High Proportion of Nuclear Phenotype Identifies Aggressive Cutaneous Squamous Cell Carcinoma.

OBJECTIVE: To develop a quantitative histopathology algorithm to predict which patients with cutaneous squamous cell carcinoma (cSCC) were likely to experience recurrence or metastases. STUDY DESIGN: This retrospective study of cSCC lesions compared patients with aggressive disease (n = 40) and those with nonaggressive disease (n = 35). Based on a previous study using nuclear karyometry, we determined that aggressive lesions had a high proportion of a specific nuclear phenotype. The proportion of those nuclei was used to derive an aggressiveness score for each lesion. The mean age of patients was similar in both groups, as were the locations of index lesions. RESULTS: The mean aggressiveness scorefor cases with aggressive lesions was 0.60 ± 0.21 and was 0.28 ± 0.35 for those with nonaggressive lesions. The overall accuracy in properly characterizing lesions was 72%. The area under the receiver operating characteristic curve was 0.80 ± 0.05. In general, the aggressive nuclear phenotype is represented by elevated levels of chromatin clumps and short linear segments of dark chromatin/intense pixels. CONCLUSION: These data suggest that discriminant functions may be utilized to distinguish between aggressive and nonaggressive lesions at the time of diagnosis.

Novel diet-related mouse model of colon cancer parallels human colon cancer.

AIM: To investigate the close parallels between our novel diet-related mouse model of colon cancer and human colon cancer. METHODS: Twenty-two wild-type female mice (ages 6-8 wk) were fed the standard control diet (AIN-93G) and an additional 22 female mice (ages 6-8 wk) were fed the control diet supplemented with 0.2% deoxycholic acid [diet + deoxycholic acid (DOC)] for 10 mo. Tumors occurred in the colons of mice fed diet + DOC and showed progression to colon cancer [adenocarcinoma (AC)]. This progression is through the stages of tubular adenoma (TA), TA with high grade dysplasia or adenoma with sessile serrated morphology, intramucosal AC, AC stage T1, and AC stage T2. The mouse tumors were compared to human tumors at the same stages by histopathological analysis. Sections of the small and large intestines of mice and humans were evaluated for glandular architecture, cellular and nuclear morphology including cellular orientation, cellular and nuclear atypia, pleomorphism, mitotic activity, frequency of goblet cells, crypt architecture, ulceration, penetration of crypts through the muscularis mucosa and presence of malignant crypts in the muscularis propria. In addition, preserved colonic tissues from genetically similar male mice, obtained from a prior experiment, were analyzed by immunohistochemistry. The male mice had been fed the control diet or diet + DOC. Four molecular markers were evaluated: 8-OH-dG, DNA repair protein ERCC1, autophagy protein beclin-1 and the nuclear location of beta-catenin in the stem cell region of crypts. Also, male mice fed diet + DOC plus 0.007% chlorogenic acid (diet + DOC + CGA) were evaluated for ERCC1, beclin-1 and nuclear location of beta-catenin. RESULTS: Humans with high levels of diet-related DOC in their colons are at a substantially increased risk of developing colon cancer. The mice fed diet + DOC had levels of DOC in their colons comparable to that of humans on a high fat diet. The 22 mice without added DOC in their diet had no colonic tumors while 20 of the 22 mice (91%) fed diet + DOC developed colonic tumors. Furthermore, the tumors in 10 of these mice (45% of mice) included an adenocarcinoma. All mice were free of cancers of the small intestine. Histopathologically, the colonic tumor types in the mice were virtually identical to those in humans. In humans, characteristic aberrant changes in molecular markers can be detected both in field defects surrounding cancers (from which cancers arise) and within cancers. In the colonic tissues of mice fed diet + DOC similar changes in biomarkers appeared to occur. Thus, 8-OH-dG was increased, DNA repair protein ERCC1 was decreased, autophagy protein beclin-1 was increased and, in the stem cell region at the base of crypts there was substantial nuclear localization of beta-catenin as well as increased cytoplasmic beta-catenin. However, in mice fed diet + DOC + CGA (with reduced frequency of cancer) and evaluated for ERCC1, beclin-1, and beta-catenin in the stem cell region of crypts, mouse tissue showed amelioration of the aberrancies, suggesting that chlorogenic acid is protective at the molecular level against colon cancer. This is the first diet-related model of colon cancer that closely parallels human progression to colon cancer, both at the histomorphological level as well as in its molecular profile. CONCLUSION: The diet-related mouse model of colon cancer parallels progression to colon cancer in humans, and should be uniquely useful in model studies of prevention and therapeutics.

Karyometry of nuclear phenotypes in cutaneous squamous cell cancer.

OBJECTIVE: To establish the karyometric characteristics of the two main nuclear phenotypes in cutaneous squamous cell cancer (cSCC) lesions. STUDY DESIGN: The clinical materials comprised 75 cases of cSCC, 38 with aggressive lesions and 37 with nonaggressive lesions. High-resolution images of 100 nuclei per case were recorded. Data were partitioned into four subgroups covering the range of lesion progression. Four discriminant functions were derived to distinguish aggressive from nonaggressive lesions. The most typical nuclei from the phenotype predominant in aggressive lesions and nonaggressive lesions were separated out by thresholding on the discriminant function score axes. For these homogeneous sets of nuclei the karyometric features were computed. RESULTS: The nuclear populations in cSCC lesions are a very heterogeneous set. There are two axes of dispersion, along the line of lesion progression and between aggressive and nonaggressive lesions. The analysis faces the difficulty that lesions from both diagnostic categories contain nuclei of the same two phenotypes with the difference between categories consisting only of differences in proportion of the two phenotypes. CONCLUSION: The nuclei of the aggressive phenotype I and nonaggressive phenotype II have substantially different chromatin patterns and can be distinguished with > 90% correct recognition rate.

Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer.

BACKGROUND: Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations. PURPOSE: To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer. RESULTS: Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million. CONCLUSIONS: The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million crypts near cancers and TVAs suggests that the tumors arose in field defects that were deficient in DNA repair and that deficiencies in Pms2, Ercc1 and Xpf are early steps, often occurring together, in progression to colon cancer.

Nuclear morphometry identifies a distinct aggressive cellular phenotype in cutaneous squamous cell carcinoma.

By identifying aggressive cutaneous squamous cell carcinoma (cSCC) in patients who are at high risk for recurrences or second primaries after resection, intensive surveillance and therapy may decrease morbidity and mortality. We investigated the role of nuclear morphometry (karyometry) in differentiating between aggressive and nonaggressive cSCC. We retrospectively analyzed cSCC lesions from 40 male patients. Twenty-two patients had evidence of aggressive cSCC (local/regional recurrence or a second primary cSCC), and 18 patients were identified with similar ages and sites of disease as control patients with nonaggressive cSCC (no evidence of recurrence, metastasis, or second primary). We carried out karyometric analysis to identify nuclear features that discriminate between aggressive and nonaggressive cSCC nuclei. We used statistically significant differences (Kruskal-Wallis test, P < 0.0001) to compose a quantitative aggressive classification score (proportion of aggressive nuclei from 0% to 100%). For comparisons, we used Fisher's exact test or Student's t test. The mean age was 79 ± 7 years for aggressive cSCC and 80 ± 9 years for nonaggressive cSCC (P = 0.66). We analyzed a mean of 96 nuclei in each group. The mean classification score for aggressive cSCC was significantly higher (69% ± 6%) than for nonaggressive cSCC (28% ± 5%, P = 0.00002). Overall, the classification score accurately categorized 80% of our patients (P = 0.0004). In most patients, karyometry differentiated between aggressive and nonaggressive cSCC. We found that classification scores, which provide information on individual lesions, could be used for risk stratification.

Loss of heterozygosity at the glutathione peroxidase 1 locus is not an early event in colon carcinogenesis.

It has been previously shown that loss of heterozygosity (LOH) at the cytosolic glutathione peroxidase (GPx-1) locus is a common event in the development of several cancer types, including colorectal cancer. GPx-1 is an antioxidant selenium-containing protein, and polymorphisms within this gene have been shown to be associated with the increased risk of cancer. In order to assess whether this genetic change was an early or late event in colon cancer development, we investigated whether LOH at this site was occurring in colorectal adenomas, a premalignant lesion. Twenty-four pairs of DNA samples, obtained from both whole-blood and adenoma tissue from the same individuals, were genotyped at 2 positions in the GPx-1 gene: a codon 198 variation resulting in either a leucine or proline at the corresponding position in the peptide, or a variable number of alanine repeat codons corresponding to the amino terminus of the GPx-1 protein. No evidence of GPx-1 LOH was observed in the examined sample sets. These data indicate that the genetic loss at the GPx-1 locus may be a late event in colon carcinogenesis.

Prognostic criteria for squamous cell cancer of the skin.

BACKGROUND: Non-well-differentiated cutaneous squamous cell carcinomas may display a more aggressive behavior. It is important to better define prognostic criteria for these tumors. METHODS: This was a retrospective case-control analysis of a squamous cell carcinoma database. Patients with non-well-differentiated and well-differentiated tumors were matched based on site of tumor, age, and immunocompromised status. Comparisons included demographics, histology, immunohistochemical protein expressions (Ki-67, p53, E-cadherin, cyclin D1), and clinical outcomes. RESULTS: Demographic features were similar between cases (n=30) and controls (n=30). Non-well-differentiated tumors were larger (1.8 cm versus 1.3 cm, P=0.08), deeper (0.81 cm versus 0.32 cm, P<0.0001), and had greater recurrence (P=0.003). Non-well-differentiated tumors showed increased proliferation rate, Ki-67 index (77% versus 61%, P=0.001); no significant difference in activity of p53, E-cadherin, and cyclin D1 between the two groups. CONCLUSIONS: Tumor differentiation and depth are important pathologic and prognostic criteria for cutaneous squamous cell carcinoma. Immunohistochemistry helps describe patterns of biomarker protein expression and may exemplify aggressive subtypes.

Progression of skin lesions from normal skin to squamous cell carcinoma.

OBJECTIVE: To assess the changes in the nuclear chromatin pattern concomitant with progressive sun damage in skin biopsies ranging from sun-exposed, normal-appearing skin to squamous cell carcinoma (SCC). STUDY DESIGN: Biopsies were taken from 140 cases with sun-exposed but histopathologically normal skin, from 20 cases visually assessed as pre-actinic keratosis (pre-AK) or early AK, from 30 cases of AK, and from 21 cases of SCC. A total of 21,094 nuclei were recorded from these biopsies. High-resolution digital imagery was recorded, and features descriptive of the nuclear chromatin pattern were computed. Both supervised learning and unsupervised learning algorithms were employed to derive progression plots. RESULTS: With increased sun exposure, the proportion of nuclei exhibiting changes in the nuclear chromatin pattern rises notably. Using karyometry, no significant differences could be substantiated between nuclei collected from early AK sites and AK lesions. Cases of SCC fell into 2 distinct groups. A larger group (approximately 66.7% of cases) had characteristics similar to AK. A smaller group (approximately 33.3% of cases) represented much more progressed lesions. CONCLUSION: Karyometric assessment can provide a numeric measure of progression for sun damage and of the deviation from normal in both AK and SCC lesions.

Regulation of deoxycholate induction of CXCL8 by the adenomatous polyposis coli gene in colorectal cancer.

Elevated deoxycholic acid (DCA), mutations in the adenomatous polyposis coli (APC) gene and chronic inflammation are associated with increased risk of colorectal cancer. APC status was manipulated to determine whether DCA mediates inflammatory molecules in normal or initiated colonic mucosa. DCA increased steady state mRNA and protein levels of CXCL8 in cells which do not express wild-type APC. Steady-state CXCL8 mRNA and protein were suppressed when cells with conditional expression of wild-type APC were exposed to DCA. Immunostaining did not detect CXCL8 in normal human colonic mucosa. CXCL8 was expressed in adenomatous polyps and adenocarcinomas. CXCL8 expression correlated with nuclear beta-catenin localization in epithelial cells of adenomas, but was associated with endothelial cells and neutrophils in the adenocarcinomas. DCA-mediated CXCL8 promoter-reporter activity was elevated in a mutant APC background. Wild-type APC suppressed this effect. Mutation of activator protein-1 (AP-1) or nuclear factor kappa B (NF-kappaB) sites suppressed the activation of the CXCL8 promoter-reporter by DCA. Chromatin immunoprecipitation revealed that AP-1 and NF-kappaB binding to the 5'-promoter of CXCL8 was induced by DCA. The beta-catenin transcription factor was bound to the 5'-promoter of CXCL8 in the absence or presence of DCA. Phenotypic assays determined that DCA-mediated invasion was blocked by antibody-directed against CXCL8 or wild-type APC. CXCL8 exposure led to matrix metalloproteinase-2 production and increased invasion on laminin-coated filters. These data suggest that DCA-mediated CXCL8 occurs in initiated colonic epithelium and neutralizing CXCL8 could reduce the invasive potential of tumors.

Sleep and non-invasive ventilation in patients with chronic respiratory insufficiency.

OBJECTIVE: Noninvasive ventilation with pressure support (NIV-PS) therapy can augment ventilation; however, such therapy is fixed and may not adapt to varied patient needs. We tested the hypothesis that in patients with chronic respiratory insufficiency, a newer mode of ventilation [averaged volume assured pressure support (AVAPS)] and lateral decubitus position were associated with better sleep efficiency than NIV-PS and supine position. Our secondary aim was to assess the effect of mode of ventilation, body position, and sleep-wakefulness state on minute ventilation (V(E)) in the same patients. DESIGN: Single-blind, randomized, cross-over, prospective study. SETTING: Academic institution. PATIENTS AND PARTICIPANTS: Twenty-eight patients. INTERVENTIONS: NIV-PS or AVAPS therapy. MEASUREMENTS AND RESULTS: Three sleep studies were performed in each patient; prescription validation night, AVAPS or NIV-PS, and crossover to alternate mode. Sleep was not different between AVAPS and NIV-PS. Supine body position was associated with worse sleep efficiency than lateral decubitus position (77.9 +/- 22.9 and 85.2 +/- 10.5%; P = 0.04). V(E) was lower during stage 2 NREM and REM sleep than during wakefulness (P < 0.0001); was lower during NIV-PS than AVAPS (P = 0.029); tended to be lower with greater body mass index (P = 0.07), but was not influenced by body position. CONCLUSIONS: In patients with chronic respiratory insufficiency, supine position was associated with worse sleep efficiency than the lateral decubitus position. AVAPS was comparable to NIV-PS therapy with regard to sleep, but statistically greater V(E) during AVAPS than NIV-PS of unclear significance was observed. V(E) was determined by sleep-wakefulness state, body mass index, and mode of therapy.

Actinic damage in histopathologically normal skin.

OBJECTIVE: To establish measures of sun damage in histopathologically normal skin. STUDY DESIGN: Biopsies were taken from the upper inner arm, representing skin with presumably minimum sun exposure, from skin of the forearm with no visible sun damage, from skin of the forearm with visible sun damage and from normal-appearing skin from the forearm of individuals who had sun exposure that had resulted in actinic keratosis (AK) lesions. In addition, a data set of nuclei from AKs was recorded. RESULTS: In histopathologically normal skin, monotonically increasing damage was observed in individuals with increased exposure to solar radiation. CONCLUSION: Karyometry can detect and statistically secure changes in skin due to solar exposure at a stage at which the skin is histopathologically determined to be normal.

Zone of traction injury of the common peroneal nerve.

The history of functional motor recovery after reconstruction of traction injury to the common peroneal nerve is poor, regardless of technique and regardless of the experience of the surgeon doing the reconstruction. The hypothesis tested is that the failure of functional motor recovery after common peroneal nerve traction injury is because the zone of injury extends beyond the visible region of peroneal nerve in continuity injury and into the muscle entry zone of the motor nerve terminations. The opportunity arose to examine pathologically this suspected distal zone of injury in one patient. Histology from this patient was compared with that from a similar zone in a leg amputated for vascular indications. With a peroneal traction injury severe enough to cause disruption of the nerve, histology using Masson trichrome stain for collagen, neurofilament and S-100 stain for nerve fibers and Schwann cells demonstrated collagen deposition between the peroneal nerve and the muscle. This fibrosis was not seen in the in the same location from the amputation specimen. These findings were corroborated by electron microscopy of the myoneural junction in both specimens. It is concluded that stretch/traction injury zone extends into the myoneural junction, preventing otherwise successful neural regeneration through nerve grafts to reinnervate muscle. This suggests that with a distal zone of injury extending into the myoneural junction region, peroneal motor function may be better achieved by direct neurotization than with nerve grafting.

MGMT promoter methylation and field defect in sporadic colorectal cancer.

BACKGROUND: Sporadic colorectal cancers often arise from a region of cells characterized by a "field defect" that has not been well defined molecularly. DNA methylation has been proposed as a candidate mediator of this field defect. The DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is frequently methylated in colorectal cancer. We hypothesized that MGMT methylation could be one of the mediators of field cancerization in the colon mucosa. METHODS: We studied MGMT promoter methylation by three different bisulfite-based techniques in tumor, adjacent mucosa, and non-adjacent mucosa from 95 colorectal cancer patients and in colon mucosa from 33 subjects with no evidence of cancer. Statistical tests were two-sided. RESULTS: MGMT promoter methylation was present in 46% of the tumors. Patients whose cancer had MGMT promoter methylation also had substantial MGMT promoter methylation in apparently normal adjacent mucosa. This methylation was seen with a quantitative assay in 50% (22/44; 95% confidence interval [CI] = 34% to 65%) of normal samples with MGMT promoter methylation in the adjacent tumors, 6% (3/51; 95% CI = 1% to 16%) of samples without MGMT methylation in adjacent tumors, and 12% (4/33; 95% CI = 3% to 28%) of control samples (P < .001 for comparison between each of the latter two groups and the first group). MGMT methylation was detected with a more sensitive assay in 94%, 34%, and 27% of these samples, respectively (P < .001). In grossly normal colonic mucosa of colon cancer patients, methylation was detected 10 cm away from the tumor in 10 of 13 cases. Tumors with MGMT promoter methylation had a higher rate of G-to-A mutation in the KRAS oncogene than tumors without MGMT promoter methylation (10/42 versus 3/46, P = .03). Using a sensitive mutant allele-specific amplification assay for KRAS mutations, we also found KRAS mutations in 12% (3/25; 95% CI = 2.5% to 31%) of colorectal mucosas with detectable MGMT methylation and 3% (2/64; 95% CI = 0.4% to 11%) of colorectal mucosas without MGMT methylation (P = .13). CONCLUSION: Some colorectal cancers arise from a field defect defined by epigenetic inactivation of MGMT. Detection of this abnormality may ultimately be useful in risk assessment for colorectal cancer.

Identification of a stem cell candidate in the normal human prostate gland.

Stem cells of the human prostate gland have not yet been identified utilizing a structural biomarker. We have discovered a new prostatic epithelial cell phenotype-expressing cytokeratin 6a (Ck6a+ cells). The Ck6a+ cells are present within a specialized niche in the basal cell compartment in fetal, juvenile and adult prostate tissue, and within the stem cell-enriched urogenital sinus. In adult normal prostate tissue, the average abundance of Ck6a+ cells was 4.9%. With proliferative stimuli in the prostate organ culture model, in which the epithelial-stromal interaction was maintained, a remarkable increase of Ck6a expression was noticed to up to 64.9%. The difference in cytokeratin 6a expression between the normal adult prostate and the prostate organ culture model was statistically significant (p<0.0001). Within the prostate organ culture model the increase of cytokeratin 6a-expressing cells significantly correlated with increased proliferation index (r = 0.7616, p = 0.0467). The Ck6a+ cells were capable of differentiation as indicated by their expression of luminal cell markers such as ZO-1 and prostate specific antigen (PSA). Our data indicate that Ck6a+ cells represent a prostatic epithelial stem cell candidate possessing high potential for proliferation and differentiation. Since the development of benign prostatic hyperplasia and prostate carcinogenesis are disorders of proliferation and differentiation, the Ck6a+ cells may represent a major element in the development of these diseases.

Mucin-rich variant of salivary duct carcinoma: a clinicopathologic and immunohistochemical study of four cases.

Salivary duct carcinoma is a relatively uncommon aggressive neoplasm, typically found in the parotid glands of older men. The histologic appearance is that of an in situ and invasive high-grade adenocarcinoma, and it closely resembles ductal carcinoma of the breast. Several variants of the latter are very well known, but only papillary, sarcomatoid, and low-grade subtypes have so far been reported in salivary duct carcinoma. This study describes the clinicopathologic and immunohistochemical findings in four examples of an additional previously undescribed variant, rich in mucin. Each tumor showed areas of typical salivary duct carcinoma, but in addition there were lakes of epithelial mucin-containing malignant cells, i.e., mucinous (colloid) carcinoma. All four tumors expressed androgen receptors, cytokeratins, epithelial membrane antigen, gross cystic disease fluid protein-15, and carcinoembryonic antigen, but S-100 protein, other myoepithelial markers, and estrogen and progesterone receptors were negative. The mucin antigen profile showed positivity for MUC2, MUC5B, and MUC6 in all cases but only rare staining with MUC5AC and MUC7. Strong immunohistochemical overexpression of HER2/neu was demonstrated in one tumor, together with amplification by fluorescence in situ hybridization; another case was weakly positive with just one antiserum, but the remaining two tumors were completely negative. Small quantities of mucin have often been described in salivary duct carcinoma but not large extracellular mucinous lakes, which though prominent in the present series, were not as extensive as in mucinous adenocarcinoma. The relatively poor clinical outcome of the patients in our study mirrored that seen in usual-type salivary duct carcinoma and emphasizes the importance of differentiating mucin-rich salivary duct carcinoma from pure mucinous (colloid) adenocarcinoma, a tumor not fully defined, but possibly with a better prognosis.

Cardiac Wegener's granulomatosis masquerading as left atrial myxoma.

A 56-year-old woman was referred with mitral regurgitation, left ventricular dysfunction, and a sessile mass on the anterior leaflet of her mitral valve. The initial impression from echocardiography was that she had a left atrial myxoma. At operation, we found an intense inflammatory process diagnosed as Wegener's granulomatosis. It also involved the aortic valve and contiguous myocardium.