Understanding Molecular Pathogenesis

C H A P T E R

12 Understanding Molecular Pathogenesis: The Biological Basis of Human Disease and Implications for Improved Treatment of Human Disease William B. Coleman, PhD1, Gregory J. Tsongalis, PhD2,3 1Department

of Pathology and Laboratory Medicine, Curriculum in Toxicology, UNC Program in Translational Medicine, UNC Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC, United States; 2Laboratory for Clinical Genomics and Advanced Technology (CGAT), Department of Pathology and Laboratory Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH, United States; 3The Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, NH, United States

O U T L I N E Consequence of the t(15;17) Translocation In Acute Myelogenous Leukemia Detection of the t(15;17) Translocation In Acute Myelogenous Leukemia

Introduction231 Hepatitis C Virus Infection Identification of the Hepatitis C Virus Risk Factors for Hepatitis C Virus Infection Hepatitis C Infection Testing for Hepatitis C Virus Infection Clinical Course of Hepatitis C Virus Infection Treatment of Hepatitis C Infection Guided Treatment of Hepatitis C Virus

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Summary235 Acute Myeloid Leukemia 235 Chromosomal Abnormalities in Acute Myelogenous Leukemia235

INTRODUCTION Disease has been a feature of the human existence since the beginning of time. Descriptions of diseases and therapeutic interventions were recorded in the earliest written histories of medicine. Over time, our knowledge

Molecular Pathology, Second Edition http://dx.doi.org/10.1016/B978-0-12-802761-5.00012-2

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Summary237 Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Gene Diagnosis of Cystic Fibrosis Abnormal Function of CFTR in Cystic Fibrosis Pathophysiology of Cystic Fibrosis

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Summary239 References240

of science and medicine has expanded and with it our understanding of the biological basis of disease. In this regard, the biological basis of disease implies that more is understood about the disease than merely its clinical description or presentation. In the last several decades, we have moved from causative factors in disease to

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studies of molecular pathogenesis. Molecular pathogenesis takes into account the molecular alterations that occur in response to environmental insults and other contributing factors, to produce pathology. By developing a deep understanding of molecular pathogenesis, we will uncover the pathways that contribute to disease, either through loss-of-function or through gain-of-function. By understanding the involvement of specific genes, proteins, and pathways, we will be better equipped to develop targeted therapies for specific diseases. Continued expansion of our knowledge base with respect to underlying mechanisms of disease has resulted in unprecedented patient management strategies. Identification of genetic variants in genes once associated with the diagnosis of a disease process is now being reevaluated as it may impact new therapeutic options. In this chapter we describe three disease entities (Hepatitis C virus [HCV] infection, acute myeloid leukemia [AML], and cystic fibrosis [CF]) as examples of our increased understanding of the pathology represented by these diseases and how novel therapeutics are being introduced into clinical practice.

HEPATITIS C VIRUS INFECTION HCV infection represents the most common chronic viral infection in North America and Europe, and a common viral infection worldwide. In the United States, the Centers for Disease Control and Prevention has periodically conducted a National Health and Nutrition Examination Survey (https://www.cdc.gov/nchs/ nhanes/index.htm). This survey initially estimated that 3.9 million people had detectable antibodies to HCV (for the period 1988–94), indicating a prior exposure to the virus, and 75% of these individuals were positive for HCV RNA, suggesting an active infection [1]. During this same period, 2.7 million individuals were estimated to have chronic HCV infection. For the period of 1999– 2002, 3.2 million people in the United States were estimated to have chronic HCV infection (representing 1.3% of the population). Most recently (2003–10), 2.7 million people in the United States were found to have chronic HCV infection (representing 1.0% of the population). Globally, 177.5 million people are infected with HCV (representing 2.5% of the population) [2]. Prevalence varies significantly by geographical location, with the highest rates of HCV infection in Africa and Asia and lower rates of infection in Europe and North America. In Egypt, the prevalence of HCV infection is estimated to be 18% [3,4]. Likewise, the prevalence of HCV infection in Mongolia is estimated at 10% [3]. In contrast, Canada has a very low prevalence of HCV infection, estimated at <1%. HCV infection has been found to be more common

in certain populations, including prison inmates and homeless people, where the prevalence of infection may be as high as 40% [5]. Worldwide, it is estimated that 340 million individuals are chronically infected with HCV [6].

Identification of the Hepatitis C Virus HCV was first recognized in 1989 using recombinant technology to create peptides from an infectious serum that were then tested against serum from individuals with non-A, non-B hepatitis [7,8]. This approach resulted in the isolation of a section of the HCV genome [7,8]. Subsequently, the entire HCV genome was sequenced [9]. HCV is a member of the family of flaviviridae. Flaviviruses are positive, single-stranded RNA viruses. The HCV genome encodes a gene for production of a single polypeptide chain of approximately 3000 amino acids. This polypeptide gives rise to a number of specific proteins. The Env proteins are among the most variable parts of the peptide chain and are associated with multiple molecular forms in a single infected person [10,11]. The mutations affecting this portion of the HCV genome (and the encoded Env protein) seem to be critical for escape of the virus from the host immune response [12,13]. The HCV protein NS5a contains an interferonresponse element. Evidence from several studies suggest that mutational variation in the HCV genome encoding this protein are associated with resistance to interferon, the main antiviral agent used in treatment of HCV [14,15]. Other proteins encoded by the HCV genome include the NS3 region that codes for a protease and the NS5b region that codes for an RNA polymerase. Drugs that target the HCV protease or polymerase are now undergoing trials as therapeutic agents to treat HCV infection [16]. There are several HCV strains that differ significantly from each other [17]. The nomenclature adopted to describe these HCV strains is based on division of the HCV RNA into three major levels: (1) genotypes, (2) subtypes, and (3) quasispecies [18,19]. There are six recognized genotypes of HCV that are numbered from 1 to 6. Among these HCV genotypes there is <70% homology in the nucleotide sequence. HCV subtypes typically display 77%–80% homology in nucleotide sequence, while quasispecies have >90% nucleotide sequence homology [18,19]. Infection of an individual with HCV involves a single genotype and subtype (except in rare instances). However, infected individuals will carry many quasispecies of HCV because these RNA viruses do not contain a proofreading mechanism and acquired mutations in the HCV genome over time are common. HCV genotype 1 is the most prevalent world-wide (49%), followed by genotype 3 (18%), genotype 4 (17%), genotype 2 (11%), and genotypes 5/6 (<5%) [2].

Hepatitis C Virus Infection

Risk Factors for Hepatitis C Virus Infection There are a number of recognized risk factors for HCV. Among the most common risk factors for HCV infection are (1) the use of injectable drugs and (2) blood transfusion or organ transplant recipient before 1992 [20]. A significant percentage of people who used recreational injectable drugs in the 1960s and 1970s became infected with HCV [21]. Less commonly, HCV infection can be transmitted by dialysis [22–24], by needlestick injury [25], and through vertical transmission from an infected mother to her child [26–28]. The likelihood of infection from needlestick injury or vertical transmission is estimated to be approximately 3%–5%.

Hepatitis C Infection The primary target cell type for HCV infection is the mature hepatocyte [29], although there is some evidence that infection can also occur in other cell types, particularly circulating mononuclear cells [30]. Following the initial HCV infection, there is a latency period of 2–4 weeks before viral replication is detectable [31]. In most cases, there is no clinical evidence of the infection even after viremia develops. In fact, only 10%–30% of individuals with HCV infection will develop the clinical symptomology of acute hepatitis [32]. When acute HCV infection develops, patients display symptoms of fever, loss of appetite, nausea, diarrhea, and specific liver symptoms, including discomfort and tenderness in the right upper abdomen, jaundice, dark urine, and pale-colored stools. Typically, these symptoms occur 2–3 months after the initial HCV infection and then gradually resolve over a period of several weeks [33]. During this time, liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferases (AST) are found at elevated levels in the blood, reflecting hepatocyte injury and death. In acute HCV infection, these enzymes are typically increased from 10-fold to 40-fold the upper reference limit of normal [34]. In the majority of individuals infected with HCV, there are no signs or symptoms that accompany the initial infection. In most of these cases, a chronic HCV infection develops, resulting in chronic hepatitis (ongoing inflammation in the liver). In general, chronic HCV infections can be clinically silent for many years without obvious symptomology associated with the infection or liver injury, or produce only mild, nonspecific symptoms such as fatigue, loss of energy, and difficulty performing tasks that require concentration. The major end-stage diseases that result from chronic hepatitis include cirrhosis and hepatocellular carcinoma. It has been estimated that 20%–30% of individuals with chronic HCV infection will progress to cirrhosis after 20 years of infection, although the fibrotic changes in the liver progress at different rates in different individuals

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[26,35]. Cirrhosis due to HCV infection has now become the most common indication for liver transplantation in the United States [5].

Testing for Hepatitis C Virus Infection Most clinical testing for HCV infection begins with detection of antibodies against HCV proteins. The sensitivity of the anti-HCV assay is reported to be in the range of 97%–99% for detecting HCV infection. Most false-negative results of the anti-HCV assay occur in the setting of immunosuppression, such as with human immunodeficiency virus (HIV) infection, or in renal failure [22,37]. Anti-HCV antibodies are detectable after 10–11 weeks of infection (on average) using the second-generation antiHCV assays, but the third-generation anti-HCV assays show improved sensitivity with positive detection of anti-HCV antibodies by 7–8 weeks after the initial infection [38–40]. At the time of clinical presentation with acute HCV infection, >40% of patients lack detectable anti-HCV [41]. In the current clinical laboratory setting, the major method employed to determine the presence of active HCV infection is HCV RNA measurement. With acute HCV infection, HCV RNA becomes detectable 2–4 weeks after infection, and viral loads climb rapidly [32,36]. Average HCV viral loads are approximately 2–3 million copies per mL. Qualitative assays are designed to determine the presence or absence of HCV RNA, without consideration of actual viral load. Two primary methodologies are used in this type of assay: (1) reverse-transcriptase polymerase chain reaction (RT-PCR) and (2) transcription-mediated amplification (TMA). The detection limit for assays of this type is <50 IU/mL. The approaches employed for qualitative determination of HCV RNA utilize a known amount of a synthetic standard to enable quantitative measurement of HCV RNA through comparison of the amounts of HCV amplified and the amount of standard amplified using a calibration curve. Determination of HCV viral load has become standard of care in evaluating patients before and during treatment for chronic HCV infection. Real-time PCR allows for reduced carryover amplification, more rapid detection of amplification, increased low-end sensitivity, and a wider dynamic range for detection and quantification [42]. Several techniques have been developed to determine the particular genotype and subtype of HCV causing infection in an individual infected patient. These techniques typically target the 5′-untranslated and/or core regions of the HCV genome, which represent the most highly conserved regions. Because most amplification methods for HCV RNA also target the 5′-untranslated region, qualitative PCR methods can provide amplified RNA for use in determination of HCV genotype. The most widely used technique is a commercial line probe

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assay [43]. In this assay, a large number of oligonucleotide sequences are immobilized on a membrane, incubated with amplified RNA, and then detected using a colorimetric reagent that detects areas of hybridization. The line probe assay enables recognition and identification of most HCV types and subtypes accurately, although there are several subtypes that cannot be distinguished from one another.

Clinical Course of Hepatitis C Virus Infection Although anyone who is infected with HCV will experience an initial infection incident, in most cases this phase of the infection will be clinically silent without obvious symptoms. Acute infection with HCV is most likely to be detected when it occurs following a needlestick exposure from a person with known HCV, or when the infection arises under other circumstances but produces symptomatic infection and jaundice (estimated to occur in less than one-third of all cases) [32,33,36]. There is some evidence to suggest that patients who develop clinical jaundice are actually more likely to clear the infection and not progress to chronic HCV infection [44]. During the initial incubation period approximately 2 weeks following infection, HCV RNA is either undetectable or can be detected only intermittently. Subsequently, there is a period of rapid increase in the amount of circulating HCV, with an estimated doubling time of <24 h [31]. HCV viral loads reach very high levels during this period of time, typically reaching values of 107 IU/mL and occasionally higher [31]. Evidence of liver injury appears after an additional 1–2 months of HCV infection. This liver injury can be detected secondary to increased serum levels of ALT and AST. Approximately 40%–50% of individuals with acute HCV infection that are clinically diagnosed are detected during this stage of infection, prior to the development of anti-HCV [41]. By 7–8 weeks following infection, anti-HCV becomes detectable using the third-generation immunoassays [40]. However, detection of anti-HCV using the second-generation immunoassay cannot be accomplished until 10–12  weeks following infection [38]. At the time of this seroconversion, the HCV viral loads decrease, sometimes to undetectable levels. In most individuals who will progress to chronic hepatitis (and in some that eventually clear the infection) the HCV viral load remains detectable, but at reduced levels. In a person suspected of having acute HCV infection, the most reliable test for proving exposure is HCV RNA. Because of the high viral loads seen, either qualitative or quantitative assays would be acceptable for this purpose. Detectable HCV RNA in the absence of anti-HCV is a strong evidence of recent HCV infection [45].

Treatment of Hepatitis C Infection In contrast to other chronic viral infections such as those associated with hepatitis B virus or HIV, treatment has been successful in eradicating replicating HCV and halting progression of liver damage. Interferon alpha-2 is the agent of choice for treatment of chronic HCV infection. There are currently two potential approaches to treatment of chronic HCV: (1) interferon alone or (2) a combination of interferon plus ribavirin. While ribavirin is ineffective as a single agent for treating HCV [46], it increases the effectiveness of interferon. Application of ribavirin in combination with interferon increases the number of patients who respond to therapy by twofold to three-fold [47]. For many years, the only form of interferon available was standard dose interferon. Using standard dose interferon, large doses (typically 3 million units) were delivered to patients infected with HCV several times each week. The short half-life of this interferon produced widely fluctuating interferon levels in these patients, diminishing its therapeutic effectiveness. In 2001, a longer-acting form of interferon was approved for use in treating HCV infection. The longeracting interferon was modified by attachment of polyethylene glycol (pegylated interferon), which resulted in increased half-life for the administered drug. Use of pegylated interferon results in sustained high levels of interferon in the patient, reducing the number of required administrations to a single injection each week. There was also an improvement in response rates among patients treated with the pegylated interferon. Currently, the preferred treatment for chronic HCV infection is the combination of pegylated interferon plus ribavirin [48]. Recent developments in the treatment of HCV infection employ direct acting agents against targets that emerge from the HCV life cycle [49–51]. For example, the NS3/4A protease represents a major target for antiviral intervention because loss of NS3/4A impairs the HCV life cycle by inhibiting maturation of the viral polyprotein. Likewise, replication of HCV genetic material is a target for antiviral drugs that function to inhibit NS5B (including both nucleotide analogues and nonnucleoside inhibitors of NS5B) [52]. Through the use of directacting agents, 90% of HCV infections can be cured, although concerns related to development of resistance remain [52].

Guided Treatment of Hepatitis C Virus The appropriate duration of treatment for HCV infection varies depending on the HCV strain (genotype) that infects the patient. HCV genotypes 2 and 3 respond much better to standard treatment regimens. Thus, only 24 weeks of therapy are needed to achieve

Acute Myeloid Leukemia

maximum benefit, compared to 48 weeks in persons infected with other HCV genotypes [48]. In current clinical practice, treatment is offered to all patients with HCV infection except those with decompensated cirrhosis, where treatment may lead to worsening of the patient’s condition [53,54]. Once treatment is initiated, the most reliable means to determine efficacy is to evaluate the response by measuring HCV RNA. Successful treatment is associated with at least two different phases of viral clearance [55]. The first phase, which occurs rapidly over the course of days, is thought to reflect HCV RNA clearance from a circulating pool through the antiviral effect of interferon. In the second phase of clearance, infected liver cells (the major site of viral replication) undergo cell turnover and are replaced by uninfected cells. The second phase of clearance is more variable in duration. First-phase clearance is less specific for detecting success of antiviral treatment; therefore, it is necessary to evaluate whether second-phase clearance has occurred.

SUMMARY HCV infection represents a relatively recently identified infectious agent that has a varied natural history from patient to patient. Intensive research efforts have characterized the phases of HCV infection and the clinical symptomology of acute and chronic HCV infection. Through improved understanding of the biology of the HCV virus and its life cycle in the infected host, effective and sensitive diagnostic tests have been developed. Unlike some other chronic viral infections, HCV infection can be effectively treated using interferon in combination with ribavirin. However, it is now recognized that effective therapy of the patient depends on knowing the genotype of the HCV causing the infection. With continued advances in the understanding of the pathogenesis of HCV infection, new treatments and/or new modes of administration of known anti-HCV drugs will emerge that provide effective control of the viral infection with minimal adverse effects for the patient.

ACUTE MYELOID LEUKEMIA The human leukemias have been classified as a distinct group of clinically and biologically heterogeneous disorders that are a result of genetic abnormalities that affect specific chromosomes and genes. In the United States, 62,130 new cases of leukemia will be diagnosed in 2017 [56]. The AML represents a major form of leukemia. Of these new cases of leukemia, approximately 21,400 will represent AML (approximately 34% of all

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leukemias) [56]. In 2017, AML will be associated with approximately 10,600 cancer-related deaths [56]. AML is characterized by accumulation of neoplastic immature myeloid cells, consisting of ∼30% myeloblasts in the blood or bone marrow and classified on the basis of their morphological and immunocytochemical features. AML can arise (1) de novo, (2) in a setting of a preexisting myelodysplasia, or (3) secondary to chemotherapy for another disorder.

Chromosomal Abnormalities in Acute Myelogenous Leukemia Various cytogenetic and/or molecular abnormalities have been associated with various types of AML. The World Health Organization recognizes several AML subtypes based upon their cytogenetic abnormalities [57]. Chromosomal translocations are the most common form of genetic abnormality identified in acute leukemias [58– 60]. Typically, these translocations involve genes that encode proteins that function in transcription and differentiation pathways [61]. As a result of chromosomal translocation, the genes proximal to the chromosome breakpoints are disrupted, and the 5′-segment of one gene is joined to the 3′-end of a second gene to form a novel fusion (chimeric) gene. When the chimeric gene is expressed, a novel protein product is produced from the chimeric mRNA. Other genetic alterations such as point mutations, gene amplifications, and numerical gains or losses of chromosomes can also be identified in the acute leukemias. The clinical heterogeneity seen in AML may be due in part to differences in the number and nature of genetic abnormalities that occur in these cancers. However, these same molecular differences define various prognostic and therapeutic characteristics associated with the specific disorder in a given patient [62]. A major chromosomal translocation in AML involves chromosomes 15 and 17. This genetic abnormality, t(15;17)(q21;q21), occurs exclusively in acute promyelocytic leukemia (APL). APL represents approximately 5%–13% of all de novo AMLs [60,63]. The presence of the t(15;17) translocation consistently predicts responsiveness to a specific treatment utilizing all-trans-retinoic acid (ATRA). Retinoic acid is a ligand for the retinoic acid receptor (RAR), which is involved in the t(15;17). ATRA is thought to overcome the block in myeloid cell maturation, allowing the neoplastic cells to mature (differentiate) and be eliminated [64,65]. Approximately 75% of patients with APL present with a bleeding diathesis, usually the result of one or more processes including disseminated intravascular coagulation, increased fibrinolysis, and thrombocytopenia, and secondary to the release of procoagulants or tissue plasminogen activator

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from the granules of neoplastic promyelocytes [65,66]. This bleeding diathesis may be exacerbated by standard cytoreductive chemotherapy. Two morphologic variants of APL have been described, typical (hypergranular) and microgranular, both of which carry the t(15;17) translocation [65–67]. In the typical or hypergranular variant, the promyelocytes have numerous azurophilic cytoplasmic granules that often obscure the border between the cell nucleus and the cytoplasm. Cells with numerous Auer rods in bundles are common. In the microgranular type the promyelocytes contain numerous small cytoplasmic granules that are difficult to discern with the light microscope but are easily seen by electron microscopy.

Consequence of the t(15;17) Translocation In Acute Myelogenous Leukemia The t(15;17) is a balanced and reciprocal translocation in which the PML (for promyelocytic leukemia) gene on chromosome 15 and the RARα gene on chromosome 17 are disrupted and fused to form a hybrid gene [68,69]. The PML-RARα fusion gene, located on chromosome 15, encodes a chimeric mRNA and a novel protein. On the derivative chromosome 15, both the PML and RARα genes are oriented in a head-to-tail orientation. The function of the normal PML gene is poorly understood. However, the gene is ubiquitously expressed and encodes a protein that contains a dimerization domain and is characterized by an N-terminal region with two zinc finger–like motifs (known as a ring and a B-box). Given its structural features, the PML protein is thought to be involved in DNA binding [65,68,69]. Furthermore, the normal PML protein appears to have an essential role in cell proliferation. The RARα gene encodes a transcription factor that binds to DNA sequences in cis-acting retinoic acid-responsive elements. High-affinity DNA binding also requires heterodimerization with another family of proteins, the retinoic acid X receptors. The RARα protein contains transactivation, DNA binding, heterodimerization, and ligand-binding domains. The normal RARα protein plays an important role in myeloid differentiation. There are three major forms of the PML-RARα fusion gene, corresponding to different breakpoints in the PML gene [70–72]. The breakpoint in the RARα gene occurs in the same general location in all cases, involving the sequences within intron 2. Approximately 40%–50% of cases have a PML breakpoint in exon 6 (the so-called long form, termed bcr1), 40%–50% of cases have the PML breakpoint in exon 3 (the so-called short form, termed bcr3), and 5%–10% of cases have a breakpoint in PML exon 6 that is variable (the so-called variable form, termed bcr2). In each form of the translocation, the PMLRARα fusion protein retains the 5′-DNA binding and dimerization domains of PML and the 3′-DNA binding,

heterodimerization, and ligand (retinoic acid)-binding domains of RARα. Recent studies indicate that the different forms of PML-RARα fusion mRNA correlate with clinical presentation or prognosis. In particular, the bcr3 type of PML-RARα correlates with higher leukocyte counts at time of presentation [71,72]. Both higher leukocyte counts and variant morphology are adverse prognostic findings, and the bcr3 type of PML-RARα does not independently predict poorer disease-free survival [72].

Detection of the t(15;17) Translocation In Acute Myelogenous Leukemia A number of methods may be used to detect the t(15;17) translocation. Conventional cytogenetic methods detect the t(15;17) in approximately 80%–90% of APL cases at time of initial diagnosis. Suboptimal clinical specimens and poor-quality metaphases explain a large subset of the negative results. Fluorescence in situ hybridization (FISH) is another useful method for detecting the t(15;17) in APL [73]. Different methods employ probes specific for either chromosome 15 or chromosome 17 (or both), and commercial kits are available. Southern blot hybridization is another method to detect gene rearrangements that result from the t(15;17) [74]. The chromosomal breakpoints consistently involve the second intron of the RARα gene, and therefore, probes derived from this region are the most often utilized. Virtually all cases of APL can be detected by Southern blot analysis using two or three genomic RARα probes. RT-PCR is a very convenient method for detecting the PML-RARα fusion transcripts [70]. Primers have been designed to amplify the potential transcripts, and each type of transcript can be recognized. Results using this method are equivalent to or better than other methods at time of initial diagnosis. Polyclonal and monoclonal antibodies reactive with the PML and RARα proteins have been generated, and immunohistochemical studies to assess the pattern of staining appear to be useful for diagnosis [75,76]. Dyck et al. [75] have studied a number of APLs and have shown that the pattern of PML or RARα immunostaining correlates with the presence of the t(15;17). APL cells immunostaining for either PML or RARα reveals a microgranular pattern. The fusion protein may prevent PML from forming normal oncogenic domains, since treatment with ATRA allows PML reorganization into these domains. For the diagnosis of residual disease or early relapse after therapy, conventional cytogenetic studies, Southern blot analysis, and immunohistochemical methods are limited by low sensitivity. Quantitative RT-PCR and FISH methods are very useful. The sensitivity and rapid turnaround time of RT-PCR makes this method very useful for monitoring residual disease after therapy [77,78].

Cystic Fibrosis

SUMMARY Acute promyelocytic leukemia is a distinct subtype of acute myeloid leukemia that is cytogenetically characterized by a balanced reciprocal translocation between chromosomes 15 and 17 [t(15;17)(q21;q21)], which results in a gene fusion involving PML and RARα. This disease is the most malignant form of acute leukemia with a severe bleeding tendency and a fatal course of only weeks in affected individuals. In the past, cytotoxic chemotherapy was the primary modality for treatment of APL, producing complete remission rates of 75%–80% in newly diagnosed patients, a median duration of remission from 11 to 25 months, and only 35%–45% of the patients were cured [79]. However, with the introduction of ATRA in the treatment and optimization of the ATRA-based regimens, the complete remission rate increased to 90%–95% and 5-year disease-free survival improved to 74% [79].

CYSTIC FIBROSIS CF is a clinically heterogeneous disease that exemplifies the many challenges of complex genetic diseases and the causative underlying mechanisms [80]. CF is the most common lethal autosomal recessive disease in individuals of European decent with a prevalence of 1:2500 to 1:3300 live births [81]. While CF occurs most commonly in the Caucasian population, members of other racial and ethnic backgrounds are also at risk for this disease. The highest prevalence of CF occurs in Irish populations where this disease occurs in 1:1400 live births [82]. In contrast, CF occurs rarely in Hispanics (1:4000 to 1:10,000 live births) and African-Americans (1:15,000 to 1:20,000), with even lower incidence rates in individuals of Asian descent [83]. In the United States, approximately 850 individuals are newly diagnosed on an annual basis, and 30,000 children and adults are affected by the disease. The majority of CF diagnoses are made in individuals who are less than 1 year of age (http:// www.genetests.org/).

Cystic Fibrosis Transmembrane Conductance Regulator Gene The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene is responsible for CF. This gene is large, spanning approximately 230 kb on chromosome 7q, and consists of 27 coding exons. The CFTR mRNA is 6.5 kb and encodes a CFTR membrane glycoprotein of 1480 amino acids with a mass of ∼170,000 Da [84–86]. CFTR functions as a cAMP-regulated chloride channel in the apical membrane of epithelial cells [87]. To date over 1000 unique mutations in the CFTR gene have been described (Cystic Fibrosis Mutation Data Base, http://

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www.genet.sickkids.on.ca/cftr/). The most common CFTR mutation is the deletion of phenylalanine at position 508 (ΔF508). This mutation affects 70% of patients worldwide. The allelic frequency of CFTR mutations varies by ethnic group. For example, the ΔF508 CFTR mutation is only present in 30% of the affected Ashkenazi Jewish population.

Diagnosis of Cystic Fibrosis A diagnosis of CF in a symptomatic or at-risk patient is suggested by clinical presentation and confirmed by a sweat test. In the presence of clinical symptoms (such as recurrent respiratory infections), a sweat chloride above 60 mmol/L is diagnostic for CF. Although the results of this test are valid in a newborn as young as 24 h, collecting a sufficient sweat sample from a baby younger than 3 or 4 weeks old is difficult. The sweat test can also confirm a diagnosis of CF in older children and adults, but is not useful for carrier detection. Mutations in the CFTR gene are grouped into six classes, including (i) Class I, characterized by defective protein synthesis where there is no CFTR protein at the apical membrane; (ii) Class II, characterized by abnormal/defective processing and trafficking where there is no CFTR protein at the apical membrane; (iii) Class III, characterized by defective regulation where there is a normal amount of nonfunctional CFTR at the apical membrane; (iv) Class IV, characterized by decreased conductance where there is a normal amount of CFTR with some residual function at the apical membrane; (v) Class V, characterized by reduced or defective synthesis/trafficking where there is a decreased amount of functional CFTR at the apical membrane; and (vi) Class VI, characterized by decreased stability where there is a functional but unstable CFTR at the apical membrane [88,89]. Of the CFTR mutations, classes I–III are the most common and are associated with pancreatic insufficiency [90]. The ΔF508 CFTR mutation (which is most common worldwide) represents a class II mutation, with varying frequency between ethnic groups [91].

Abnormal Function of CFTR in Cystic Fibrosis CFTR is a member of an ATP-binding cassette family with diverse functions such as ATP-dependent transmembrane pumping of large molecules, regulation of other membrane transporters, and ion conductance. Mutations in the CFTR gene can lead to an abnormal protein with loss or compromised function that results in defective electrolyte transport and faulty chloride ion transport in apical membrane epithelial cells affecting the respiratory tract, pancreas, intestine, male genital tract, hepatobiliary system, and the exocrine system, resulting in complex multisystem disease. The loss of CFTR-mediated anion conductance explains a variety

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of CF symptoms including elevated sweat chloride, due to a defect in salt absorption by the sweat ducts, and meconium ileus, a defect in fluid secretion by intestinal crypt cells [92]. The malfunction of CFTR as a regulator of amiloride-sensitive epithelia Na+ channel leads to increased Na+ conductance in CF airways, which drives increased absorption of Cl− and water. Most of the symptoms associated with CF, such as meconium ileus, loss of pancreatic function, degeneration of the vas deferens, thickened cervical mucus, and failure of adrenergically mediated sweating are due to the role CFTR plays in Cl-driven fluid secretion. CFTR is an anion channel that functions in the regulation of ion transport. It plays multiple roles in fluid and electrolyte transport, including salt absorption, fluid absorption, and anion-mediated fluid secretion [92]. Defects in this protein lead to CF, the morbidity of which is initiated by a breach in host defenses and propagated by an inability to clear the resultant infections [93]. Since inflammatory exacerbations precipitate irreversible lung damage, the innate immune system plays an important role in the pathogenesis of CF. Respiratory epithelial cells containing the CFTR also provide a crucial environmental interface for a variety of inhaled insults. The local mucosal mechanism of defense involves mucociliary clearance that relies on the presence and constituents of airway surface liquid (ASL). The high salt in the ASL found in CF patients interferes with the natural antibiotics present in ASL such as defensins and lysozyme [94]. Bals et al. categorized the role of CFTR in the pathogenesis of CF-related lung disease by dividing patients into two groups [93]. The first describes defects in CFTR that result in altered salt and water concentrations of airway secretions. This then affects host defenses and creates a milieu for infection. The second is associated with CFTR deficiency that results in biologically and intrinsically abnormal respiratory epithelia. These abnormal epithelial cells fail as a mechanical barrier and enhance the presence of pathogenic bacteria by providing receptors and binding sites or failing to produce functional antimicrobials. Much debate exists regarding the relative biologic activity of antibacterial peptides such as beta-defensins and cathelicidins in human ASL and their role in the pathogenesis in CF-related lung disease [92,93,95]. It is possible that the innate immune system provides a first line of host defense against microbial colonization by secreting defensins, small cationic antimicrobial peptides produced by epithelia. The innate antibiotics are thought to possess salt-sensitive bacteriocidal capabilities. Hence, these innate antibiotics demonstrate altered (impaired) function in the lungs of CF patients [95]. Mannose-binding lectin represents another antimicrobial molecule that is present in ASL and is thought to be inactivated by high salt concentrations in the lungs

of CF patients. Mannose-binding lectin, an acute phase serum protein produced in the liver, opsonizes bacteria and activates complement. Common variations in the mannose-binding lectin gene (MBL2) are associated with increased disease severity, increased risk of infection with B. cepacia, poor prognosis, and early death [96]. The understanding that such naturally occurring peptide antibiotics exist has resulted in the pharmacologic development of these peptides for therapeutics.

Pathophysiology of Cystic Fibrosis The occurrence of CF leads to clinical, gross, and histologic changes in various organ systems expressing abnormal CFTR, including the pancreas, respiratory, hepatobiliary, intestinal, and reproductive systems. In addition, pathologic changes have been observed in organ systems that do not express the CFTR gene (such as the rheumatologic and vascular systems). The current age of individuals affected with CF ranges from 0 to 74 years, and the predicted survival age for a newly diagnosed child is 33.4 years. The increasing age of survival of CF has led to increased manifestation of pulmonary and extrapulmonary disorders (gastrointestinal, hepatobiliary, vascular, and musculoskeletal) associated with the disease. The extent and severity of disease tends to correlate with the degree of CFTR function. Although all these organ systems are affected, the pulmonary changes are the most pronounced and the major cause of mortality in most cases [97]. Lung infection remains the leading cause of morbidity and mortality in CF patients. It is currently recognized that CF-related lung disease is the consequence of chronic pulmonary consolidation by the well-known opportunistic pathogens Pseudomonas aeruginosa (mucoid and nonmucoid), Burkholderia cepacia, Staphylococcus aureus, and Haemophilus influenzae [98]. Morbidity and mortality due to persistent lung infection despite therapeutic advances focus attention toward the expanding microbiology of pulmonary colonizers. These increasingly prevalent flora include Burkholderia cepacia complex (genomovar I-IX), methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Mycobacterium abscessus, Mycobacterium-avium complex, Ralstonia species, and Pandoraea species [95,99]. Inflammatory exacerbation precipitates progressive irreversible lung damage, of which bronchiectases are the landmark changes. Bronchial mucous plugging facilitates colonization by microorganisms. Repetitive infections lead to bronchiolitis and bronchiectasis. Other pulmonary changes include interstitial fibrosis and bronchial squamous metaplasia. Often, subpleural bronchiectatic cavities develop and communicate with the subpleural space with resultant spontaneous secondary pneumothorax, the incidence of which increases later in life.

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Summary

Exocrine pancreas insufficiency is present in the majority of patients with CF. This clinically manifests by failure to thrive and fatty bulky stools owing to deficiency of pancreatic enzymes. However, pancreatic lesions vary greatly in severity, and the pancreas may be histologically normal in some patients who die in infancy [100]. Early in the postnatal development of the pancreas, patients with CF have a deficiency of normal acinar development. Increased secretory material within the ducts and increased duct volume also contribute to progressive degradation and atrophy of pancreatic acini. These factors result in duct obstruction and progressive pancreatic pathology [101,102]. Exocrine pancreatic disease appears to develop as a result of deficient ductal fluid secretion due to decreased anion secretion. Coupled to normal protein load derived from acinar cell secretion, this then leads to pancreatic protein hyperconcentration within the pancreatic ducts. The protein hyperconcentration increases susceptibility to precipitation and finally obstruction of the duct lumina [103,104]. Hence, the characteristic lesion is cystic ductal dilation, atrophy of pancreatic acini, and severe parenchymal fibrosis. The manifestation of CF in the hepatobiliary system is directly related to CFTR expression. The liver disease in CF is considered inherited liver disease due to impaired secretory function of the biliary epithelium [105]. While defective CFTR may be expressed, males are more likely to be affected than females and the risk for developing liver disease is between 4% and 17% as assessed by yearly exams and biochemical testing [106,107]. CFTR is expressed in epithelial cells of the biliary tract. Therefore, any or all cells of the biliary tree may be affected. While a variety of liver manifestations exist [108,109], including fatty infiltration (steatosis), common bile duct stenosis, sclerosing cholangitis, and gallbladder disease, the rare but characteristic liver lesion in CF is focal biliary cirrhosis, which develops in a minority of patients and is usually seen in older children and adults [110]. With the increasing life expectancy in patients with CF, liver-related deaths have increased and may become one of the major causes of death in CF [110]. The associated liver disease usually develops before or at puberty, is slowly progressive, and is frequently asymptomatic. There is negligible effect on nutritional status or severity of pulmonary involvement [111]. Only a minority of patients go on to develop a clinically problematic liver disease with rapid progression. Abnormal bile composition and reduced bile flow ultimately lead to intrahepatic bile duct obstruction and focal biliary cirrhosis [110]. Diagnosis of CF-associated liver disease is based on clinical exam findings, biochemical tests, and imaging techniques. Although liver biopsy is the gold standard for the diagnosis of most chronic liver diseases, only rarely is it employed in the diagnostic workup, mainly due to sampling error [110,112].

The gastrointestinal manifestations of cystic fibrosis are seen mainly in the neonatal period and include meconium ileus, distal intestinal obstruction syndrome (DIOS), fibrosing colonopathy, strictures, gastroesophageal reflux, rectal prolapse, and constipation in later childhood [106,113–116]. Throughout the intestines CFTR is the determinant of chloride concentration and secondary water loss into the intestinal lumen. Decreased water content results in viscous intestinal contents, with a 10%–15% risk of developing meconium ileus in babies born with cystic fibrosis. This also accounts for DIOS and constipation in older children [117]. DIOS (formerly meconium ileus equivalent) is a recurrent partial or complete obstruction of the intestine in patients with CF and pancreatic insufficiency [116]. Arthritis is a rare but recognized complication of cystic fibrosis that generally occurs in the second decade [118–121]. Three types of joint disease are described in patients with cystic fibrosis: (1) cystic fibrosis arthritis or episodic arthritis, (2) hypertrophic pulmonary osteoarthropathy, and (3) co-existent or treatment-related arthritis [118,119,122,123]. The most common form, episodic arthropathy, is characterized by episodic, self-limited polyarticular arthritis with no evidence of progression to joint damage [118]. Histologic features are minimal with prominent blood vessels and interstitial edema occurring most commonly, or rarely lymphocytic inflammation [124]. Infertility is an inevitable consequence of cystic fibrosis in males occurring in >95% of patients and is due to congenital bilateral absence or atrophy of the vasa deferentia (CBAVD) and/or dilated or absent seminal vesicles [125]. Spermatogenesis and potency remain normal. Mutations in the CFTR gene are present in up to 70% of the patients with CBAVD [126]. Diagnosis of obstructive azoospermia may be diagnosed by semen analysis; however, it must be confirmed by testicular biopsy and no other reason for azoospermia. Fertility in females may be impaired due to dehydrated cervical mucus, but their reproductive function is normal [127]. Advances in techniques such as microscopic epididymal sperm aspiration (MESA) and intracytoplasmic sperm injection have allowed males with cystic fibrosis the ability to reproduce [97].

SUMMARY CF is a complex multiorgan system disease that results from mutation in the CFTR gene. Advances in the understanding of the pathogenesis of this disease and related complications (such as recurrent lung infection) have led to improvement in diagnosis and treatment of affected individuals, resulting in improved life expectancy. With continued expansion of our understanding of the

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molecular pathogenesis of this disease and the variant manifestations of CF-related disorders, it is expected that new treatments will emerge that attempt to counteract or correct the pathologic consequences of CFTR mutation.

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