuous along the other template strand (the lagging strand) (5). The lagging strand is thus composed of many Okazaki fragments that are initiated from discontinuous primers (Figure 1), and DNA ligase joins each of these fragments to form one continuous strand. Figure 1. Events at the replication fork. Replication is terminated when the entire length of a chromosome has been synthesized, resulting in two complete copies of each chromosome. In circular bacterial chromosomes, termination occurs simply when the origin of replication has been reached. The mechanisms that control termination of linear eukaryotic chromosomes are far more complex. DNA Damage: Errors of DNA Replication Maintaining the integrity of DNA is essential for normal cellular function. Chemical carcinogens or ultraviolet radiation can directly induce structural alterations, such as thymidine dimers, single- or double-stranded breaks, and covalent cross-linking. The DNA excision repair system recognizes and excises such structurally altered nucleotides; specific details are available in a recent excellent review (6). The current review focuses instead on a different type of error that occurs during replication. Errors of DNA replication are observed primarily during the elongation phase. The most common error is a simple mispairing of nucleotides. Patterns of hydrogen bonding govern the pairings of nucleotides. An adenine (A) is always paired with a thymidine (T), and a cytosine (C) is always paired with a guanine (G). The DNA polymerase may incorrectly pair an adenine with a guanine, for example, and such mispairings are predicted to occur once every 103 to 104 base pairs. Repair mechanisms keep the actual error rate much lower. In bacteria, de novo mutations occur only once every 1010 base pairs (7). A second type of error can occur during DNA replication. There can be slippage of the DNA polymerase complex, a process analogous to a loose bicycle chain transiently slipping but then reengaging. This slippage occurs during the replication of microsatellite DNA sequences, which are defined as short dinucleotide (5-TCAATGCCACACACACACACACCTGAGGC-) or mononucleotide (5-TCTAGGCTAAAAAAAAATGCCGAGT-) repeats. The daughter strands may then contain either too many or too few copies of these repeated sequences. With intact mismatch repair mechanisms, these errors of slippage are quickly corrected, and microsatellite DNA sequences are considered stable. However, in the presence of deficient mismatch repair function, these errors are not corrected. This phenomenon is called microsatellite instability (MSI) (Figure 2). Figure 2. Slippage during DNA replication. MSS MSI The DNA polymerase complex itself provides the first line of defense against errors of replication. Through poorly defined mechanisms, DNA polymerase can immediately recognize a mismatched pair of nucleotides. An intrinsic enzyme subunit with 3 to 5 exonuclease activity excises the mispaired nucleotide from the newly synthesized strand, and the excised sequences are replaced with the correct nucleotides. The DNA Mismatch Repair System For errors that are not immediately corrected by DNA polymerase, the DNA mismatch repair system provides a secondary system of proofreading. This system corrects not only single base-pair mismatches but also small mispaired loops of DNA that result from replication errors of microsatellite tracts. Studies in yeast have demonstrated that defective mismatch repair specifically leads to a 100- to 700-fold increase in the instability of these microsatellite tracts (8). The DNA mismatch repair system requires the cooperation of many genes from the mutS (hMSH2, hMSH3, hMSH6) and mutL (hMLH1, hMLH3, hPMS1, and hPMS2) families. Briefly, hMSH2 serves as the scout that recognizes and binds directly to the mismatched DNA sequence (9, 10). It forms a heterodimeric complex with hMSH6 if a single base-pair mismatch is recognized or with hMSH3 if there is a larger two- to eight-nucleotide insertion or deletion (Figure 3). A second heterodimeric complex of hMLH1 and hPMS2 is then recruited to excise the mismatched nucleotides. Although heterodimers of hMLH1/hPMS1 and hMLH1/hMLH3 also form, their specific roles remain to be defined. Figure 3. Components of the DNA mismatch repair system. Mutations in DNA Mismatch Repair Genes A mutation in one of several mismatch repair genes results in deficient DNA mismatch repair activity. The extent of the mismatch repair deficiency depends on the specific gene that is altered. If hMSH2 or hMLH1 is inactivated, then a high level of MSI is typically observed, the so-called MSI-H phenotype (11). Mutations in genes such as hMSH6 result in only a partial deficiency of mismatch repair and low levels of MSI (the MSI-L phenotype) (12, 13). The U.S. National Cancer Institute defines the MSI-H phenotype as present when two of five microsatellite markers from a standard panel display instability and the MSI-L phenotype as present when one of the five markers is unstable. Mutations in mismatch repair genes may occur in germline or somatic DNA. Of interest, an inherited germline alteration does not result in widespread developmental anomalies. One possible explanation for this is that the second wild-type allele may provide sufficient DNA mismatch repair function. The observed phenotype is the predisposition to early-onset tumors, primarily of the colon and endometrium. The biological basis for this organ specificity is unknown. In each tumor that forms, the second copy of the affected DNA mismatch repair gene has been somatically mutated, thereby leading to bi-allelic gene inactivation (14, 15). The direct consequence of defective DNA mismatch repair is the so-called mutator or replication-error phenotype. Rather than directly causing malignant transformation, DNA mismatch repair deficiency creates the milieu that permits mutations to accumulate in other growth-regulatory genes. Colon tumors that develop in persons with germline mutations in hMSH2 or hMLH1 characteristically display the MSI-H phenotype (16, 17). Most microsatellite sequences in the genome are located within noncoding, or intr

AAV2 Gene Therapy Readministration in Three Adults with Congenital Blindness

Repeat administration of gene therapy to the contralateral retina of three congenitally blind patients was safe and resulted in improved vision. Shining a Light with Gene Therapy Gene therapy has great potential for treating certain diseases by providing therapeutic genes to target cells. Administration of a gene therapy vector carrying the RPE65 gene in 12 patients with congenital blindness due to RPE65 mutations led to improvements in retinal and visual function and proved to be a safe and stable procedure. In a follow-up study, the same group of researchers led by Jean Bennett set out to discover whether it would be possible to safely administer the vector and the therapeutic transgene to the contralateral eye of the patients. A big concern was whether the first gene therapy injection might have primed the patients’ immune system to respond to the adeno-associated virus (AAV) vector or the product of the therapeutic transgene that it had delivered. To test the safety and efficacy of a second administration of gene therapy to the second eye, the authors demonstrated that readministration was both safe and effective in animal models. Then, they selected 3 of the original 12 patients and readministered the AAV vector and its RPE65 transgene to the contralateral eye. They assessed safety by evaluating inflammatory responses, immune reactions, and extraocular exposure to the AAV vector. Efficacy was assessed through qualitative and quantitative measures of retinal and visual function including the ability to read letters, the extent of side vision, light sensitivity, the pupillary light reflex, the ability to navigate in dim light, and evidence from neuroimaging studies of cortical activation (which demonstrated that signals from the retina were recognized by the brain). The researchers did not discover any safety concerns and did not identify harmful immune responses to the vector or the transgene product. Before and after comparisons of psychophysical data and cortical responses provided the authors with evidence that gene therapy readministration was effective and mediated improvements in retinal and visual function in the three patients. The researchers report that the lack of immune response and the robust safety profile in this readministration gene therapy study may be due in part to the immune-privileged nature of the eye, and the low dose and very pure preparation of the AAV vector. Demonstration of safe and stable reversal of blindness after a single unilateral subretinal injection of a recombinant adeno-associated virus (AAV) carrying the RPE65 gene (AAV2-hRPE65v2) prompted us to determine whether it was possible to obtain additional benefit through a second administration of the AAV vector to the contralateral eye. Readministration of vector to the second eye was carried out in three adults with Leber congenital amaurosis due to mutations in the RPE65 gene 1.7 to 3.3 years after they had received their initial subretinal injection of AAV2-hRPE65v2. Results (through 6 months) including evaluations of immune response, retinal and visual function testing, and functional magnetic resonance imaging indicate that readministration is both safe and efficacious after previous exposure to AAV2-hRPE65v2.

An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia

The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a central role in the transcriptional response to oxygen flux. To gain insight into the molecular pathways regulated by HIF-1, it is essential to identify the downstream-target genes. We report here a strategy to identify HIF-1-target genes based on an integrative genomic approach combining computational strategies and experimental validation. To identify HIF-1-target genes microarrays data sets were used to rank genes based on their differential response to hypoxia. The proximal promoters of these genes were then analyzed for the presence of conserved HIF-1-binding sites. Genes were scored and ranked based on their response to hypoxia and their HIF-binding site score. Using this strategy we recovered 41% of the previously confirmed HIF-1-target genes that responded to hypoxia in the microarrays and provide a catalogue of predicted HIF-1 targets. We present experimental validation for ANKRD37 as a novel HIF-1-target gene. Together these analyses demonstrate the potential to recover novel HIF-1-target genes and the discovery of mammalian-regulatory elements operative in the context of microarray data sets.

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.