The esophagus is not immune to disease, despite its superficial simplicity. The particular disease state of interest for my project addresses Barrett’s esophagus (BE). Barrett’s esophagus is defined as a change in replacing the normal squamous epithelium lining of the esophagus with specialized columnar epithelium that has goblet cells, typically in the distal esophagus. Metaplasia and dysplasia of the tissue lining are evidence of BE. The change in tissue type is an aberrant malady that puts the individual at an elevated risk for developing esophageal adenocarcinoma (EAC), or cancer of the epithelium in the distal portion of the tube. As such, 90 – 95 percent of BE patients will not develop EAC over their lifetime. Supporting this low risk of progression, it has been suggested that BE is merely the act of natural selection at the cellular level because BE tissue is highly specialized towards providing mucosal defense against acid reflux. More on this adaptive, protective nature will be covered later. The key identifying traits and stages of BE are depicted in Fig. 4. BE classification may be broken down into short segmented (up to 3 cm. in length) or long segmented (greater than 3 cm. in length). Most commonly, gastroesophageal reflux disease (GERD) plays a pivotal role in the development metaplasia and dysplasia of the epithelium. The following sections will discuss the disease state in greater detail, starting with the history and coining of BE, epidemiology, and continuing with an elucidation of the pathophysiology and a discourse on the risk factors most associated with BE.
Figure 4. Advancing from left to right, the typical progression of epithelial changes in the esophagus lining to columnar metaplasia followed by low and high grades of dysplasia. Adenocarcinoma begins in the mucosa and advances deeper into the esophageal tissue layers.
History
The literature states that inflammation of the esophagus has been in study since the mid-1800’s. Studies from the early Victorian era were interested in discerning how esophagitis led to the ulceration of the epithelial lining and the role that acid and pepsin had in the process. During this initial study period, the hypothesis of acid and pepsin playing a large role was not widely accepted due to reflux patients not usually having a low enough pH value. Not until the mid-1900’s was an animal model established that could replicate the conditions of chronic reflux esophagitis, leading the way to studies for a better understanding of GERD and the litany of complications it can cause. The man for which Barrett’s gets its name was Norman Barrett, a thoracic surgeon who was born in Australia and went on to lecture and practice in London, U.K. In 1950, it is written that he described reflux oesophagitis when he distinguished that ulcers of the esophagus were not peptic ulcers, but rather, gastric ulcers that arise in a gastric mucous membrane. In 1953, Allison and Johnstone referred to Barrett’s finding as “Barrett’s ulcer,” where chronic ulceration occurs in the presence of gastric mucosa lining the esophagus. It was believed that the condition was a congenital abnormality, and not until later research was it discovered that the condition is acquired with constant reflux being a primary cause. Barrett’s seminal paper “The Lower Esophagus Lined by Columnar Epithelium” was published in Surgery in 1957. It elucidated the pathological occurrence of the signature red columnar epithelium of the distal esophagus. Following in the 1960’s through the 1980’s was an effort to establish a viable canine model for esophageal metaplasia, which would help researchers clarify how intestinal epithelial cells became metaplastic and how GERD had a role in the morphing process. More recent research has focused on genetic factors that influence the development of BE, along with changes in the expression of genes that can serve a biomarkers indicating disease.
Pathology
Pathology is the science of understanding the nature of disease, including the aberrant anatomical and physiological processes that define the disease. For BE, the signature salmon-colored patches of epithelium seen during endoscopy contrast against the normal pallid coloring of the esophagus and are a prime indication that the disease state is present (Fig. 5). Going back to Figure 4, the progression of phenotypic change can be expected as the pathogenesis of the disease carries on with time. Histologically identifying the cellular changes is a large component of understanding the disease and determining risk, which will be discussed later. The following sections will explore the disease state further by covering the metaplastic changes to the cell lining, the grades of dysplasia, and esophageal adenocarcinoma.
Figure 5. The hallmark of identifying Barrett’s Esophagus is salmon-colored tissue emanating upwards from the gastroesophageal junction. It is evident either circumferentially or in strips measured in centimeters. It will clash in color with normal squamous epithelium that is pallid and normally seen throughout the entirety of the esophagus. Goblet cells indicate metaplastic tissue. BE tissue may take on the appearance of undulating waviness that characterizes intestinal tissue lining.
Metaplasia
The adult cells in the body normally do not experience any further significant change in cell type through normal processes and living one matured. When the adult cells do change, however, and get replaced by another different sort of cell type, then this process is called metaplasia. Metaplasia is usually a consequence of chronic aggravation or injury to the tissue—some sort of repeated event that causes a chain of disturbances to the cells. In the case of BE, the most frequent disturbance to the stratified squamous epithelium of the mucous membrane is the reflux of stomach fluid through the LES and into the distal esophagus. The epithelial lining will shift in cell morphology that gradually becomes more glandular and has mucous-secreting columnar cells becoming the dominant cell type (Fig. 6).
The process can be thought of as occurring in stages of increasing change in cell morphology. A multilayered columnar transitional epithelium is the first lesion stage, which then turns into a single layered non-intestinal type of columnar epithelium over time. The final metaplastic state is the specialized intestinal type of columnar epithelium containing goblet cells. The cells are similar to small intestine mucosa cells, but lack the well-defined brush borders. The wave-like pattern, including crypts, becomes the overall tissue architecture; and simple mucous secreting glands are present in far greater numbers in the lamina propria. Differing mucins, like neutral and acid-nonsulfated, help to characterize the tissue as being specialized. BE, then, resembles a combination of intestinal, gastric, small intestinal, and colonic epithelium. Diagnosis of the metaplastic tissue normally depends on oral white light endoscopy, usually an esophagogastroduodenoscopy procedure; but transnasal endoscopy is also gaining acceptance. Different dyes, or narrow-band imaging that selectively filters out red light, also help to contrast the metaplastic tissue and reveal more tissue structure versus normal tissue. For the diagnosis procedure, an emphasis is placed on identifying relevant landmarks because the squamocolumnar junction is no longer situated within the gastroesophageal junction, but rather, extends upwards in the esophageal tube. The squamocolumnar junction is the point where squamous epithelium turns into columnar epithelium for the rest of the GI tract. A palisade network of vessels in the lower esophagus is identified, along with the top of the gastric folds and diaphragmatic pinch. Any metaplasia superior to these points is considered BE. Biopsy is then performed leading to a histologic analysis to determine the presence of goblet cells that will confirm BE. The genesis of the original columnar cells is still uncertain, however, and further research to trace the origin of the columnar cells is ongoing. Before moving onto dysplasia, a detour into cell origins, gastroesophageal reflux, and acids will be taken.
Figure 6. Metaplastic changes usually begin with the introduction of bile and acid refluxing into the esophagus. Over short periods, it will cause inflammation and lead to esophagitis. Over prolonged periods, it will cause a phenotypic change in the lining resulting in a single layered columnar layer containing goblet cells.
Origins of BE Cells
Several theories currently abound about the origin of the cells implicit in the development of BE in humans. Two such theories, transdifferentiation and transcommitment, include differing mechanisms affecting the local cellular population. Transdifferentiation’s premise (number 4 in Fig. 7) is that repeated acidic insult to the tissue lining activates a different set of cell transcription factors that causes the already differentiated adult cells to morph into another cell type. Essentially, transcription factors that promote cellular differentiation are expressed in greater amounts, whereas transcription factors that lead to normal squamous cell development are somehow inhibited. Transcription factors normally found in quantifiable amounts in the intestinal tissues also become expressed in the metaplastic tissue. Interestingly, occurring in the fetal esophagus between 20 to 25 weeks, transdifferentiation mechanistically acts to have stratified squamous epithelium replace the developmental stratified columnar epithelium. In the event of BE, it could be claimed that a reversal of this fetal transdifferentiation occurs.
Transcommitment, on the other hand, would involve immature progenitor cells of the basal epithelial layer being forced onto a different track of development towards columnar cells instead of squamous cells. This would occur through GERD and chronic inflammation compromising the superficial epithelial layers and presenting more opportunities for stomach acids and bile salts to reach deeper towards the basal layer, acting on the undifferentiated cells. Transcription factors like caudal homeobox genes CDX1 and CDX2 would become expressed and push the cells towards committing to an intestinal tissue phenotype. Transcommittment is viewed as more plausible than transdifferentiation and has been generally accepted for the past 30 years.
Figure 7. Depiction of origin sources for BE cells. Cells may transdifferentiate (4) or be recruited from locally sourced stem cells in submucosa glands (5). Cells may migrate up from the cardiac stomach (3) or the squamo-columnar junction (1). Hematopoietic stem cells originating in the bone marrow may also account for BE cells.
Other theories consider the regenerative and multipotent capabilities of stem cells from differing locations in the body. Animal models have observed circulating hematopoietic stem cells (#2) that travel from the bone marrow to the sights of injury and differentiate into columnar cells, or that stem cells travel from the nearby stomach cardia tissue to then differentiate (#1). Other possible locations of origin include the proximal esophagus, where submucosal glands have stem cells that could migrate out and proliferate (#5); and the squamocolumnar junction that has been shown to harbor an embryonic population of multipotent cells (#3). Barrett’s specialized tissue requires that cell renewal proceed as normal after metaplastic changes have occurred. This factor means that transdifferentiation is unlikely, and that stem cells with enduring proliferative properties are integral to the maintenance of BE, thus, supporting theories where a local stem cell population’s expression profile is changed. Many theories are being put to the test in animal models, however, and a definitive answer for human BE cases remains elusive.
Transcription Factors
Transcription factors and epigenetic changes in the BE tissue are under heavy study to help determine the origins and differentiation of BE tissue. Embryologic signaling pathways are being evaluated for their roles in BE tissue formation. As mentioned previously, CDX1 and CDX2 can push epithelial cells towards developing into an intestinal phenotype. These genes are part of the HOX family, meaning that they drive embryological development by relaying information about cell positioning for different body segmentation. It is known that metaplasia commonly reverts tissue to a form that was present in the developing embryo, which in the esophagus was columnar epithelium. It can be assumed that developmental factors involved with the changing of embryonic to adult tissue can be implicated in BE metaplasia, like CDX1, CDX2, and Sox9, as seen in Fig.8.
Figure 8. Genes that are active during embryonic formation but are silenced during maturation are thought to play a role in BE. Those genes become activated again in the presence of chornic acid reflux or injury, causing a reversion back to the columnar epithelial tissue previously seen only in the embryo.
CDX1, CDX2, and Sox9 are the potential “master genes” that are common in intestinal tissue and routinely expressed in BE, but not found in normal esophageal squamous epithelium. In mouse studies, Cdx1 directs cells to become columnar and Cdx2 directs tissue to become intestinal. CDX1 expression in BE is exhibited by demethylation on the gene promoter, allowing for genetic freedom to initiate gene products. CDX1 expression is considered to precede CDX2 in squamous epithelium exposed to bile acids, yet both can induce each other’s promoter activity; and a higher level of CDX2 has a limiting effect on CDX1. CDX2 expression in BE tissue has been found to be 400 times higher than that of normal tissue samples. CDX2 expression also precedes the expression of intestinal markers, like mucin-2 (MUC2), where it is believed that hyper-expression of CDX2 leads to MUC2 expression in keratinocytes. The MUC2 protein is secreted by goblet cells in the colon and small intestine, and is part of a defensive mucous gel-like coating on the outer epithelial layer that serves to mitigate damage from microbes and toxins. Its presence in the esophagus is a strong indicator of metaplasia and columnar differentiation processes.
It has been found, however, that acid and bile salt exposure to normal biopsied esophageal squamous epithelial cells was found to not cause CDX2 mRNA expression in patients who only had GERD, whereas expression did occur in GERD patients that also had BE. Such a distinction may mean that there are variances among individuals, perhaps in their innate genetic configurations, that put others at a greater disposition for developing BE. Or it could mean that CDX2 expression is reliant on some other trigger that needs to be activated first. CDX gene expression may ultimately be induced by the acid compounds or by the subsequent inflammatory cytokines.
Sox9’s expression alone is enough to convert squamous epithelium to a columnar morphology that has cytokeratins specific to columnar differentiation. It is mediated by Sonic hedgehog (SHH) and is a product of expression by progenitor cells within the trough of the intestinal crypts. It also directs the formation of goblet cells and Paneth cells. Sox9 and Cdx2 expressed in conjunction produce the same effect on squamous epithelium as Sox9 by itself. The general signaling pathway occurs via SHH ligand expression upon exposure to acids and bile. SHH targets bone morphogenetic protein 4 (BMP4), which then acts on Sox9 genes. SHH and BMP4 are highly important factors in the development of the early columnar tissue in the embryo, so their role in BE sort of mirrors early development stages. On the other hand, Wingless integrated (Wnt) and Notch are involved with more specific differentiation of the embryological gut development. Their interplay leads to the development and maintenance of crypts and villi in the intestine. While a detailed analysis of all pathways and transcription factors implicated in BE is beyond the scope of this thesis, it is important to understand that a vast network of players is involved in metaplastic BE. Essentially, BE tissue differentiation is the confluence of the transcription factors shown in Table 1 that are normally expressed in regular intestinal tissue now playing a role in the esophagus, along with the developmental pathways of the embryo resurfacing in adulthood as a result of repeated acid and bile exposures.
Table 1. Summary of transcription factors and signaling pathways linked to metaplasia of squamous epithelium and development of Barrett’s Esophagus.
Transcription Factors & Signaling Description Activity Related to Barrett’s Esophagus
SOX9 (sex determining region Y-BOX-9) Expressed in fetal and adult stem and progenitor cells; has roles in cell fate and maintenance Expressed in embryonic esophageal columnar tissue; activated by SHH & BMP4; intestinalizes squamous tissue in esophagus
CDX1, CDX2 (caudal-type homeoboxes) Involved in intestinal development, cell differentiation, and maintaining intestinal phenotype into adulthood Activated by bile acids; upregulated in BE tissue to cause intestinalization; CDX1 leads to goblet cells, CDX2 leads to overall tissue intestinalization
Sonic Hedgehog (SHH) Highly involved in developmental processes in embryonic esophagus; in adults, it promotes stem cell expansion and tissue regeneration, expressed in adult gut and intestine for homeostasis Expressed in embryonic esophageal columnar tissue and adult BE
Bone Morphogenetic Protein 4 (BMP4) Determines cell fate; drives embryo columnar tissue to form squamous epithelium Expressed in embryonic esophageal columnar tissue and adult BE; target of SHH; expressed in stromal fibroblasts of lamina propria; activates expression of Sox9 in epithelial cells
NF-kB (nuclear factor kappa B) Largely related to inflammatory responses, pro- and anti-; also regulates genes involved with cell proliferation and survival by initiating anti-apoptotic pathway Activated by DCA; causes release of inflammatory cytokines IL-6 & IL-8; TNF-α release increased; imbues BE cells with apoptosis resistance
Notch Cell-cell communication for cellular differentiation; maintenance of stem cell populations; esophageal basal layer homeostasis Down-regulated upon bile salt exposure; inhibited by CDX2 in BE metaplasia, leading to goblet cell formation
Wnt (wingless integrated) Determines cell fate and positioning in embryo development; tissue regeneration in intestine; stem cell renewal Upregulated in BE and EAC tissue; acts on Notch pathway
Gastroesophageal Reflux Disease (GERD)
GERD is the condition of chronic reflux of the stomach contents and acids returning up through the lower esophageal sphincter and into the distal portion of the esophagus. The contents of the reflux mixture may include pepsin, gastric acid, water, conjugated bile salts, pancreatic enzymes, and food. Extreme cases may see reflux reach all the way to the beginning portion of the esophagus or into the larynx and lungs. All people in very small amounts experience some reflux every day, but it will usually not turn into a pathologic condition if the LES, diaphragm muscles, and stomach are anatomically functioning properly. These benign reflux episodes are 90% cleared by a secondary peristalsis wave and 10% cleared by saliva neutralizing the remaining substance; and tend not to pose a danger for heartburn or inflammation. Thus, 80% to 90% of reflux episodes go unnoticed or are asymptomatic. It can be considered, then, that GERD patients are categorized into two subgroups that have different clinical characteristics: 1) the erosive esophagitis (EE) group that experiences inflammation of the mucosal lining with chronic hypotensive LES; and 2), the non-erosive reflux disease (NERD) group that has the common heartburn symptom with pathological acid reflux but without evidence of mucosal damage on endoscopy.
Heartburn is the stimulation of chemoreceptors in the mucosa layer by gastric acid and bile salts where the pH is less than four; and it presents as the sensation of pain or burning in the thoracic heart region. NERD is estimated to comprise about 50% to 70% of the GERD population, and the pathogenic risks of going from profiles of NERD to EE to BE are assumed to be very low across all categories and is still under investigation. Essentially, once found to be in either of the three categories, the patient state will remain in that specific category for the remainder of their lifetime. The patient profile for both NERD and erosive esophagitis are similar in that the mean age at diagnosis is about 50, but a 60/40 split for women to men comprises the NERD group, while 59% of the EE group is men. EE patients also present with a higher body mass index and a higher prevalence of hiatal hernia.
It is when the episodes of reflux become more frequent and protracted (longer than five minutes) that the normal esophageal clearance mechanisms begin to lose their effectiveness and lead to esophagitis and BE. Unsurprisingly, comparing healthy individuals to NERD, esophagitis, and BE produces an escalating increase in the number of reflux events and longer exposure periods to acids less than pH 4.0 across the sequence. The persistence of acid in the range of pH 2.0 to 4.0 combined with pepsin induces damage to the mucosal lining, causing the presence of dilated intercellular spaces that are evidence of the first lesion. The dilated spaces between the cells allow for the penetration of acid deeper into the mucosal lining, which compromises the mucosal resistance to acid. Deeper acid penetration also activates or recruits more peripheral nociceptors to transmit pain signals. Esophageal damage is assessed according to the Los Angeles Classification of Esophagitis :
• Grade A: One (or more) mucosal break no longer than 5 mm that does not extend between the tops of two mucosal folds
• Grade B: One (or more) mucosal break more than 5 mm long that does not extend between the tops of two mucosal folds
• Grade C: One (or more) mucosal break that is continuous between the tops of two or more mucosal folds but which involve less than 75% of the circumference
• Grade D: One (or more) mucosal break which involves at least 75% of the esophageal circumference
The greatest chances for reflux occurring are 30 to 60 minutes after eating a meal, during bending over, lying down, and during sleep. Pain may also be caused by mechanical means like esophageal distention and peristalsis motility disorders. Numerous causes to the sensation of pain can obfuscate the true nature of GERD, though, and may mean a combination of anatomic and physiologic disturbances. Other symptoms from GERD may include dysphagia (difficulty swallowing), laryngitis, pharyngitis, asthma, chronic cough, or dental erosion. Reflux reaching all the way back up the esophagus and into the pharynx may cause inflammation in the pharyngeal tissue or erode teeth enamel; and if it escapes into the larynx it can irritate the vocal cords and trachea.
Malfunctioning of the LES plays a key role in the reflux of stomach fluid in the esophagus. Normally, the LES’s resting pressure is 15 mm Hg to 30 mm Hg greater than that of the intra-abdominal pressure and will adequately inhibit the reflux of stomach fluids. The pressure at the LES does not stay at a constant rate, though, and will differ depending on the body position, breathing, movement, and time of day; where it is low during the day and higher at nighttime. The LES pressure also falls under the influence to different outside compounds (Table 2) like medications, hormones, and food chemicals.
Table 2. Compounds that irritate conditions within the gastric chamber.
Foods Peppermint, chocolate, caffeine, alcohol, tobacco, citrus fruits and juices, spicy foods, raw onions, tomatoes
Hormones & Neurotransmitters Secretin, cholecystokinin, glucagon, progesterone, vasoactive intestinal polypeptide, gastric inhibitory polypeptide, serotonin, dopamine
Medications Nitrates, morphine, barbiturates, diazepam, calcium channel blockers, atropine, tricyclic antidepressants, ganglion blockers
A second line of control comes into play with the left and right crural muscles of the diaphragm, which connect to the LES via the phrenoesophageal ligament. These diaphragm muscles normally aid in preventing reflux during times of heavy lifting, pregnancy, and any other causes for increased abdominal pressure. Transient LES relaxations (TLESRs) are relaxations of the LES moderated by the vagus nerve and usually caused by the buildup of gastric gases or stomach distension, as in, they are unrelated to swallowing or peristalsis. These relaxations of the LES can last anywhere between 10 to 35 seconds and reduce the pressure of the LES to levels aligned with the gastric pressure. During TLESRs, the crural muscles of the diaphragm are inhibited, releasing pressure on the sphincter opening. TLESRs are more frequent in GERD by contributing to almost 90% of reflux episodes, and hiatal hernias exacerbate the problem even further by leading to increased sphincter openings. Hiatal hernias are when a portion of the stomach pushes upwards past the diaphragm. Ninety percent of hiatal hernias are Type 1, where the LES moves upwards and pushes through the diaphragm. Rarer forms involve the squeezing of the fundus portion of stomach past the diaphragm and results in a pouch conducive for lingering stomach acid. Type 1 causes a reduction of LES pressure and length, limiting acid clearance and increasing mucosa exposure to acid compounds. Obesity and weakening of the surrounding muscles are the main causes for hiatal hernias, with increasing age contributing to the weakening of muscle structures. Older people typically have a higher body mass index, as well. The phrenoesophageal ligaments may also lose elasticity with age and contribute to the formation of the hernias. When hiatal hernias exceed two centimeters, then the incidence of esophagitis and BE is higher.
Factors that can contribute to the development of GERD involve several modes of dysfunction. Peristaltic dysfunction hinders acid clearance from the distal esophagus and comes in the forms of failure for peristaltic contraction or low-amplitude contraction waves. Dysfunction in the emptying of the stomach can occur through gastroparesis, neuromuscular diseases, pyloric dysfunction, duodenal motility disorders, and even duodenogastric reflux (DGR). DGR is the reflux of bile from the duodenum back into the stomach. Some bile reflux is normal, due to proximity, but reflux may occur in larger quantities and mix with the contents of the stomach. Upon gastric reflux, the gastric acid and bile mixture travels up into the esophagus. DGR is found to play a role in inflammation and injury of the esophagus. In those with esophagitis, DGR is much more common and means bile acid along with gastric acid are compounding injury to the mucosal lining (Fig. 9). With patients exhibiting the Los Angeles esophagitis rating system from A-D in increasing mucosal breaks, DGR has been in found in 67, 68, 80, and 100% of patients in those categories, respectively. This finding establishes a clear link between the interplay of DGR, bile acid, and esophagitis leading up to metaplastic change. Other factors are Zollinger-Ellison syndrome (where acid is secreted in above normal amounts), ulcers or strictures that obstruct the gastric outlet, and connective tissue disorders like scleroderma.
Figure 9. Comparing acid-bile profiles among different GERD subgroups shows that having acid and bile together leads to greater risk for complicated BE, or the precancerous condition of dysplasia.
Stomach Acids & Bile
As has been mentioned previously, stomach acids and bile acids play a central role in the development of metaplastic BE tissue. Under normal conditions, stomach acids are pivotal to the breakdown of foods and the proper functioning of the digestive system. Our nervous system is highly sensitive to stimuli involving food, where the sight, smell, touch, and taste of food trigger the acid regulatory pathways in the stomach: acetylcholine, gastrin, and histamine. The gastric glands of the stomach lining regulate the acid pathways and produce numerous substances like mucus, gastrin, pepsinogen, histamine, and intrinsic factor. Mucus secreting cells are located superficially in the glands and are meant to protect the stomach lining from its very own acid through a bicarbonate buffer. The cells located deeper in the glands are parietal cells, which secrete hydrochloric acid (HCL) based on surface receptor activation. Those receptors are sensitive for histamine, acetylcholine, and gastrin; and will cause a flood of HCL that can lower the pH of the gastric fluid to 1 or less. Upon a stimulus, gastrin is released from G cells in the antrum of the stomach. The gastrin travels through the blood stream to gastrin receptors on parietal cells in the gastric body and fundus; and begins the chemical activation of proton pumps located on the opposite side of the cells. Parietal cells will also activate the HCL pathway through receptors for histamine and acetylcholine. Pepsinogen is released by chief cells and breaks down proteins. It becomes converted to pepsin upon exposure to low pH. If the pH becomes too low, gastrin production is shut down via a negative feedback loop, halting HCL secretion.
The scientific literature describes inflammation of the esophagus due to acid reflux that is a consequence of GERD. Continued exposure of the esophageal lining to HCL and pepsin begin the breakdown and inflammation of the mucosal lining. The pepsin will destroy proteins securing the tight junctions between cells, allowing hydrogen ions to reach deeper into the lining. Enhancing this process is that the esophagus simply lacks the necessary amount of mucus to negate the acid. Subsequent to a reflux episode, mass mitosis may occur to compensate for the cell deaths that acids cause on the esophageal lining. Basal cells may experience hyperplasia and inflammatory cells will be attracted to the area in vast quantities. They will release cytokines, or interleukins, that have an influence over the proliferation and regeneration of cells. Reactive oxygen species are also released by the immune cells that wreak havoc on cell membranes and clash with DNA to cause breaks and other deleterious effects.
Along with stomach acids, bile acids may become refluxed back into the stomach. Bile acids have received much scientific attention recently in relation to GERD and BE. It normally serves as a detergent compound that enables the absorption and transport of vitamins, nutrients, and lipids in the small intestine. Bile is an amalgamation of numerous different substances. Mainly, it is 95% water, but is also composed of bile acids, bilirubin, amino acids, steroids, heavy metals, medicine, and toxins. Bile serves to transport cellular waste from the body, like old red blood cells, hormones, and harmful lipophilic substances that cannot be excreted through urine. It also removes cholesterol from the body and helps protect against GI infection. Originating from cholesterol, the synthesis of bile in the liver is the primary pathway for cholesterol catabolism into amphipathic (hydrophobic & hydrophilic) molecules and serves to release up to half the body’s cholesterol. After being synthesized in the liver, bile collects in the gallbladder for temporary storage until food matter enters the duodenum. The food triggers the gallbladder to contract and release bile into the duodenum to combine with the food.
The current literature describes how increasing exposure to both stomach acid and bile on the esophagus is highly compounding of injury and inflammation. The composition of bile acid is roughly 40% cholic acid, 40% chenodeoxycholic acid (CDCA), and 20% deoxycholic acid (DCA). The DCA comes from bacterial conversion of cholic acid in the intestine, thus, making it a secondary bile acid. DCA is a hydrophobic compound that has destructive qualities towards the membranes of cells, causing randomized damage with increasing inflammation and severity based on concentration. Recent findings support that bile acids are a primary contributing factor in the development of BE, where they can exert greater influence than gastric acid alone. CDX2, Muc2, and BMP4 have been found in elevated levels in the esophageal cells of rats exposed to BA, and suggest that esophagitis that leads to metaplasia and dysplasia is dependent on BA. DCA has also been shown to act through an array of pathways and mechanisms, like stimulating ROS activity, which activates the nuclear factor kappa beta (NF-kB) pathway while also leading to DNA damage. This means that the response of intestinal cells and BE cells to DCA is incredibly different, despite similar morphologies. The NF-kB pathway is oriented towards ensuring cell survival and anti-apoptosis; properties that would enable the survival of BE tissue subjected to frequent reflux episodes. It is theorized that the metaplastic specialized tissue of BE has resistance to apoptosis so that the repairing of damaged DNA caused by reflux may occur. However, the anti-apoptotic quality has a caveat: it is also a factor in the development of EAC, where a mutation can be passed on to daughter cells.
Dysplasia
The literature has seen extensive studies on dysplasia. It often follows the initial metaplastic development of specialized intestinal epithelium and means an identifiable change in the structure of the cell. Dysplasia, in general, is defined as precancerous neoplastic epithelium growth that is isolated in the basement membrane of the associated gland. The observed change in cells is characterized by a larger cell nucleus, which helps to identify cytologic atypia. Termed hyperchromasia, the increased DNA within the nucleus allows for it to draw up more staining chemicals and the nucleus will appear darker under the microscope. The degree of the cytologic atypia is what can separate dysplasia into two subgroups of identification, low-grade and high-grade. Low-grade dysplasia presents as varying hyperchromasia, a small degree of nuclear clumping, and abnormal nuclear contours. The cells still exhibit polarity where the nucleus is oriented towards the basal end. Goblet cells are nominal compared to regular metaplastic BE tissue. On the other end, high-grade dysplastic cells feature more extreme atypia than low-grade and have more pronounced architecture abnormalities. The villiform surface of the tissue is much more evident with cribriform glands. The nuclei of the cells have increased hyperchromasia along with less cytoplasm space and loss of nuclear polarity. Abnormal mitosis of the cells at the epithelial surface occurs (when it usually does not), and the cells never completely mature as the surface cells highly resemble those in the bottom of the crypts. High-grade dysplasia is the immediate form of lesion before esophageal adenocarcinoma, yet the cells do not have the capability to penetrate deeper into the epithelial tissue layers or metastasize. Diagnosis of dysplasia requires endoscopy and histologic examination. Using narrow-band imaging during endoscopy, irregular vasculature and mucosal patterning abnormalities help to identify dysplastic tissue. Some cases of dysplasia are deemed indefinite if it is unclear whether the enlargement of nuclei and atypia are due to tissue repair. Even when having experience diagnosing lesions of dysplasia, consensus amongst doctors can be a toss-up in identifying histological tissue states, where, when shown three images of a biopsied lesion, pathologists voted: No dysplasia – 3, Indefinite – 3, Low-grade – 8, High-grade – 9, and invasive carcinoma – 1.
Adenocarcinoma
Esophageal adenocarcinoma arises through the long-term continued exposure of reflux acids to the esophageal lining. As mentioned previously, adenocarcinoma follows high-grade dysplasia and consists of varying hyperchromasia and irregularly shaped nuclei clumping together into undifferentiated cells. Generally, cancer cells and tumors display a shared list of characteristics that sets a base of understanding for any malignant lesion. Eight features have been identified as keys to the proliferation of cancer :
1. Continued cell signaling
2. Escaping growth-limiting factors
3. Resistance to apoptosis
4. Achieving cellular immortality
5. Activating blood vessel growth
6. Spreading locally and to distant segments of body
7. Conversion of energy metabolism for needs
8. Harmony with immune system
For EAC, the literature describes the accumulation of genetic and epigenetic alterations that lead to the development of the malignant tissue. The genetic changes found in BE tissue addresses somatic mutations that can be passed on to daughter cells due to the anti-apoptotic pathway of NF-kB. Loss of heterozygosity in BE has been found on tumor suppressor gene loci 17p for p53 and 9p for CDKN2A, meaning the loss of that gene’s corresponding allele on the other chromatid. p53 is intimately involved in the G1/S checkpoint and can call in for DNA repair or signal for apoptosis if repair is futile.
CDKN2A is part of the cell-cycle regulation dynamic. The mutation of p53 is known to be a later event compared to uncomplicated BE, where over two thirds of patients with high-grade dysplasia and EAC have been found to harbor mutation, usually seen with tetraploidy as the genome careens towards further dysfunction. Mutations to tumor suppressor genes have been found in nondysplastic BE patients, but conflicting data leave that development profile unclear while whole exome and genome research continues to unravel genome alterations.
The control of the genetic environment regulates the state of the chromatin and access to DNA sites where transcription factors and other influential proteins can position for activation. Epigenetic mechanisms involved in this control include :
• The methylation of cytosine bases in DNA sequences abundant in CG dinucleotides (CpG) – the use of sequencing points towards a general tendency for more hypomethylation of CpG sites in both BE and EAC, but hypermethylation also presents
• Posttranslational alteration to the histone packaging proteins
• MicroRNAs (miRNA) and noncoding RNA – miRNA 21 is upregulated in BE and EAC, 34 other differentially expressed miRNAs
• Nucleosome positioning
Figure 10. Different staging categories for the distinction of esophageal adenocarcinoma in surrounding mucosal layers.
EAC staging uses 2010 American Joint Committee on Cancer guidelines. Under these guidelines, high-grade dysplasia is classified as an in situ carcinoma, or Tis (Fig. 10). T1a category tumors are limited to the mucosal layers, with spreading to the third layer, the muscularis mucosa, the extent of the superficial tumor development. Also to note, at this stage the regional lymph system becomes a component for possible tumor interaction. A designation of N0 means no metastasis to the local lymph nodes, whereas an N1 means one to two nodes show tumor characteristics. Increasing from N1 through N3 means more regional lymph nodes are recruited into the metastasis of tumor tissue. T1b tumors have moved beyond the mucosal layers and into the submucosa. At T2 stage, the tumor is within the muscular layers, either the circular or longitudinal. T3 reaches the outermost adventitia and T4 is when the tumor has started to invade adjacent local tissues, like the aorta, diaphragm, vertebrae, or trachea. If the tumor reaches higher levels of N2, N3, or T4; the patient is at higher risk for M1 metastasis to distant regions of the body due to having advanced penetration into vessel network structures.
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