Determination of stromal signatures in breast carcinoma

Many soft tissue tumors recapitulate features of normal connective tissue. We hypothesize that different types of fibroblastic tumors are representative of different populations of fibroblastic cells or different activation states of these cells. We examined two tumors with fibroblastic features, solitary fibrous tumor (SFT) and desmoid-type fibromatosis (DTF), by DNA microarray analysis and found that they have very different expression profiles, including significant differences in their patterns of expression of extracellular matrix genes and growth factors. Using immunohistochemistry and in situ hybridization on a tissue microarray, we found that genes specific for these two tumors have mutually specific expression in the stroma of nonneoplastic tissues. We defined a set of 786 gene spots whose pattern of expression distinguishes SFT from DTF. In an analysis of DNA microarray gene expression data from 295 previously published breast carcinomas, we found that expression of this gene set defined two groups of breast carcinomas with significant differences in overall survival. One of the groups had a favorable outcome and was defined by the expression of DTF genes. The other group of tumors had a poor prognosis and showed variable expression of genes enriched for SFT type. Our findings suggest that the host stromal response varies significantly among carcinomas and that gene expression patterns characteristic of soft tissue tumors can be used to discover new markers for normal connective tissue cells.     

Nuclear DNA in histologic sections from squamous-cell carcinoma and adenocarcinoma of the lung

The nuclear DNA content in morphologically identified tumor cells was analyzed in 4-micron histologic sections from 58 patients with lung carcinoma who survived for at least five years. Thirty-three of the carcinomas were invasive squamous bronchial carcinomas and 25 were pulmonary adenocarcinomas. In all squamous carcinomas, the majority of tumor cells were found to exhibit DNA values exceeding the normal tetraploid and/or diploid region. In contrast, some of the pulmonary adenocarcinomas were found to be composed of a majority of tumor cells with DNA values in the normal diploid region. The results indicate that invasive squamous bronchial carcinomas, in general, are tumors with aneuploid DNA patterns indicative of a high malignant potential and that malignancy grading based on DNA measurements does not add any significant prognostic information to that obtained by morphologic diagnosis.     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

Frequency of hyperdiploid chromosome complements in endometrioid tumors of the endometrium whereas similar tumors in the ovary tend to show hypodiploidy: a significant difference that may not be distinguishable by flow cytometry of DNA content

Recent interest has focused on endometrioid carcinomas of the endometrium in view of the frequent occurrence of microsatellite stability, accompanied by a favorable prognosis, and by near-diploidy when studied by flow cytometry. The latter feature fails to address the question whether (and to what extent) the karyotypes of the tumor cells may or may not be truly diploid, an important feature on which there is virtually no information. A reconsideration of earlier published and unpublished work in this laboratory on near-diploid carcinomas of the endometrium, and comparable studies on near-diploid ovarian carcinomas (a site where endometrioid carcinomas are also commonly found) has therefore been undertaken. In the endometrium, these studies have clearly shown that carcinomas with near-diploid chromosomes are in fact commonly hyperdiploid, often of endometrioid histology, and in many instances show a single additional chromosome, differing in different tumors, as the apparently sole chromosome change. By contrast, similar studies on the ovary (which also included several endometrioid carcinomas) revealed a tendency towards hypodiploidy, with loss of a few chromosomes, as well as the presence of structural chromosome changes and a generally poor prognosis.     

Ploidy disturbances in endometrial and ovarian carcinomas. A review

A survey of DNA and chromosome aberrations in human endometrial and ovarian carcinomas is presented, including data obtained by chromosome analysis, absorption cytophotometry and flow cytometry. Nearly all of the cancers showed some structural or numerical chromosomal abnormalities. Endometrial carcinomas were reported to have a near-diploid DNA content in two-thirds of the cases, as opposed to ovarian carcinomas, in which only one-third of the tumors were near diploid. DNA aberrations in tumors of both origins seemed to be associated with an unfavorable prognosis, indicating that DNA measurements may supply a valuable additional criterion for biologic malignancy. Evaluable data on ploidy correlated to histologic subtypes was not available; in general, the number of patients in each study was small. Tumors of low differentiation tended more often to be aneuploid, but this was not a consistent finding. Further research comparing disease stage, tumor grade and tumor type in larger series of patients is therefore mandatory.     

Proliferative characteristics of primary and metastatic human solid tumors by DNA flow cytometry

The percentage of cells in S-phase (S-index) was calculated from DNA histograms of 453 primary and metastatic human solid tumors (predominantly bladder, breast, colorectal, renal, prostate, ovarian and lung carcinomas, melanomas, and sarcomas). S-indices varied widely among both primary and metastatic tumors (1-48%); there was no significant difference in S-indices between primary and metastatic tumors. The S-indices for aneuploid tumors were significantly higher than for diploid tumors. When data for all aneuploid tumors were analyzed collectively, there was no significant relationship between S-index and DNA ploidy index. However, for colorectal and ovarian carcinomas S-indices increased, and for lung carcinomas S-indices decreased with elevation in the degree of DNA-ploidy. Lung carcinomas had the highest S-indices. Comparison of flow cytometry (FCM) and cytology data indicated that for most diploid tumors S-indices reflect the proportion of S-phase cells among a mixed population of normal and tumor cells. For most aneuploid tumors, the proportion of tumor cells estimated cytologically was similar to the proportion of aneuploid cells estimated by FCM. For a small proportion of aneuploid tumors a comparison of cytology and FCM data indicated the presence of a predominant diploid tumor stemline and a minor stemline with aneuploid DNA content. There was a wide spread in the values of S-indices within tumor groups defined by degree of differentiation and stage of disease at surgery.     

DNA and nuclear protein measurement in isolated nuclei of human endometrium

Feulgen-DNA and nuclear light green-protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates. The DNA content of the G0/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium. The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and post-menopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for post-menopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.     

Colorectal carcinoma. DNA ploidy pattern and prognosis with reference to tumor DNA heterogeneity.

Nuclear DNA content was measured in 72 colorectal carcinomas using single-cell microspectrophotometry on Feulgen-stained smears. Four samples were analyzed from each tumor. Patients were followed for 41-65 months (average, 53). DNA heterogeneity (both aneuploid and nonaneuploid patterns) was present in 44% of the cases. Sixty-eight percent of the tumors showed an aneuploid DNA pattern in at least one of the samples. Patients with nonaneuploid tumors tended to have a survival advantage over patients with homogeneously aneuploid tumors and demonstrated a significantly longer disease-free survival. The DNA ploidy pattern is of potential value in conjunction with histopathologic prognostic parameters in colorectal carcinoma. Since colorectal tumors exhibit pronounced DNA heterogeneity, multiple samples are required from each tumor to permit a proper evaluation of its DNA pattern. The DNA heterogeneity may represent tumor progression and can partly explain the conflicting results reported concerning DNA pattern and prognosis in colorectal carcinoma.     

ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome

Application of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems, we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas, high ALDH activity identifies the tumorigenic cell fraction, capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas, expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts.     

Routine detection of the replication error phenotype in clinical tumor specimens using fluorescence-SSCP

Almost all tumors from patients with hereditary non-polyposis colon carcinoma and approximately 10%-15% of sporadic colon and gastric carcinomas contain widespread deletions within mono- and dinucleotide repeat sequences in their DNA. This is referred to as the replication error (RER+) phenotype and, in the case of colon carcinoma, is often associated with an improved tumor prognosis and possibly also with response to chemotherapy. The RER+ status of tumors is usually determined by examining several dinucleotide and mononucleotide repeats for size variations when compared with the matching normal DNA. Alternately, the identification of deletions within BAT-26, a quasi-monomorphic polyadenine tract within the hMSH2 gene, has been shown to establish the RER+ status of tumors with greater than 99% accuracy and without the need for normal DNA. Here, we use fluorescent PCR in combination with single strand conformation polymorphism analysis to detect deletions in BAT-26. This technique provides a sensitive, rapid, reproducible and inexpensive assay suitable for the routine identification of RER+ status in clinical tumor specimens.     

Flow DNA analysis in human lung cancer: potentiality and limitations due to sampling methods

Flow DNA analysis was performed on samples from 71 surgically removed lung cancers and from 145 patients undergoing bronchoscopy. Abnormal DNA stem lines, characterized by their DNA index (DI), were frequently observed in operated lung carcinomas (87%). Two or three abnormal DNA stem lines were discovered simultaneously in 10% of the samples. The mean DI of all abnormal tumor stem lines was lowest for rare tumor cell types and highest for adenocarcinomas. Intermediate mean DI values were found for epidermoid and small cell carcinomas, which were among the most proliferative tumors. The high rate of false negative results suggests poor diagnostic reliability of flow DNA analysis on bronchoscopic samples. However, the method appears to provide a promising objective tool capable of evaluating tumor behavior and prognosis.     

G-Cimp Status Prediction Of Glioblastoma Samples Using mRNA Expression Data

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.     

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.     

Classifying breast carcinomas with DNA measurements and morphometry

The relation between the DNA distribution pattern and the variables DNA content and nuclear area was investigated in 73 breast carcinomas. According to the DNA patterns, the tumors were subclassified into groups I, II, III, and IV (Auer's classification). The Auer's classes correlate with prognosis. In this investigation it was shown that Auer's classes strongly correlate with the standard deviation of DNA content and mean nuclear area. The carcinomas were subclassified using data of DNA content and nuclear area. For the establishment of this subclassification, multivariate analysis was applied. It was evident that Auer's group III was a hybrid group. Therefore a second classification rule was calculated without the data of Auer's group III. This second classification rule was even more successful in determining the Auer's group of each individual case. Our investigation shows that it is possible to subclassify breast carcinomas objectively using DNA content and nuclear area of the tumor cells.     

The Role of Stem and Pluripotent Cells in Differentiation and the Neoplastic Transformation of Pancreatic Cells

Certain differentiation-specific antigenic determinants are characteristic of pancreatic cells at each stage of embryonic development. Using immunohistochemical markers, we have demonstrated that the cells of several pancreatic tumors express some fetal proteins characteristic of embryonic cells of the pancreas. We propose that the presence of fetal antigens and proteins in the tumor is a sign of a lower tumor differentiation and a less favorable prognosis.     

Diagnostic value of flow cytometric DNA determination combined with fine needle aspiration biopsy in thyroid tumors

The nuclear DNA content and the numbers of cells in the S and G2M phases of the cell cycle were determined by flow cytometry (FCM) in fine needle aspirates of 187 thyroid tumors to evaluate the diagnostic value of nuclear DNA content determination in combination with aspiration cytology. DNA aneuploidy was present in 4 of 5 follicular carcinomas, 2 of 3 anaplastic carcinomas, 5 of 15 excised follicular adenomas and 2 of 20 excised adenomatous goiters; all 7 papillary carcinomas and 4 lymphomas were diploid in the aspirate. Aneuploid carcinomas had easily distinguishable S and/or G2M phases, unlike the benign aneuploid tumors. None of the histologically benign tumors or the nonexcised tumors had greater than 6% S-phase cells, and only one benign tumor had greater than 9% G2M-phase cells. In contrast, all lymphomas had greater than 10% S-phase cells and four of seven papillary carcinomas had greater than 9% G2M-phase cells. The use of FCM determination in combination with fine needle aspiration biopsy cytology improved the diagnostic potential of the latter technique.     

Altered methylation at microRNA-associated CpG islands in hereditary and sporadic carcinomas: a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA)-based approach

MicroRNAs (miRNAs) are small noncoding RNAs that contribute to tumorigenesis by acting as oncogenes or tumor suppressor genes and may be important in the diagnosis, prognosis and treatment of cancer. Many miRNA genes have associated CpG islands, suggesting epigenetic regulation of their expression. Compared with sporadic cancers, the role of miRNAs in hereditary or familial cancer is poorly understood. We investigated 96 colorectal carcinomas, 58 gastric carcinomas and 41 endometrial carcinomas, occurring as part of inherited DNA mismatch repair (MMR) deficiency (Lynch syndrome), familial colorectal carcinoma without MMR gene mutations or sporadically. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assays were developed for 11 miRNA loci that were chosen because all could be epigenetically regulated through the associated CpG islands and some could additionally modulate the epigenome by putatively targeting the DNA methyltransferases or their antagonist retinoblastoma-like 2 (RBL2). Compared with the respective normal tissues, the predominant alteration in tumor tissues was increased methylation for the miRNAs 1-1, 124a-1, 124a-2, 124a-3, 148a, 152 and 18b; decreased methylation for 200a and 208a; and no major change for 373 and let-7a-3. The frequencies with which the individual miRNA loci were affected in tumors showed statistically significant differences relative to the tissue of origin (colorectal versus gastric versus endometrial), MMR proficiency versus deficiency and sporadic versus hereditary disease. In particular, hypermethylation at miR-148a and miR-152 was associated with microsatellite-unstable (as opposed to stable) tumors and hypermethylation at miR-18b with sporadic disease (as opposed to Lynch syndrome). Hypermethylation at miRNA loci correlated with hypermethylation at classic tumor suppressor promoters in the same tumors. Our results highlight the importance of epigenetic events in hereditary and sporadic cancers and suggest that MS-MLPA is an excellent choice for quantitative analysis of methylation in archival formalin-fixed, paraffin-embedded samples, which pose challenges to many other techniques commonly used for methylation studies.     

P63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues.

p63 is essential for epithelial cell survival and may function as an oncogene. We examined by immunohistochemistry p63 expression in human normal and tumor salivary gland tissues. In normal salivary glands, p63 was expressed in the nuclei of myoepithelial and basal duct cells. Among 68 representative salivary gland tumors, 63 displayed p63 reactivity. In all tumor types differentiated towards luminal and myoepithelial lineages (pleomorphic adenomas, basal cell adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas), p63 was expressed in myoepithelial cells, whereas luminal cells were always negative. Similarly, in mucoepidermoid carcinomas, basal, intermediate, and squamous cells expressed p63, in contrast to luminal mucous cells. p63 reactivity was also restricted to basal cells in Warthin tumors and oncocytomas. Myoepitheliomas and myoepithelial carcinomas all expressed p63. The only five negative tumors were three of four acinar cell carcinomas and two of three adenocarcinomas. In conclusion, p63 is expressed in the nuclei of normal human salivary gland myoepithelial and basal duct cells. p63 expression is retained in the modified myoepithelial and basal cells of human salivary gland tumors, which suggests a role for p63 in oncogenesis of these complex tumors.     

The clinical usefulness of determining ploidy patterns in human tumors as measured by slide-based Feulgen microspectrophotometry

Some aspects of the clinical value of the Feulgen microspectrophotometric assessment of DNA ploidy patterns in human tumors are reviewed. This method has been shown to be of predictive value for a number of tumor sites and may be independent of other prognostic indicators, such as the histopathologic grade. The association between ploidy and prognosis probably reflects the degree of chromosomal changes in the tumor cells; while it is probable that all malignant tumors are aneuploid, there is a tendency for the changes to be more extensive in more aggressive tumors. Thus, tumors with DNA modes that depart significantly from the diploid and tetraploid levels may have a worse prognosis than do tumors whose modes are at or close to these levels. This has clearly been shown for tumors of the breast, ovary, endometrium and several other sites. For some sites, including the cervix uteri and the large bowel, such a relationship is less clear, probably because tumors at these sites have frequently undergone extensive chromosomal changes that do not result in a significant deviation of the DNA mode from the euploid levels. The use of slide-based DNA analysis systems, in which the morphology of the cells being measured can be assessed, has advantages over flow cytometry that may be crucial in some situations.(ABSTRACT TRUNCATED AT 250 WORDS)