Feulgen DNA cytophotometry in histologic sections of mammary neoplasms

Because of a lack of suitable archival material, it is rarely possible to make retrospective studies of the correlation between the prognosis for a patient with mammary carcinoma and the distribution of nuclear DNA in the cells of the neoplasm. An investigation of the possibility of using sections cut from paraffin-embedded specimens showed that such sections are not suitable for use in retrospective studies of breast carcinoma. Because of such factors as the heterogeneity in size and shape of the nuclei from neoplastic cells and their tendency to mold around each other, determinations of DNA content of cells in sections were extremely difficult; in this particular carcinoma it was found that the distribution of nuclear DNA as obtained from a Feulgen-stained histologic section was not the same as that obtained from a Feulgen-stained imprint smear, and some polyploid tumors were erroneously classified as aneuploid.     

Nuclear DNA content as an indicator of chemosensitivity in small-cell carcinoma of the lung

The relationship between the nuclear DNA histogram pattern of tumor cells obtained by bronchoscopic brushing and the response to combination chemotherapy (Cytoxan + Adriamycin + Vincristine) was studied in 28 patients with small-cell carcinoma of the lung. Microspectrophotometric analysis of the tumor cells showed a near-diploid nuclear DNA pattern in 18 patients and a hyperdiploid pattern in 10 patients. Eight of the ten patients with the hyperdiploid pattern showed a good response (complete or partial response) to the chemotherapy. However, of the 18 patients with the near-diploid DNA pattern, only 2 displayed a good response; the remaining 16 patients exhibited no response. The hyperdiploid DNA pattern of tumor cells was thus associated with a better response to chemotherapy than was the near-diploid pattern. These results indicate that the nuclear DNA histogram pattern may be an indicator for predicting the degree of response to chemotherapy in small-cell carcinoma of the lung.     

Nuclear DNA cytophotometry in prostate carcinoma

Eighty patients with prostate carcinoma underwent fine-needle aspiration biopsy for cytologic grading and DNA-single-cell fluorescence photometry before and at 6-month intervals after endocrine treatment. The histograms of DNA values showed single peaks and bimodal and scattered distributions which correlated to the different tumor grades before therapy. The DNA values were significantly different from the controls with benign prostatic hypertrophy. After start of therapy, regressive changes of the DNA-histograms were increases of diploid and hypodiploid DNA values and disappearance of secondary peaks. Progressive changes were increased scattering of DNA values and appearance of secondary peaks. Progressive changes in the histograms were closely related to clinical remission and stable disease, but related poorly to clinical progression. The survival correlated with the pretherapeutic DNA-histograms and with the DNA-median, third-quartile, and maximum parameters.     

Integrated analyses of copy number variations and gene differential expression in lung squamous-cell carcinoma

Background

Although numerous efforts have been made, the pathogenesis underlying lung squamous-cell carcinoma (SCC) remains unclear. This study aimed to identify the CNV-driven genes by an integrated analysis of both the gene differential expression and copy number variation (CNV).

Results

A higher burden of the CNVs was found in 10–50 kb length. The 16 CNV-driven genes mainly located in chr 1 and chr 3 were enriched in immune response [e.g. complement factor H (CFH) and Fc fragment of IgG, low affinity IIIa, receptor (FCGR3A)], starch and sucrose metabolism [e.g. amylase alpha 2A (AMY2A)]. Furthermore, 38 TFs were screened for the 9 CNV-driven genes and then the regulatory network was constructed, in which the GATA-binding factor 1, 2, and 3 (GATA1, GATA2, GATA3) jointly regulated the expression of TP63.

Conclusions

The above CNV-driven genes might be potential contributors to the development of lung SCC.     

Nuclear DNA content, cytomorphologic features and clinical characteristics of small cell carcinoma of the lung

The relationship between the cytomorphologic features, the nuclear DNA patterns and the clinical prognosis of patients with small cell carcinoma of the lung was studied. In cases in the long-survival group (greater than or equal to 24 months), bronchial brushing smears contained a relatively high frequency of nuclei with large, irregular shapes and finely granular chromatin patterns, in comparison with patients in the short-survival group (less than or equal to 9 months); the correlation was not statistically significant, however. The incidence of cells with round or oval nuclei and finely granular chromatin patterns was higher in patients whose cells had hyperdiploid DNA patterns than for patients whose cells had near-diploid patterns; again, the difference was not statistically significant. Patients whose tumor cells had hyperdiploid DNA patterns had significantly shorter survival times than did patients whose tumor cells had near-diploid patterns. These results indicate that (1) judging the nuclear DNA pattern from the cytomorphologic features of small cell carcinoma is unreliable and (2) the nuclear DNA patterns are related to the clinical prognosis of patients with small cell carcinoma of the lung.     

Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq

Although the accuracy of quantitative real-time polymerase chain reaction （qRT-PCR） is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing （RNA-Seq） to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression proffies of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes （B2M, GAPDH, GUSB, HMBS, HPRT1, IP08, PGK1, POLR2A, PPIA, RPLPO, TBP, TFRC, UBC, and YWHAZ）, the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes （ACTB） and the 5 additional candidate reference genes （EEFIA1, FAU, RPS9, RPSll, and RPSI4） were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEFIAI, FA U, RPS9, RPSl l, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.     

Cervicovaginal cytology in uterine adenocarcinoma and adenosquamous carcinoma. Comparison of cytologic and histologic findings

To investigate the diagnostic accuracy and to characterize the findings in false-negative cases, the results of cervicovaginal cytology in 56 adenocarcinomas and 25 adenosquamous carcinomas (42 cervical, 36 endometrial, 2 metastatic and 1 arising synchronously from both cervix and endometrium) were reviewed, including review of the actual slides in 56 cases. Overall, 80% of the initial cytologic diagnoses resulted in diagnostic curettage (i.e., cytology was effectively positive); 84% of the postreview diagnosis were effectively positive. Nine cytology slides showed no malignant cells; eight of these negative smears showed repair, five were atrophic, two showed a high estrogen effect and one had enlarged atypical bare nuclei. These false-negative diagnoses were associated with an endometrial primary site (P less than .01), endometrioid histology (P less than .005), low-grade or intermediate-grade histology (P less than .005), small size of tumor (P less than .05) and absence of cervical involvement (P less than .005) in those cases in which a hysterectomy was performed. False-negative diagnoses were not associated with an absence of endocervical cells or with scanty cellularity. Of 39 cervical and 28 endometrial carcinomas with a positive cytologic diagnosis (initially or after review of the available slides), cytology correctly identified the primary site in 18% and 54% of the cases, respectively. Cytology incorrectly classified the anatomic site of four cervical and three endometrial carcinomas and considered one case arising in both the endometrium and cervix to be endometrial. Routine cervicovaginal cytology does have a role in screening for uterine glandular carcinoma; to maximize its diagnostic sensitivity, we suggest using a recommendation for curettage in the report of positive cases so that all of the varied cytologic diagnoses associated with glandular carcinomas will receive a uniform clinical response. In those cases with preserved cancer cells, a correlation can be made with the histologic type of the carcinoma, rather than with the anatomic site.     

The effect of sampling on quantitative nuclear image features in histologic sections of lung carcinomas

A feasibility study showed that quantitative nuclear image (QNI) analysis, in which the morphology of the nucleus is described by a number of mathematical parameters, can be used to make the therapeutically and prognostically important distinction between small cell lung carcinoma (SCLC) and non-SCLC, which can be difficult to make with subjective histologic typing. In the present study, the effects of sample size and sample site on the QNI features were investigated. For all sample sites in a given tumor, comparison was made between the histologic classification, the ultrastructural findings and the classification based on the QNI features. Using a running mean, it was found that a sample size of 25 nuclei is sufficiently large. Histologic and quantitative classifications of samples from different sites of the same tumors were in agreement with regard to the separation of SCLC and non-SCLC in 19 of 20 sections. In the case in which disagreement occurred in one section, the ultrastructural findings supported the quantitative classification. These data indicate that sampling from different sites has no essential influence on the QNI classification of lung carcinomas.     

Prognostic value of bronchioloalveolar carcinoma component in lung adenocarcinoma

BAC is a common pattern in conventional lung adenocarcinoma. In the past, however, there were no well-defined criteria for BAC. As a result, it was difficult to evaluate the prognosis on this type of lung adenocarcinoma. Though the 1999 WHO classification of BAC provides a useful framework, it does not provide detailed enough information to predict prognosis in lung adenocarcinomas with BAC feature. The aim of this study was to address the prognostic value of bronchioloalveolar carcinoma (BAC) component in lung adenocarcinoma. Ninety-one consecutive surgically treated patients with adenocarcinoma exhibiting various degrees of BAC features and complete follow-up records were retrospectively studied. According to the percentage of BAC component designed as less than 50%, 50%-79%, 80%-99%, and 100%, tumors were classified as type I, type II, type III, and type IV respectively. The overall 5-year survival rate was 64.84%. Multivariate analysis revealed that the four classified types are independent prognostic factors (P=0.0008), as is tumor stage (P=0.0000). The 5-year survival rates were 39.29%, 58.82%, 81.25%, 85.71% for the four classified types respectively, and were 88.89% for stage I, 46.15% for stage II, and 23.81% for stage III. However, the size of tumor (>2 cm) was significant only in the univariate analysis (P=0.0275). In the patients with tumor size exceeding 2 cm in diameter, the BAC component was also significant to predict prognosis (p=0.0008). In lung adenocarcinoma, the BAC component may prove to be useful to predict the outcome of the patients, and the percentage of BAC pattern and pathological stage appear to be two independent prognostic factors.     

Neural network-based segmentation and classification system for automated grading of histologic sections of bladder carcinoma

OBJECTIVE: To develop an image analysis system for automated nuclear segmentation and classification of histologic bladder sections employing quantitative nuclear features. STUDY DESIGN: Ninety-two cases were classified into three classes by experienced pathologists according to the WHO grading system: 18 cases as grade 1, 45 as grade 2, and 29 as grade 3. Nuclear segmentation was performed by means of an artificial neural network (ANN)-based pixel classification algorithm, and each case was represented by 36 nuclei features. Automated grading of bladder tumor histologic sections was performed by an ANN classifier implemented in a two-stage hierarchic tree. RESULTS: On average, 95% of the nuclei were correctly detected. At the first stage of the hierarchic tree, classifier performance in discriminating between cases of grade 1 and 2 and cases of grade 3 was 89%. At the second stage, 79% of grade 1 cases were correctly distinguished from grade 2 cases. CONCLUSION: The proposed image analysis system provides the means to reduce subjectivity in grading bladder tumors and may contribute to more accurate diagnosis and prognosis since it relies on nuclear features, the value of which has been confirmed.     

DNA methylation changes in atypical adenomatous hyperplasia, adenocarcinoma in situ, and lung adenocarcinoma

Background

Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation.

Methodology/Principal Findings

We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma), and invasive lung adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML, RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer.

Conclusions/Significance

The gradual increase in DNA methylation seen for numerous loci in progressively more transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to lay out a possible roadmap for aberrant DNA methylation events in tumor development. In addition, it identifies which DNA methylation changes might be used as molecular markers for the detection of preinvasive lesions.     

The influence of variations in the measuring procedure on quantitative nuclear image features in histologic sections of lung tissue

The qualitative and quantitative features of cell nuclei in tissue sections play an important role in diagnostic histopathology; variations in staining intensity and measuring procedures may interfere with their proper evaluation. To identify nuclear features that are relatively insensitive to these technical variables, the influence of critical steps in a scanning-stage densitometer measuring system was studied on 87 quantitative nuclear image (QNI) features in histologic sections of lung tissue. The influences of the following measuring variations were evaluated: interactive segmentation (with and without median filtering; with and without 5% uniform distributed noise added); scanning (with and without median filtering); calibration of the photomultiplier (different background localizations and different intensity levels); and time. In addition, the influence of artificially changed intensity variations was investigated. The results showed that, while the coefficient of variation (CV) induced by variations in the measuring system was usually low (below 10%), for some QNI features the CV can be high (up to 216%). The influence of artificial variations in intensity was restricted: only a minority of the QNI features showed a significant difference. Of the 87 QNI features, 35 had a CV of less than 10%, and 25 of these were significantly correlated with each other. Thus, only ten uncorrelated, low-CV QNI features remained; these belonged to all of the different QNI feature categories used. These features may be diagnostically important since they may best describe the morphologic properties of the nuclei. The results of this study should help in selecting quantitative nuclear image features that are less sensitive to variations in the measuring procedure and staining intensity.     

Aspiration biopsy of salivary gland tumors. VI. Morphologic studies on smears and histologic sections from mucoepidermoid carcinoma.

The cytologic findings in fine-needle aspiration biopsy of 24 primary mucoepidermoid carcinomas of salivary glands are reviewed. The morphologic details which permit recognition of this tumor in smears of the aspirate are described.     

Colon carcinoma metastatic to the lung. Cytologic manifestations and distinction from primary pulmonary adenocarcinoma.

Based upon a review of cytologic specimens obtained from 46 patients, the morphologic characteristics of metastatic colon carcinoma and primary pulmonary adenocarcinoma were compared and contrasted. A monoclonal antibody (D-14) reported to be helpful in identifying colorectal carcinoma was also evaluated. Colon brushings and washings from 10 patients with colonic carcinoma and a variety of respiratory tract cytology samples from 16 and 20 patients with metastatic colonic and primary pulmonary adenocarcinomas, respectively, formed the basis of this review. The presence of well-formed glands and dirty necrosis was significantly more characteristic of colon carcinoma. Other cytologic features and D-14 staining were not sufficiently distinctive to allow a separation from primary pulmonary adenocarcinoma.     

Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells

Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.     

Comparison of quantitative histomorphometry and DNA ploidy in tissue sections of prostate carcinoma

OBJECTIVE: To examine the ability of quantitative histomorphometry to predict DNA ploidy of prostate carcinoma in biopsy tissue sections assigned after quantitation by nuclear digital image analysis. STUDY DESIGN: Thirty-five diploid, 35 tetraploid and 35 aneuploid prostatic carcinomas in biopsies, assessed by the CAS 200 image analyzer (Bacus Laboratories, Lombard, Illinois, U.S.A.), were reevaluated by the Bacus Laboratories Incorporated Slide Scanner, a microscope that quantifies histologic images. Thirty-one histomorphometric features from cancer cells were captured at 40 x magnification, averaged across tilesfor each case and incorporated into a multivariate discriminant model to determine which features predicted ploidy interpretation by nuclear image analysis using the CAS 200. RESULTS: On average, 60 and 15 minutes were required to perform nuclear image analysis and histomorphometry, respectively. The multivariate discriminant model identified configurable run length, difference variance, contrast, inverse difference moment, sum entropy and diagonal variance as histomorphometry features capable of distinguishing diploid from nondiploid tumors (P < .05). Cross-validation studies showed the model correctly classified 74.3% of the diploid and 57.1% of the nondiploid cases. CONCLUSION: Quantitative histomorphometry can predict the ploidy of prostate carcinoma in biopsy tissue sections. Quantitative histomorphometry has potential as a method of rapidly assessing DNA ploidy otherwise earmarked for nuclear image analysis, resulting in savings of time and expense.