Formalin-fixed Paraffin-embedded Material Research Articles

Sequential Flow Cytometry and Fluorescence In Situ Hybridization for the Study of Formalin-Fixed, Paraffin-Embedded Breast Cancer Cells

Both flow cytometry and fluorescence in situ hybridization (FISH) are useful techniques in the analysis of cancer tissues. When the two are used in the study of the same specimens, they are usually performed in parallel, separately. This is problematic where there is a scarcity of material, making completion of both studies impossible. Fluorescence in situ hybridization procedures that will utilize excess material discarded from flow cytometry would be advantageous. The present report describes an optimized protocol for performing sequential flow cytometry and FISH using formalin-fixed paraffin-embedded archival material. Although breast cancer tissues were used in this initial study, the protocol is applicable to other cancer tissues as well.

A newly identified pattern of K-ras mutations at codons 12 and 13 is associated with long-term survival in colorectal cancer

Background . Although K-ras mutations reportedly occur in 40% to 60% of all colorectal carcinomas, the relationship between specific mutations and clinical outcome is unclear. The purpose of this study was to assess the frequency and types of K-ras mutations in 89 colorectal cancer patients, comparing groups with short-term (less than 5 years) and long-term (more than 10 years) survival. Methods . The group was divided into four cohorts by survival and modified Dukes classification (Dukes B 2 and C 2). DNA was extracted from formalin-fixed paraffin-embedded archival material. Mutational status was analyzed using a modification of allele-specific-polymerase chain reaction. Results . Mutations in codon 12 were found in 11.2% of tumors, and 83% of tumors had mutations in codon 13. Gly > Asp accounted for 85.2% of the mutations. Tumors with mutations in both codon 12 and codon 13 occurred significantly more frequently in the long-term (21.3%) versus the short-term (2.4%) survival group. Gly > Asp mutations in either codon were related to long-term survival, and 80% of long-term survivors with mutations in both codons had Gly > Asp mutations in both. Conclusions . Simultaneous mutation in codons 12 and 13 of the K-ras gene appears to be a positive prognostic indicator in colorectal cancer.

Prognostic significance of Ki-67 antigen and p53 protein expression in pancreatic duct carcinoma: a study of the monoclonal antibodies MIB-1 and DO-7 in formalin-fixed paraffin-embedded tumour material.

Formalin-fixed paraffin-embedded material from 57 patients in whom curative resection of pancreatic carcinoma had been attempted was analysed by an immunohistochemical procedure to estimate proliferation and p53 protein expression. Using the monoclonal antibody (MAb) MIB-1, which recognizes a Ki-67 epitope, the proliferating cell index (PCI, percentage of immunoreactive tumour nuclei) and proliferating cell area (PCA, percentage of immunoreactive tumour nuclear area) were calculated using an interactive image analysis system and were compared with semiquantitative scoring of stainability. MAb DO-7, which recognizes both wild- and mutant-type p53 protein, was used to assess p53 expression in the same material. MIB-1 stainings were of high quality in 53 tumours. The median PCI was 29.7% (range 0.5-82.1%) and the median PCA was 10.6% (range 0.0-36.5%). There was a close correlation between PCI and PCA (P < 0.0001). PCI and PCA values were in conformity with the semiquantitative scoring (P < 0.0001). The p53 immunohistochemical stainings were successful in 48 tumours and the protein was expressed in 22 (46%). High PCI values (> 45%, n = 14) correlated with shorter survival time (P < 0.01). PCA (P < 0.05) and the expression of p53 protein (P < 0.001) were independent prognostic variables.

Implications of prostate micrometastases in pelvic lymph nodes: An archival tissue study

Objectives In the United States, radical retropubic prostatectomy for adenocarcinoma usually includes a staging pelvic lymphadenectomy. If frozen section analysis of the lymph nodes fails to reveal any evidence of metastases, the prostate is removed. We have previously noted that as many as 56% of patients undergoing radical prostatectomy demonstrate rising serum prostate-specific antigen (PSA) levels by 4 years postoperatively. This report was designed to determine whether micrometastases undetectable by conventional pathologic methods could have accounted for these biochemical failures. Methods A retrospective analysis of formalin-fixed paraffin-embedded pelvic lymph node material was undertaken using a reverse transcription-polymerase chain reaction (RT-PCR)-based assay designed to amplify messenger RNA from PSA. All specimens were obtained from a group of 57 patients with prostate cancer who had undergone staging pelvic lymphadenectomy at the time of radical prostatectomy, and whose long-term follow-up was known. Results Although all of these nodes appeared to be free of tumor by conventional pathologic methods, a RTPCR assay was used to identify evidence of prostate metastases in 44% of évaluable samples. Of these, 14 of 16 went on to manifest rising serum PSA values by 5 years postoperatively. Conclusions These results suggest that molecular staging of pelvic lymph nodes prior to planned therapy for clinically organ-confined prostate cancer may better distinguish between patients with local disease and those for whom local therapy alone will not be curative. To our knowledge, this is the first large-scale retrospective gene expression study published.

Immunohistochemical Characterization of 22 Monoclonal Antibodies against the CA125 Antigen: 2nd Report from the ISOBM TD-1 Workshop

We evaluated the immunohistological (IH) characteristics of 22 different antibodies that were submitted for study in the frame of the TD-1 ISOBM Workshop on monoclonal antibodies against CA125. Information on relative affinities and epitope similarities was obtained from a parallel immunochemical study. Antibodies were tested at concentrations of 10 and 1 micrograms/ml on frozen and paraffin sections. Paraffin sections were stained according to the streptavidin-biotin complex protocol, and frozen sections according to a two-step immunoperoxidase technique. Aminoethylcarbazole served as the chromogen. The tissues were from normal proliferative endometrium (formalin-fixed paraffin-embedded) material and clear-cell adenocarcinoma of the ovary (formalin-fixed paraffin-embedded and frozen material). Sections were scored for staining in epithelial cells, basal, apical and diffuse cytoplasmic and in stromal components. Intensity was graded as 1, 2 or 3 for epithelial cells and as -1, -2 or -3 for stroma. The cumulative scores for each antibody expressed the discriminative properties of specific epithelial staining against background. M11 and M11-like antibodies, as well as OC125 and OC125-like antibodies, in general showed good staining results. Although there was a trend for high-affinity antibodies to show higher scores, there was no clear relationship between affinity and staining result. For nine antibodies (ZR45, MA602-1, K102, K94, K90, OV185, K97, K96, OV198), the reactions in paraffin and frozen sections were of similar intensity. Most of these were of low affinity with one exception: antibody ZR45, a rat monoclonal antibody (MAb) which had a high relative affinity. For eight antibodies (M11, K101, MA602-6, ZS33, B27.1, B43.13, K93, OC125), a loss of specific staining was observed in frozen sections. All but two of these antibodies (MA602-6 and OC125) were of high relative affinity. With four antibodies (K91, ZR38, K95, K100), the reverse situation was observed. One (K100) was of low affinity, two (K95 and K91) of high affinity and the fourth (ZR38) was a rat MAb of high affinity. Mainly due to the increased cytoplasmic staining in carcinoma, the reactivity in paraffin sections was less extensive in normal endometrium compared to ovarian carcinoma for the majority of antibodies, irrespective of their affinity or epitope group. The IH characterization of these antibodies may be of help in selecting antibodies with specific properties for further comparative studies. The reactivity of normal endometrium with all useful antibodies makes it a good candidate for standard external IH tissue control.

TP53 gene mutations in gastric carcinoma detected by polymerase chain reaction/single-strand conformation polymorphism analysis of archival material.

TP53 gene mutations, one of the most common alterations described in human tumors, have also been detected in gastric carcinoma, and shown to occur rather late in disease progression. A better assessment of the prognostic value of TP53 gene mutations can be obtained by examining archival material, as this allows stored cases with well-defined histories to be monitored. We performed immunohistochemical and polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analyses of formalin-fixed paraffin-embedded material from nine selected cases of gastric carcinoma at different pathological stages. PCR-SSCP analysis of TP53 exons 5-8 detected missense point mutations in two out of five immunostain(PAb1801)-positive tumors, and a deletion (allowing for a premature stop codon) in one of the remaining four immunostain-negative tumors. Thus, PCR-SSCP analysis represents a feasible strategy for the detection of TP53 alterations in archival material of gastric carcinoma cases.

The expression of proliferating cell nuclear antigen (PCNA) immunostaining in renal pelvic and ureteral cancers and its prognostic value

The expression of the proliferating cell nuclear antigen (PCNA) was analyzed in formalin-fixed paraffin-embedded materials of 20 primary renal pelvic and ureteral cancers. The relation between PCNA positive rate and clinical pathological stage and prognosis were investigated. The fraction of PCNA positive nuclei ranged between 1.6-64.4% (mean 25.1%). The PCNA positive rate was found to be related to established prognostic factors, but not to patient age, sex and length of time for paraffin-embedded. Grade 3 cancers showed a higher fraction of PCNA positive nuclei than grade 1 and 2 cancers (p < 0.05). Superficial cancers showed a significantly lower fraction on nuclei positive for PCNA than invasive cancers (p < 0.01). The cancers with lymph node metastases showed a higher fraction (p < 0.05). The cancers with distant metastases postoperatively, also had a higher fraction (p < 0.05). As PCNA immunostaining can be applied to routinely processed paraffin-embedded specimen, this method is very useful to assess the cell proliferation. The results suggest that the fraction of PCNA positive nuclei would be useful for investigating the malignant potential, of renal pelvic and ureteral cancers thereby providing prognostic factors of these diseases.

DNA histogram typing in a series of 707 tumors of the central and peripheral nervous system.

The diagnostic value of ploidy level was investigated in a series of 707 tumors (from 707 patients) of the central and peripheral nervous system. The series contained 91 nerve sheath tumors (NSTs), 222 meningiomas (MNGs), 37 primitive neuroectodermal tumors (PNETs), 293 glial tumors of the adult (ATTs), and 64 brain metastases (MTTs). The ploidy level of each tumor was obtained by means of its DNA histogram type (DHT), which was computed (digital cell image analysis) on Feulgen-stained nuclei from archival formalin-fixed paraffin-embedded materials. The data reveal that the measurement of the ploidy level showed a diagnostic value in some of these tumor cases. Indeed, the proportion of highly aneuploid cases significantly (p < 0.05-0.01) decreased according to the sequence PNETs (72%)-->MTTs (64%)-->ATTs (40%)-->NSTs (23%)-->MNGs (9%). Nevertheless, this significant diagnostic value in terms of a distinction between benignity and malignancy only appears when all the tumors are taken into consideration. Indeed, at the level of one individual patient, our study shows that, like traumatic neuromas, schwannomas, or classical meningiomas, completely benign tumors can exhibit a high aneuploidy level. In contrast, some tumors from histopathological groups like brain metastases or PNETs, which are known to be clinically aggressive, could be completely diploid.

A new tumor-associated antigen defined by a monoclonal antibody directed to gastric adenocarcinoma

To develop useful markers for the gastrointestinal neoplasms, the authors have established a new monoclonal antibody (MoAb), FU-MK-1, directed to gastric adenocarcinoma. The MoAb was produced by a mouse hybridoma technique by immunizing a BALB/c mouse with cancerous ascites derived from a poorly differentiated adenocarcinoma of the stomach. FU-MK-1 did not react with the normal adult gastric mucosa, but did react with most carcinomas of the gastrointestinal tract. In addition, the MoAb recognized cholangiocarcinomas (CC), but it did not react with hepatocellular carcinomas (HCC). Furthermore, in the combined type tumor consisting of a mixture of HCC and CC, the MoAb react only with CC element, but not with pseudoglandular structures in the HCC areas. These results indicate that FU-MK-1 is a useful antigenic marker for distinguishing HCC from CC in the liver. Furthermore, because this MoAb retains its reactivity with formalin-fixed paraffin-embedded material, it may become a useful reagent for routine or retrospective immunohistologic studies. The molecular weight of the FU-MK-1 antigen was estimated to be ca. 41,000 dalton by the Western blot analysis. Periodic acid and trypsin treatment on the antigen suggested that the antigenic determinant is a glycoprotein. This MoAb may contribute to the histopathologic and immunologic studies of the digestive system.

Rapid in situ hybridization for the diagnosis of latent Epstein-Barr virus infection

Latent Epstein-Barr virus (EBV) infection is associated with a variety of malignancies. Rapid in situ hybridization techniques have been described for various lytic viral infections because of limited gene expression. However, EBERS (E̱pstein Ḇ arr e̱ arly Ṟ NAs) are expressed in abundance in tumour cells which are latently infected with EBV. We have targeted these transcripts in a rapid (3 h) in situ hybridization assay of the detection of latent EBV in clinical specimens, including formalin-fixed paraffin-embedded material. EBER RNA was detected in control cell lines which have two copies of the EBV genome and in paraffin-embedded biopsy specimens from patients with nasopharyngeal carcinoma, EBV-associated Hodgkin's disease, Burkitt's lymphoma and post-transplant lymphoma. The technique did not detect EBER RNA in oral hairy leukoplakia, a pathologic process previously characterized as associated with lytic EBV infection. The sensitivity, specificity and rapidity of this technique make it ideal for the diagnostic detection of EBV in latently infected clinical specimens.

Immunohistochemical assessment of cellular proliferation in the developing human CNS using formalin-fixed paraffin-embedded material

Assessment of cell proliferation in the developing human brain has been limited to counting mitotic cells, since the other procedures currently used for determination of the mitotic activity either depend on experimental manipulation of the tissue, e.g., labeling with radioactive thymidine precursors or with bromodeoxyuridine, or on frozen sections of the tissue, e.g., Ki-67 immunoreactivity. As most of the developing human brain material available is formalin fixed and paraffin embedded, these procedures are not practical. An immunohistochemical technique based on a monoclonal antibody directed against a proliferating cell nuclear antigen (PCNA) was used for detecting cellular proliferation in the developing human brain. Known progenitor cells in proliferating zones showed distinct PCNA-reactivity, but the method also revealed new areas of cell proliferation in fixed paraffin-embedded tissue blocks from the developing human brain.

Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis.

Loss of function of both copies of the RB1 gene is a causal event in the development of retinoblastoma. The predisposition to this tumor can be inherited as an autosomal dominant trait. Direct detection of the genetic defect is important for presymptomatic DNA diagnosis and genetic counseling in families with hereditary retinoblastoma. We have used multiplex polymerase chain reaction and high-resolution polyacrylamide gel electrophoresis to detect RB1 gene deletions as small as one base pair. By using three independent sets of amplification reactions, which cover 26% of the RB1 gene coding region, we identified RB1 gene deletions in the DNA of peripheral blood cells in 3 out of 24 (12.5%) unrelated patients with hereditary retinoblastoma. In one case, formalin-fixed paraffin-embedded tumor material was also used to detect the mutation. Sequencing of the mutated alleles revealed deletions of 1, 3 and 10 base pairs. Each deleted region was flanked by direct repeats.

C- erbB-2 oncogene expression in hepatocellular carcinoma and cholangiocarcinoma

The c- erbB-2 proto-oncogene encodes a transmembrane protein which is homologous to the epidermal growth factor receptor. This protein can be localized immunohistochemically in formalin-fixed paraffin-embedded material using a monoclonal antibody NCL-CB11; positive membrane staining correlates with gene amplification and protein overexpression in breast cancer. Using this technique we have shown that only 2 26 (8%) of hepatocellular carcinomas, 0 10 (0%) of cholangiocarcinomas and 0 2 (0%) hepatoblastomas overexpressed c- erbB-2 as evidenced by membrane staining. Moreover c- erbB-2 mRNA was not detected in seven hepatocellular carcinomas examined by Northern blot analysis. c- erbB-2 overexpression is, therefore, unlikely to be contributing to the malignant phenotype in hepatocellular carcinoma and cholangiocarcinoma.

Cytomegalovirus infection of renal allografts. Detection by polymerase chain reaction.

Early laboratory diagnosis of acute cytomegalovirus infection in renal transplant recipients is desirable but often difficult. The polymerase chain reaction (PCR) technique for detecting CMV DNA, although it promises a high sensitivity, risks the possibility of detecting latent CMV infection and leading to false-positive results. To address this issue and the feasibility of applying PCR to renal biopsy specimens, we analyzed 37 renal allografts by PCR. Formalin-fixed or Bouin-fixed paraffin-embedded materials were employed, and primers from the LA (late-antigen) region of CMV were used. Amplified products were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot analysis. Of 21 nephrectomy samples, three showed CMV-specific amplified products by PCR, but CMV inclusion bodies were detected histologically in only one of the three. Of 16 renal biopsies, three specimens were positive by PCR, with rare viral inclusions histologically identified in only one. All PCR-positive patients had clinically significant CMV disease as evidenced by positive CMV culture and/or seroconversion. In contrast, all CMV-seropositive patients without active viral disease had PCR-negative allografts. We conclude that PCR positivity in the renal allograft strongly correlates with active CMV disease but not latent infection. For the diagnosis of active CMV disease in patients with a renal allograft, PCR provides a means that is more sensitive and objective than histologic examination, more specific than serology, and faster than viral culture.

Altered expression of ABO (H) carbohydrate antigens is seen in pleomorphic adenomas.

Cell surface carbohydrate antigens show changes in relation to differentiation, maturation and malignant transformation. The expression of type 2 chain ABH carbohydrate structures of the ABO histo-blood group system was investigated in 28 pleomorphic adenomas (PA) and normal parotid glands in order to study possible changes in the glycosylation pattern. The distribution of carbohydrate structures was investigated by immunohistological stainings of formalin-fixed paraffin-embedded material using monoclonal antibodies (MAbs) with well-defined specificity. A strong interindividual variation was found in the normal tissue as well as in the tumors. In normal tissue, acinus and duct cells all expressed elongated carbohydrate structures. The yoepithelial cells did not stain with any of the MAbs investigated. In the PAs, staining was seen in the ductular structures and myoepithelial cells. In contrast to normal tissue, the tumors expressed the short precursor molecule sialylated N-acetyllactosamine. Furthermore, the PAs showed loss of H and A antigens, and a reduced expression of Le(y) compared to normal tissue. The ductular structures as well as the modified myoepithelial cells expressed binary N-acetyllactosamine, which in the normal tissue could only be found in the striated and excretory ducts. Thus our study has shown that aberrant glycosylation is not only a feature of malignant neoplasms but also occurs in pleomorphic adenomas.

Parathyroid Hormone-Like Peptide in Normal and Neoplastic Human Endocrine Tissues*

PTH-like peptide (PLP) is produced by tumors commonly associated with hypercalcemia as well as nonneoplastic tissues and several endocrine glands and tumors. To characterize the distribution of PLP in human endocrine tissues and tumors, we localized PLP in formalin-fixed paraffin-embedded material using the avidin-biotin-peroxidase technique with polyclonal antisera. Among peptide hormone-producing tissues, PLP was identified in nontumorous adenohypophysis and pituitary adenomas; medullary thyroid carcinomas; normal, hyperplastic, and adenomatous parathyroids; adrenal medulla and pheochromocytomas; normal pancreatic islets; and endocrine tumors of pancreas, gut, and lung, including small cell carcinomas. In other endocrine tissues PLP was identified in nontumorous thyroid follicular epithelium, colloid nodules, and follicular neoplasms; normal adrenal cortex, adrenocortical adenomas, and carcinomas; nontumorous testicular Leydig cells; normal ovarian granulosa and thecal cells; an ovarian thecoma and a granulosa cell tumor; placental trophoblast; and decidua. These results demonstrate that PLP is localized in many normal and neoplastic endocrine cells, including those not known to influence extracellular calcium homeostasis. The presence of PLP in a variety of endocrine tissues suggests that it may play a local physiological role in the growth or function of endocrine cells.

Identification of EBV-DNA in lymph nodes from patients with lymphadenopathy and lymphomas associated with AIDS

Fourteen examples of non-Hodgkin's lymphoma (NHL) and four of Hodgkin's disease in patients with AIDS as well as lymph nodes exhibiting changes related to the lymphadenopathy syndrome (LAS) from 11 HIV-positive individuals were studied for the presence of Epstein-Barr virus (EBV) genome both by in situ DNA hybridization and blotting techniques. Both methods were performed using formalin-fixed paraffin-embedded material. All the NHLs were of high malignancy and all but one were of the B-cell type. Of the four examples of Hodgkin's disease, two were lymphocytic predominant, one of mixed cellularity and one of the nodular sclerosing variety. The lymph nodes of patients with LAS were mostly stage I with marked follicular hyperplasia. In 7 of the 14 NHLs the presence of EBV-DNA was clearly demonstrated by dot-blotting and by in situ hybridization. All lymph nodes from the patients with LAS and AIDS-related Hodgkin's disease were negative for EBV by dot-blot and in situ hybridization assays. We conclude that EBV plays a role in the development of AIDS-related lymphomas, but the fact that half these lymphomas are EBV-negative suggests that other mechanisms such as polyclonal stimulation of B-cells by HIV products may also be important.

Methodological Aspects of DNA Image Cytometry in Formalin-Fixed Paraffin-Embedded Material from Pancreatic Adenocarcinoma

Deparaffinized and disintegrated material from conventionally formalin-fixed and paraffin-embedded surgical specimens of 100 cases of ductal adenocarcinoma of the pancreas was Feulgen-stained, and the cytochemical DNA distribution patterns of at least 100 single tumour cells and 50 "control" cells (fibrocytes) were assessed by means of image cytometry (ICM). In 77 cases a sufficient number of neoplastic cells could be obtained for these DNA assessments. The fairly high number (23) of cases that had to be excluded due to too small amounts of disintegrated cells or cell nuclei may be explained by the high content of connective tissue stroma in these pancreatic adenocarcinomas. The tumour cell nuclei in 76 of these 77 cases showed cytochemically a clear-cut "non-diploid" DNA distribution pattern. This observation reflects the well-known highly malignant growth potential of this carcinoma. Despite the fact that about 1/4 of the tumours had to be excluded, the main result of our methodological study is, after all that conventionally formalin-fixed paraffin-embedded specimens of most pancreatic adenocarcinomas can be successfully used for the deparaffinization-disintegration procedure preceding the nuclear DNA assessments by means of ICM. Additional studies are, however, required to obtain the diagnostic and prognostic impact of the results of such cytochemical analyses of the DNA distribution pattern in adenocarcinomas of the pancreas.

Lectin histochemistry of the mast cell: a light microscopical study.

The purpose of this study was to determine if human mast cell granules contain non-repeating oligosaccharide sequences. The binding of lectins to human mast cell granules was studied using a panel of 11 lectins variously selective for both N- and O-linked oligosaccharide sequences. The tissues were principally derived from cutaneous neurofibromata and benign and malignant breast diseases, that is, readily available human material with a known high content of mast cells. Lectin-binding sites in the formalin-fixed paraffin-embedded or resin-embedded material was visualized by means of biotinylated lectins and an avidin-peroxidase technique for light microscopy. The results indicate that human mast cell granules contain abundant N-linked sequences, but few or no O-linked residues. These sequences appear to be mostly in the form of non-bisected highly branched or smaller biantennate sequences, although variable positive binding with erythrophytohaemagglutinin was observed, indicating some degree of bisection.

Stromal cells in cerebellar haemangioblastomas: an immunocytochemical study

The nature of the stromal cells in formalin-fixed paraffin-embedded material from 23 cerebellar haemangioblastomas was investigated using antisera to intermediate filaments (glial fibrillary acidic protein, vimentin and desmin), histiocytic markers (alpha 1-antitrypsin, alpha 1-antichymotrypsin and lysozyme), glycolytic enzymes (alpha and gamma enolase and aldolase C4) and the endothelial markers, factor VIII related antigen and Ulex europaeus I lectin. Most stromal cells stained positively for vimentin and the glycolytic enzymes. Occasional process-bearing cells within the stroma stained strongly for glial fibrillary acidic protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. No stromal cell staining for desmin, lysozyme or the endothelial markers was observed, although the latter stained the vascular endothelium within all neoplasms. The findings do not support previous suggestions of an endothelial or histiocytic origin for the stromal cells. They appear to be a heterogeneous population including entrapped reactive astrocytes and locally-derived non-angiogenic cells of neuroectodermal (pial) origin.