Henry D. Appelman

Aldehyde Dehydrogenase 1 Is a Marker for Normal and Malignant Human Colonic Stem Cells (SC) and Tracks SC Overpopulation during Colon Tumorigenesis

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted, mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC), investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly, aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1(+) cells are sparse and limited to the normal crypt bottom, where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma, ALDH1(+) cells increased in number and became distributed farther up the crypt. CD133(+) and CD44(+) cells, which are more numerous and broadly distributed in normal crypts, showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic-severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor(-) cells did not), (b) generated them after implanting as few as 25 cells, and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44(+) or CD133(+) serially) only modestly increased enrichment based on tumor-initiating ability. Thus, ALDH1 seems to be a specific marker for identifying, isolating, and tracking human colonic SC during CRC development. These findings also support our original hypothesis, derived previously from mathematical modeling of crypt dynamics, that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development.

Achalasia: A morphologic study of 42 resected specimens

Achalasia is characterized by failure of relaxation of the lower esophageal sphincter and absence of progressive peristalsis in the esophageal body. Few data are available regarding the morphologic features of achalasia, in particular its histologic progression. The esophagi of 42 patients with achalasia treated with total thoracic esophagectomy were examined histologically in order to systematically identify morphologic features of clinically unresponsive achalasia and to determine what could be learned about the diseases evolution. In all cases, myenteric ganglion cells within the esophageal body were markedly diminished, with 20 specimens having none. Twenty specimens had residual ganglion cells in the proximal esophagus, and 15 specimens had a few randomly distributed ganglion cells in the mid- and distal portions of the esophagus. Inflammation within myenteric nerves, present in all cases, generally consisted of a mixture of lymphocytes and eosinophils, occasionally with plasma and mast cells. Focal replacement of myenteric nerves by collagen occurred in all cases, and there was almost complete replacement in several cases. Actual destruction of the residual ganglion cells was not seen. The resected esophagi also shared extramyenteric morphologic features. Some features probably stemmed from physiologic obstruction, such as muscular hypertrophy, mainly of the muscularis propria (all cases), with secondary degeneration and fibrosis (29 cases), and eosinophilia of the muscularis propria (22 cases). Other changes, probably resulting from chronic stasis of ingested materials in the lumen, included diffuse squamous hyperplasia (all cases), lymphocytic mucosal esophagitis (28 cases), lymphocytic inflammation of the lamina propria and submucosa with prominent germinal centers (all cases), and submucosal periductal or glandular inflammation with complete loss of submucosal glands in half of the cases. One patient had high-grade squamous dysplasia, and another had superficially invasive squamous cell carcinoma. A third group of changes was probably due to previous esophagomyotomy, including abnormal gastroesophageal reflux, as shown by pH reflux testing (13 cases) and Barretts mucosa (four cases). In one case of Barretts there was low-grade dysplasia. Clinically unresponsive, surgically resected achalasia has almost total loss of ganglion cells, and widespread destruction of myenteric nerves has already occurred. The only active component is myenteric inflammation. However, it cannot be determined whether this inflammation is a manifestation of ongoing nerve destruction or whether it is a secondary phenomenon.

Microallelotyping defines the sequence and tempo of allelic losses at tumour suppressor gene loci during colorectal cancer progression.

Microallelotyping of many regions from individual colorectal tumours was used to determine the sequence and tempo of alleiic loss on 5q, 17p and 18q during neoplastic progression. No alleiic losses were found in normal tissues surrounding colorectal neoplasms, but losses occurred abruptly on 5q at the transition from normal colonic epithelium to the benign adenoma, and on 17p at the transition from adenoma to carcinoma, indicating an essential role for these losses in tumour progression. Alleiic losses were uniform throughout extensively microdissected benign adenomas and carcinomas. However, substantial alleiic heterogeneity was found in high–grade dysplasia, the transition lesion between adenoma and carcinoma. Thus, alleiic losses on 5q and 17p are associated with abrupt waves of clonal neoplastic expansion, and high–grade dysplasia is characterized by a high degree of alleiic heterogeneity.

Clinical, Epidemiologic and Morphologic Comparison Between Adenocarcinomas Arising in Barrett's Esophageal Mucosa and in the Gastric Cardia

Forty-nine cases of surgically resected adenocarcinomas of the gastric cardia and 23 cases of resected adenocarcinomas arising in Barretts columnar lined lower esophagus were compared histologically and clinically. Morphologically, the two groups were almost identical in terms of pattern of growth (expansile or infiltrative), degree of differentiation, and extent of spread at the time of operation. These similarities are not surprising, as the two carcinomas arise close to each other from almost identical mucosas. The major histologic difference was the finding of much more frequent dysplasia in the adjacent Barretts mucosa than in the surrounding cardiac mucosa, probably a reflection of the larger surface area covered by Barretts than by cardiac mucosa. The groups differed in certain epidemiologic parameters that possibly reflected independent carcinogenetic mechanisms. Thus, although the age ranges and median ages were the same, there was a higher male predisposition among the patients with cardiac carcinoma. In addition, patients with cardiac carcinoma had a significantly higher history of heavy smoking and history of alcohol intake, whereas those with Barretts carcinoma were much more likely to have hiatal hernias.

Histopathology differentiates acute self-limited colitis from ulcerative colitis

Acute self-limited colitis (ASLC) must be distinguished from chronic ulcerative colitis (CUC) for the proper early management of patients with the acute onset of bloody diarrhea. This study was undertaken to determine if any clinical, endoscopic, microbiologic, or histologic parameters can be used to make this distinction reliably and quickly. Forty-eight patients with ASLC, 36 patients with chronic ulcerative colitis during their first attack [CUC(F)], and 84 patients with recurrent flares of chronic ulcerative colitis [CUC(R)] were studied prospectively. The presence of fever (temperature greater than 100 degrees F), abdominal pain, or the time from onset of bloody diarrhea to presentation were not discriminatory. Overall clinical and endoscopic severity were identical among the three groups. Microbiologic studies identified an infectious agent in only 42% of patients with ASLC. Histopathologic features always distinguished patients with CUC from those with ASLC. No case of ASLC was misdiagnosed histologically as CUC or vice versa. Plasmacytosis in the lamina propria extending to the mucosal base and mucosal distortion were present in all cases of CUC(F) and CUC(R), but were absent in all cases of ASLC. The finding of focal cryptitis during the resolving phase of ASLC could be confused with similar lesions in biopsy specimens from patients with Crohns disease and mandates clinical follow-up. Histopathology is thus the only reliable diagnostic tool for the rapid differentiation of ASLC from CUC. However, biopsy specimens are only diagnostic when obtained during the acute phase of illness; that is, usually within the first 4 days from the onset of symptoms.

Aldehyde Dehydrogenase–Expressing Colon Stem Cells Contribute to Tumorigenesis in the Transition from Colitis to Cancer

Patients with chronic ulcerative colitis are at increased risk of developing colorectal cancer. Although current hypotheses suggest that sporadic colorectal cancer is due to inability to control cancer stem cells, the cancer stem cell hypothesis has not yet been validated in colitis-associated cancer. Furthermore, the identification of the colitis to cancer transition is challenging. We recently showed that epithelial cells with the increased expression of aldehyde dehydrogenase in sporadic colon cancer correlate closely with tumor-initiating ability. We sought to determine whether ALDH can be used as a marker to isolate tumor-initiating populations from patients with chronic ulcerative colitis. We used fluorescence-activated cell sorting to identify precursor colon cancer stem cells from colitis patients and report both their transition to cancerous stem cells in xenografting studies as well as their ability to generate spheres in vitro. Similar to sporadic colon cancer, these colitis-derived tumors were capable of propagation as sphere cultures. However, unlike the origins of sporadic colon cancer, the primary colitic tissues did not express any histologic evidence of dysplasia. To elucidate a potential mechanism for our findings, we compared the stroma of these different environments and determined that at least one paracrine factor is up-regulated in the inflammatory and malignant stroma compared with resting, normal stroma. These data link colitis and cancer identifying potential tumor-initiating cells from colitic patients, suggesting that sphere and/or xenograft formation will be useful to survey colitic patients at risk of developing cancer.

Loss of CDX2 Expression and Microsatellite Instability Are Prominent Features of Large Cell Minimally Differentiated Carcinomas of the Colon

Most large bowel cancers are moderately to well-differentiated adenocarcinomas comprised chiefly or entirely of glands lined by tall columnar cells. We have identified a subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance that we term large cell minimally differentiated carcinomas (LCMDCs). These tumors likely include a group of poorly differentiated carcinomas previously described by others as medullary adenocarcinomas. To better understand the pathogenesis of these uncommon neoplasms, we compared molecular features of 15 LCMDCs to those present in 25 differentiated adenocarcinomas (DACs) of the colon. Tumors were examined for alterations commonly seen in typical colorectal carcinomas, including increased p53 and beta-catenin immunoreactivity, K-ras gene mutations, microsatellite instability, and loss of heterozygosity of markers on chromosomes 5q, 17p, and 18q. In addition, tumors were evaluated by immunohistochemistry for CDX2, a homeobox protein whose expression in normal adult tissues is restricted to intestinal and colonic epithelium. Markedly reduced or absent CDX2 expression was noted in 13 of 15 (87%) LCMDCs, whereas only 1 of the 25 (4%) DACs showed reduced CDX2 expression (P < 0.001). Nine of 15 (60%) LCMDCs had the high-frequency microsatellite instability phenotype, but only 2 of 25 (8%) DACs had the high-frequency microsatellite instability phenotype (P = 0.002). Our findings provide support for the hypothesis that the molecular pathogenesis of LCMDCs is distinct from that of most DACs. CDX2 alterations and DNA mismatch repair defects have particularly prominent roles in the development of LCMDCs.

Ulcerative Colitis: Patterns of involvement in colorectal biopsies and changes with time

Chronic inflammation, both endoscopic and histologic, in a contiguous and symmetric distribution is said to be important in distinguishing ulcerative colitis (UC) from Crohns disease. Little is known whether this rule holds during the course of the disease and whether endoscopic/histologic correlation persists. In this study, we analyzed histologic patterns of UC in sequential sets of biopsy specimens to assess whether endoscopic and histologic findings correlate with time and treatment and to see whether distribution changes. Two hundred seventeen sets of colorectal biopsy specimens from 797 sites from 41 patients with clinical UC were studied and correlated with endoscopic findings. Each biopsy specimen was classified as definite or suspicious for chronic colitis or normal. Two histologic patterns of disease were identified: (1) diffuse, when all areas in all pieces from a biopsy segment had clear-cut colitis and (2) nondiffuse, when not all pieces were involved or single pieces had disease and normal mucosa both. Of 41 patients, the maximal extent of histologic disease was pancolitis in 30; 25 had less extensive disease at some point in the course. The maximal extent was left-sided in eight patients, seven of whom had less extent at some point. Of the three patients in whom the maximal extent was proctosigmoiditis, in one the inflammation disappeared. Seventy percent of the biopsy sites had diffuse patterns and 30% had nondiffuse. Histologic and endoscopic disease reverted to normal in 22 and 24 of 41 patients, respectively. Endoscopic and histologic findings were similar in 65% of the biopsy sites. Our results indicate that in long-standing UC (1) histologic disease may revert to normal mucosa, (2) because endoscopy alone may be insufficient to identify the mucosa as normal, biopsies should also be performed on the endoscopically normal mucosa, (3) the full extent of UC often is not established by a single set of biopsies, and (4) nondiffuse chronic inflammation and rectal sparing occurs in UC and are not necessarily markers of Crohns disease.

The histopathologic spectrum of acute self-limited colitis (acute infectious-type colitis)

Acute self-limited colitis (ASLC) is a self-limiting diarrheal illness which is often caused by known infectious agents (Campylobacter, Salmonella, and Shigella), but many cases are of unknown etiology. This report describes the histopathologic features of acute self-limited colitis as related to its natural history. The extent of inflammation and regeneration varies with the duration of the disease. In the peak activity stage (within 0–4 days of onset of bloody diarrhea) there is mucosal edema, cryptitis, crypt ulcers, and abcesses. At the time of resolution (within 6–9 days of onset of bloody diarrhea), regenerative features become apparent along with residual focal neutrophilic cryptitis. In the later stages of resolution, along with some regenerative features, occasional crypts with transmigrating lymphocytes may be present.A rectal biopsy is diagnostic only in the early stages of the disease. Later in the course, the rectal biopsy from patients with ASLC may be nondiagnostic or may be confused with Crohns disease due to the persistence of focal cryptitis. In our experience, the presence of crypt distortion and basal plasmacytosis are the two most useful criteria to differentiate chronic ulcerative colitis from ASLC.

Sofosbuvir and Daclatasvir Combination Therapy in a Liver Transplant Recipient With Severe Recurrent Cholestatic Hepatitis C

Recurrent HCV infection following liver transplantation can lead to accelerated allograft injury that is difficult to treat with interferon. The aim of this study is to describe the first ever use of an interferon‐free, all oral regimen in a liver transplant recipient with severe recurrent HCV. A 54‐year‐old male with HCV genotype 1b developed severe cholestatic HCV at 6 months posttransplant with ascites, AST 503 IU/mL, alkaline phosphatase of 298 IU/mL, HCV RNA of 12 000 000 IU/mL, and histological cholestasis with pericellular fibrosis. Sofosbuvir, an HCV polymerase inhibitor (400 mg/day), and daclatasvir, an HCV NS5A replication complex inhibitor (60 mg/day), were co‐administered for 24 weeks. Within 4 weeks of initiating treatment, serum HCV RNA levels became undetectable and liver biochemistries normalized with concomitant resolution of ascites. The patient achieved a sustained virological response with undetectable HCV RNA at 9 months posttreatment. During and following treatment, the daily dose and blood level of tacrolimus remained stable and unchanged. The rapid and sustained suppression of HCV replication in this liver transplant recipient provides great promise for the use of combination oral antiviral regimens in other immunosuppressed and interferon refractory HCV patients.