Ernst Heinmöller

Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12 of Ki-ras by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR. The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 30%) and five cells (efficiency rate I-PEP-PCR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 30 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses in diagnostic and experimental tumor pathology.

Molecular Analysis of Microdissected Tumors and Preneoplastic Intraductal Lesions in Pancreatic Carcinoma

Little or no data exist concerning the inactivation of tumor suppressor genes in intraductal lesions surrounding invasive ductal pancreatic carcinomas. Using a novel improved primer extension and preamplification polymerase chain reaction, we analyzed microdissected paraffin-embedded specimens of pancreatic carcinoma (n = 29) and their corresponding pancreatic intraductal lesions (PIL, n = 331) for loss of heterozygosity (LOH) of p16(INK4), DPC4, and p53 by microsatellite analysis and for p53 protein by immunohistochemistry. LOH at the p16(INK4) locus (9p21) was found in nine of 22 informative tumors (41%), in 15 of 25 tumors (60%) at the DPC4 locus (18q21.1), and in 22 of 27 tumors (81%) at the p53 locus (17p13). Homozygous deletions of p16(INK4) and DPC4 were found in eight of 22 (36%) and four of 25 tumors (16%), respectively. Furthermore, 24 of 29 tumors (83%) revealed considerable intratumoral genetic heterogeneity. In 165 of 277 PILs (60%) having suitable DNA for microsatellite analysis, alterations in at least one tumor suppressor gene were found. In individual PILs, up to three alterations were detected, and p53 LOH occurred even in morphologically normal-appearing ductal epithelium near the tumor. Although deletions of all three tumor suppressor genes were found in PILs without nuclear atypia, there was a tendency toward earlier LOH of p16(INK4) compared to DPC4 and p53 in these lesions. LOH in tumors accompanied positive p53 immunohistochemistry in 81% but only in 38% in PILs.

Studies on tumor-cell-induced platelet aggregation in human lung cancer cell lines.

We investigated the ability of human lung cancer cells of different histological subtypes to cause platelet aggregation. Tumor-cell-induced platelet aggregation (TCIPA) was studied in vitro in 13 human lung cancer cell lines [small-cell lung cancer (SCLC), squamous-cell lung cancer, large-cell lung cancer, adenocarcinoma and alveolar-cell lung cancer]. Three tumor cell lines failed to aggregate platelets in plateletrich plasma, whereas platelet aggregation was induced by 12 cell lines when added to washed platelets and minimal amounts of platelet-poor plasma (0.5% v/v). The thrombin antagonist hirudin inhibited TCIPA in non-small-cell lung cancer cell lines (NSCLC). In SCLC, TCIPA was fully abolished only when the ADP scavenger apyrase was added to hirudin. Thus ADP and thrombin generation by these tumor cell lines are responsible for platelet aggregation. The ability to activate platelets independently of coagulation factors VII and X was demonstrated for 8 cell lines. Electronmiicroscopically, direct tumor-cell/platelet contact was found to be the initiating mechanism of TCIPA in SCLC, whereas tumor-cell/platelet contacts in NSCLC could only be observed at the peak of the aggregation curve. Lung cancer cells activate platelets in vitro by generation of thrombin and/or ADP.

Toward efficient analysis of mutations in single cells from ethanol-fixed, paraffin-embedded, and immunohistochemically stained tissues.

Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments <200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage.

Pitfalls in diagnostic molecular pathology – significance of sampling error

Abstract Today, molecular diagnostic tests are widely used in clinical medicine with polymerase chain reaction (PCR)-based techniques being of particular interest. In tissue specimens, however, false-positive and false-negative results can be obtained if pathomorphological and processing aspects are not considered. We therefore studied the impact of tissue sampling in three widely used diagnostic tests: (1) assessment of clonality in B-cell non-Hodgkins lymphoma, (2) analysis of microsatellite instability (MSI) in colorectal neoplasia, and (3) demonstration of mycobacterium tuberculosis. Tissue sections of routinely formalin-fixed and paraffin-embedded diagnostic specimens were taken, and the significance of sampling was systematically investigated using laser microdissection or processing of the entire section. PCR analyses were done according to standard protocols. False-positive pseudo-monoclonality was obtained in small gastrointestinal biopsies and in laser microdissected lymph follicles of non-neoplastic tonsils. False negativity (pseudo-stability) could be demonstrated in a case with hereditary rectal adenoma if whole tissue sections were taken without microdissection of the most dysplastic subareas. Demonstration of mycobacteria failed in tissue sections of a formalin-fixed lymph node that was positive after complete digestion or in fresh frozen material of the same patient. In diagnostic molecular pathology, sampling error is an important source of false-positive and false-negative results, particularly if disease- and tissue-specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity, are ignored.

The genetic basis of sporadic pancreatic cancer

Adenocarcinomas of the pancreatic gland are the fifth leading cause of cancer death in the USA and western Europe with a 5-year survival rate of less than 5% and a median survival of less than 6 months [76]. They bear histological resemblance to the pancreatic ducts and are referred to as pancreatic ductal adenocarcinoma [5,34,53]. Due to the late occurrence of symptoms and the high metastatic potential, pancreatic adenocarcinomas have a dismal prognosis. Since the pancreaticoduodenectomy, a surgical resection that includes the pancreatic head, the duodenum, common bile duct and the gallbladder was introduced in 1912 by Kausch, surgery still remains the primary curative treatment. However, the 5-year survival for patients undergoing resection, is only about 20% [76] and about 80% of all cancers are unresectable at the time of diagnosis [2]. In order to improve the clinical management of cancer patients, it is mandatory to understand the genetic basis of pancreatic cancers. This review describes the histological progression of pancreatic adenocarcinomas and summarizes the underlying chromosomal and genetic alterations.

Genomic instability at both the base pair level and the chromosomal level is detectable in earliest PanIN lesions in tissues of chronic pancreatitis.

Objective: Chronic pancreatitis (CP) is a predisposing disease for pancreatic carcinoma (PC), however, precise molecular mechanisms of cancer development in the background of CP are ill defined. Methods: A total of 443 laser-microdissected pancreatic intraepithelial neoplasias (PanINs), acinar-ductal metaplasia (ADM), and normal ducts from 21 patients with CP were analyzed for loss of heterozygosity (LOH) and immunohistochemical protein expression of p53, p16, and DPC4. Pancreatic intraepithelial neoplasias were analyzed for mutations in p53, p16, and Ki-ras genes by ABI sequencing. Aneuploidy was determined by fluorescence in situ hybridization with probes for chromosomes 3, 7, 8, and 17. Results: Loss of heterozygosity rate in PanIN-1 and ADM was between 1.7% (p53) and 5.8% (p16). In PanIN-3, p53 protein overexpression and loss of expression for p16 and DPC4 protein were seen. Heterozygous mutations of p53 and p16 without LOH were found in PanIN-1A and ADM, whereas homozygous mutations were found in PanIN-3. Aneuploidy increased from PanIN-1A to PanIN-3. Ki-ras mutations were discovered first in PanIN-1. Conclusions: Heterozygous mutations of p53- and p16 genes together with chromosomal instability occur early in CP and are clonally expanded, but final inactivation mostly by LOH happens later in pancreatic carcinogenesis. Determination of aneuploidy in pancreatic juice may be of value for early detection and risk assessment in patients with long-standing CP.

Laser microdissection of small tissue samples--application to chronic pancreatitis tissues.

Laser microdissection is considered to be the gold standard of tissue sampling, especially if a defined small tissue area consisting of single or few cells within a heterogeneous tissue compartment is of interest. This sophisticated technique offers the opportunity of rapid and contamination-free tissue sampling for RNA- or DNA-based molecular genetic studies. We have applied laser microdissection to a molecular genetic study of pancreatic intraductal lesions (PanINs) in tissues of chronic pancreatitis, where an exact microdissection of small ducts within a dense fibrous tissue is of paramount importance for following analysis. From nine patients suffering from chronic pancreatitis, formalin-fixed, paraffin-embedded tissue specimens were laser microdissected, and a total of 202 normal ducts and PanINs of grade PanIN-1A to grade PanIN-2 were harvested. After whole genome amplification by improved primer extension and preamplification PCR (I-PEP-PCR), microsatellite-PCR based loss of heterozygosity analysis (LOH) of the tumor suppressor gene loci TP53, p16INK4, and DPC4 was performed. One of 85 informative duct lesions (1.2%) had LOH of TP53, 1 of 76 duct lesions (1.3%) had LOH of DPC4, and 2/29 duct lesions (6.9%) showed LOH of p16INK4. Microsatellite instability (MSI) was seen in 2 of 178 duct lesions (1.1%). Immunohistochemical staining of p53 protein and DPC4 protein revealed no aberrant expression. These preliminary data indicate that LOH of tumor suppressor genes, important in pancreatic cancer genesis or MSI, can be found in chronic pancreatitis tissues, but their incidence is low.

Microdissection and molecular analysis of single cells or small cell clusters in pathology and diagnosis - Significance and challenges

Colour figures can be viewed on http://www.esacp.org/acp/2002/24‐4_5/heinmoeller.htm

Pankreaskarzinom — Bedeutung der Laparoskopie und der intraoperativen Spülzytologie für das perioperative Staging

ZusammenfassungGrundlagen: Operabilität bei Pankreaskarzinom erfordert auch heute noch trotz sensitiver Diagnostik häufig eine Staging-Laparotomie. Methodik: Von September 1992 bis Dezember 1995 wurde in einer prospektiven Studie an 70 Patienten mit Pankreaskarzinom die Wertigkeit von perioperativer Laparoskopie und peritonealer Zytologie zur Sicherung der Operabilität überprüft. Ergebnisse: Trotz Durchführung sensitiver Staging-Untersuchungen (EUS, Spiral-CT, ERCP) wurden in 15% unerwartete subglissonische Lebermetastasen gefunden. Bei 46 von 55 operierten Patienten wurde eine peritoneale Zytologie perioperativ gewonnen. Alle Patienten mit Tumorzellnachweis (n=11) waren inoperabel. Bei 10 Patienten dieser Gruppe war die ventrale Pankreasorgangrenze durchbrochen. 9 Patienten zeigten präoperativ Aszites, hierin konnte jedoch nur bei 2 Patienten der Tumorzellnachweis geführt werden. Zur Prädiktion der Inoperabilität lagen für Zytologie alleine die Sensitivität bei 60 und die Spezifität bei 80%. Schlußfolgerungen: Nach Einführung der diagnostischen Laparoskopie und vermehrtem Einsatz kombinierter minimal invasiver Verfahren (Endoprothese/PTCD, laparoskopische Gastrojejunostomie) sank die Letalität der chirurgischen Palliation bei Pankreaskarzinom von 16,8 auf 5,8%. Laparoskopie und Peritonealzytologie sagten mit einer Wahrscheinlichkeit von 80% die Inoperabilität voraus. Da Lebermikrometastasen in 5 bis 10% zu erwarten sind, ist eine perioperative Laparoskopie zur Sicherung der Operabilität sinnvoll. Bei bereits drainierten Gallenwegen sollte zur Verbesserung der Lebensqualität eine minimal invasive Palliation angestrebt werden.SummaryBackground: Demonstration of operability even nowadays often requires diagnostic laparotomy in pancreatic carcinoma. Methods: In a prospective study on 70 patients with pancreatic carcinoma from September 1992 till December 1995 the impact of laparoscopy and peritoneal cytology was measured. Results: Although sensitive diagnostic procedures where practized (endoscopic sonography, spiral CT-scan, ERCP) we found subglissonian metastasis in 15%. In 46 patients peritoneal washings were performed, all patients with detected peritoneal tumor cells (n=11) were inoperable. In 10 patients of this group the ventral integrity of pancreas was damaged. In 9 patients with preoperative existant ascites only in 2 cases peritoneal tumor cells could be detected. Sensitivity at 60% and specifity at 80% were the values for cytology as a predictor of inoperability. Conclusions: With diagnostic laparoscopy and combined palliative procedures (endoprosthesis via ERCP or PTCD, laparoscopic gastrojejunostomy) operative letality of diagnostic laparotomy with palliation decreased from 16.8% to 5,8%. Together with peritoneal cytology prediction of inoperability was possible at a security range of 80%. As unexpected subglissonian micrometastasis may be present in 5 to 10% perioperative laparoscopy is advisable to demonstrate operability. If endoscopic biliary drainage has been installed minimally invasive palliation procedures should be prefered.