Microdissection molecular copy-number counting (microMCC)--unlocking cancer archives with digital PCR.

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.

A 190-kilodalton protein overexpressed in non-P-glycoprotein-containing multidrug-resistant cells and its relationship to the MRP gene.

BACKGROUND: A 190k (190-kilodalton) membrane protein has been identified in several multidrug-resistant (MDR) cell lines that show decreased drug accumulation without expression of P-glycoprotein. It is not clear whether this 190k protein is involved directly in drug efflux. Recently, a gene for a putative transporter protein, MRP (multidrug resistance-associated protein) has been sequenced and localized to chromosome 16. The protein encoded by this gene contains a 7-amino-acid sequence present in the synthetic peptide used to generate the antiserum recognizing the 190k protein. PURPOSE: The study was undertaken to clarify the relationship of the 190k protein to MRP gene expression in non-P-glycoprotein-containing MDR cells of the large-cell and adenocarcinoma lung cancer lines, COR-L23 and MOR. METHODS: Expression of the 190k protein was determined by Western blot analysis and that of the MRP gene by polymerase chain reaction amplification of complementary DNA reverse transcribed from RNA. Abnormalities of chromosome 16 were investigated in chromosome spreads by fluorescence in situ hybridization. RESULTS: The amount of detectable 190k protein is closely associated with degree of drug resistance. Cell lines surviving in higher drug concentrations have greater amounts of protein, and revertant lines grown without drug for up to 28 weeks show reduced expression of the protein together with enhanced drug sensitivity. The 190k protein appears to be one of the major proteins differentially expressed in membranes of drug-resistant cells. The amount of MRP messenger RNA correlates closely with that of the 190k protein. The MDR cells contain amplified chromosome 16 material with many double minutes in the large-cell lung tumor lines and an enlarged chromosome 16 in the adenocarcinoma lines. CONCLUSION: The 190k protein detected immunologically is likely to be the protein, encoded by the MRP gene, which becomes overexpressed in these cells as a consequence of chromosomal amplification and fragmentation. IMPLICATION: Though associated with drug resistance, enhanced drug efflux, and decreased drug accumulation in cell lines, the role of this protein in clinical resistance has yet to be determined.

Clonal evolution of lung tumors.

Lung tumors, particularly squamous cell carcinomas, are believed to develop through a series of morphological abnormalities, driven by underlying somatic genetic changes. One way of studying this process is to analyze candidate somatic genetic changes in samples of squamous metaplasia and bronchial dysplasia of varying degrees of severity as well as tumor from the same patient. This assumes a clonal relationship between these lesions. In this article, we provide evidence that adjacent, physically distinct bronchial abnormalities are clonally related. This has been achieved using a plaque assay technique to detect the same p53 mutation, present throughout a tumor specimen, in a small proportion of cells in an adjacent squamous metaplasia. In addition, we have obtained two dysplasia samples from a tumor-free patient over a 9-month interval. The earlier sample had one p53 mutation, whereas the later sample has to p53 mutations on different alleles. Thus, the pattern of clonal evolution detected in the parallel samples mimics the pattern seen in longitudinal samples and supports the analysis of synchronously collected samples for the study of tumor progression.

Production of neuromedin B and neuromedin B gene expression in human lung tumor cell lines.

Gastrin-releasing peptide (GRP), a mammalian bombesin-like peptide, has been shown to be an important autocrine growth factor for small cell lung cancer (SCLC). However, not all SCLC cell lines express the GRP gene or respond mitogenically to GRP stimulation, suggesting the existence of other autocrine pathways in this tumor. Neuromedin B (NMB), the mammalian counterpart of amphibian ranatensin, has been shown to be a mitogen for SCLC cell lines in vitro. To determine whether NMB is a potential autocrine growth factor for lung tumors, NMB gene expression, peptide synthesis, and secretion have been investigated in a panel of SCLC and non-SCLC (NSCLC) cell lines. Northern blot analysis and enzymatic amplification from mRNA by polymerase chain reaction showed that the NMB gene was expressed in all SCLC and NSCLC cell lines examined. In contrast, the GRP gene was expressed in four of six classic SCLC cell lines but not in variant SCLC or NSCLC cell lines. Immunoreactive NMB was detected by radioimmunoassay in the majority of classic SCLC, in one of three variant SCLC and in one of three NSCLC cell lines, and secreted NMB was detected in medium conditioned by a SCLC and a NSCLC cell line. The present study also demonstrated the presence of immunoreactive GRP in the absence of detectable GRP gene expression. The antiserum used in the GRP radioimmunoassay failed to cross-react with NMB but showed some cross-reactivity with amphibian phyllolitorin raising the possibility that certain SCLC cell lines may produce a phyllolitorin-like peptide.

A gene from human chromosome region 3p21 with reduced expression in small cell lung cancer.

A combination of cytogenetic and molecular studies has implicated the p21 region of human chromosome 3 as the probable site of a gene the loss of which contributes to the development of small cell lung cancer. We report here the isolation of a gene from this region which is expressed in normal lung tissue and in cell lines derived from a number of different types of tumor, but the expression of which in small cell lung cancer cell lines is undetectable by RNA blot analysis. Although the more sensitive polymerase chain reaction did detect transcripts, a novel quantitative polymerase chain reaction assay showed that their concentration in small cell lung cancer cell lines is less than 3% of that in normal lung.

Sequence of a germ-line N-myc gene and amplification as a mechanism of activation.

Amplification of the N-myc gene has been reported in a number of tumour types including neuroblastoma, retinoblastoma and small cell lung carcinoma. Two amplified human N-myc genes have been sequenced. To determine whether any sequence changes accompany gene amplification and hence to define more precisely the mechanism of activation of the N-myc gene, we have sequenced a germ-line N-myc gene. This sequence differs from the amplified gene sequences by only a single base substitution, which may represent a polymorphic site. This comparison indicates that neither changes in gene structure nor nucleotide sequence accompany gene amplification. Analyses of four small cell lung carcinoma cell lines with amplification of the N-myc gene using restriction endonucleases and Northern blotting reveal no structural aberrations of the majority of the gene copies, substantiating the concept that amplification of the N-myc gene results in high levels of structurally normal N-myc mRNA.

Sequential molecular genetic changes in lung cancer development.

Epithelial tumours develop through a sequence of pre-invasive lesions of increasing disarray driven by underlying somatic genetic changes. We have studied the occurrence of the two most common somatic genetic changes associated with lung cancer in a series of premalignant bronchial lesions representing different stages in lung tumorigenesis. We present evidence that allele loss on chromosome 3 precedes damage to the p53 gene. Damage to chromosome 3 itself appears to be sequential in that the pattern of allele loss seen in dysplasia is often much more discrete than in invasive tumours. This implies that preneoplastic lesions may be a useful source of material for deletion mapping studies aimed at localising the position of tumour suppressor genes. We illustrate this by the comparison of an interstitial deletion described in this study with a homozygous deletion we have described previously, which has resulted in a better definition of the localisation of a tumour suppressor gene believed to be involved in lung cancer development.

Expression of the multidrug resistance-associated protein (MRP) gene in human lung tumours and normal tissue as determined by in situ hybridisation.

In a number of cell lines with a multidrug resistant phenotype, there is no overexpression of the putative efflux pump, P-glycoprotein. Some such lines do, however, overexpress the MRP gene which encodes a protein bearing considerable amino acid homology to P-glycoprotein. We have used in situ hybridisation to study expression of the MRP gene in human cell lines, lung tumours (representing all the major histologies) and normal lung tissue. Considerable heterogeneity of expression was seen in parental cell line COR-L23/P whereas relatively uniform high-level expression was seen in the resistant line COR-L23/R. Normal bronchial epithelium was strongly positive, but the major epithelial component of all eight lung tumours studied showed only a negative to weak signal. However, the leading edge of the tumours consistently produced a more intense signal similar to that in normal epithelium. Areas of lymphocytic infiltrate were more strongly positive than the tumour epithelium. These results suggest that expression of the MRP gene may be a significant factor determining response of lung tumours to chemotherapy, but that considerable caution is needed in the interpretation of expression studies carried out on homogenised tissue biopsies.

A combined approach of conventional and molecular cytogenetics for detailed karyotypic analysis of the small cell lung carcinoma cell line U2020.

Until recently the ability to analyze complex karyotypic rearrangements was totally dependent upon light microscopy of G-banded chromosomes. Developments in the area of molecular cytogenetics have revolutionized such analysis, making it possible to determine the nature of complex rearrangements. An extensive analysis has been made of the small cell lung carcinoma (SCLC) cell line U2020, using a combined approach of conventional and molecular cytogenetics, enabling a highly detailed karyotype to be constructed revealing rearrangements previously undetected by G-banding alone. This approach offers the opportunity to reassess other tumor karyotypes, particularly those of high complexity found in solid tumors, for tumor-specific consistent rearrangements indecipherable by conventional karyotyping.

Elucidation of the mechanism of homozygous deletion of 3p12-13 in the U2020 cell line reveals the unexpected involvement of other chromosomes.

Homozygous deletions in tumor cells have been useful in the localization and validation of tumor suppressor genes. We have described a homozygous deletion in a lung cancer cell line (U2020) which is located within the most proximal of the three regions on the short arm of chromosome 3 believed to be lost in lung cancer development. Construction of a YAC contig map indicates that the deletion spans around 8 Mb, but no large deletion was apparent on conventional cytogenetic analysis of the cell line. To investigate this paradox, whole chromosome, arm-specific, and regional paints have been used. This analysis has revealed that genetic loss has occurred by complex rearrangements of chromosomes 3, rather than simple interstitial deletion. These studies emphasize the power of molecular cytogenetics to disclose unsuspected tumor-specific translocations within the extremely complex karyotypes characteristic of solid tumors.

Cytogenetic abnormalities in human small cell lung carcinoma: cell lines characterized for myc gene amplification.

Nine cell lines established from various malignant tissues of patients with small cell lung carcinoma (SCLC) were examined for chromosomal abnormalities and myc gene amplification. Cytogenetic studies revealed that all cell lines were aneuploid, often with a bimodal distribution with modal concentrations in the hypodiploid and hypertriploid range. With respect to chromosome #3, deletions of 3p were confined to six of nine SCLC "classic" lines. The region of overlap of the observed 3p deletions lies within 3p21-3p24 which is in agreement with previous assignments. Six of the nine lines tested with c-, N-, and L-myc probes showed an increase of between ten- and 100 fold in myc gene copy number. Coamplification of two or more of these genes was not observed in any cell line. Five of the six lines with myc gene amplification had cytogenetic markers of gene amplification either in the form of homogeneously staining regions (HSR) or double minutes (DM). Our results confirm that cytogenetically visible deletions of 3p are often present in cell lines established from patients with SCLC, and that mutually exclusive c-, L-, or N-myc gene amplification is also a common event in SCLC cell lines.

Clonal divergence in lung cancer development is associated with allelic loss on chromosome 4.

Patients who receive curative treatment for lung cancer can develop additional lung tumors that may or may not be related to the original tumor and thus require different clinical management. If a subsequent tumor has a pattern of allele loss, revealed by allelotype analysis, overlapping that of the first tumor, it is believed to be a local recurrence or metastasis. In this case history, we present loss of heterozygosity analyses of the original primary tumor, and two second primary tumors occurring in the ipsilateral and the contra-lateral lungs. The allelotyping suggests that these tumors are all clonally related but concordance is not complete. Our interpretation is that the original primary tumor and the two new primary tumors have developed to full malignancy independently, but are clonally related, possibly via a clone of motile progenitor cells. Deletion mapping of DNA from biopsies of this patient delineated a region in 4p16 that we had previously shown to be lost in the transition from carcinoma in situ to invasive tumor. We identified a minimally deleted region encompassing six genes including two candidate tumor suppressor genes, CRMP1 a lung cancer metastasis-suppressing gene and PPP2R2C a gene for a regulatory subunit of the PP2 complex known to suppress tumorigenesis, particularly viral induced transformation.

Genetic changes in the development of lung cancer.

Both inherited predisposition and acquired somatic genetic changes contribute to lung tumour development but the former is likely to be the minor component. The relative ease with which lung tumours can be established as cell lines has resulted in extensive cytogenetic analysis, and, this together with molecular techniques which assess loss of genetic material has revealed consistent patterns of genetic damage. Some of these characteristic somatic genetic changes have been traced back to the pre-invasive stage of tumour development and this information may contribute to the future management of this currently intractable disease.

Multi-step evolution of lung cancer.

In an attempt to improve the poor survival rates for lung cancer, therapeutic strategies require a deeper understanding of the biological events contributing to the formation and progression of the disease. In particular, the importance of studying the different stages of lung cancer including early pre-neoplasia is being recognised and studies examining genetic changes in pre-invasive and invasive lesions are being used to identify key events in tumorigenesis.

PCR-RFLP studies on chromosome 3p in formaldehyde-fixed, paraffin-embedded cervical cancer tissues.

Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p21-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues.

Lung cancer . 3: Fluorescence bronchoscopy: clinical dilemmas and research opportunities.

Recent developments in the detection of pre-invasive lesions of the large airways by fluorescence bronchoscopy are reviewed and the clinical and biological implications discussed.

Amplification and expression of mdr1 gene in a multidrug resistant variant of small cell lung cancer cell line NCI-H69.

Amplification and expression of the mdr1 gene encoding P-glycoprotein have been studied in H69/LX4 a multidrug resistant variant (MDR) of small cell lung cancer (SCLC) cell line NCI-H69. Recently a second independently derived MDR variant of this cell line designated H69/AR was found by others not to show amplification, rearrangement or over-expression of the mdr1 gene. The present study reports that in marked contrast to H69/AR, H69/LX4 shows amplification and expression of the P-glycoprotein gene and raises the possibility that P-glycoprotein hyperexpression may be a clinically relevant component of MDR in some SCLC tumours.

Localization of polymorphic DNA probes frequently deleted in lung carcinoma.

Five polymorphic DNA segments from human chromosome 3, that are frequently deleted in lung carcinoma were mapped by non-isotopic in situ hybridization to metaphase chromosomes. The DNA segment D3S3 mapped to 3p13-p14.2, D3S6 to 3p14.3-p14.5, D3S48 to distal 3p21-p22, ERBA beta to 3p24.3 and ERBA2 to 3p24.3. The map location of ERBA beta and ERBA2 was confirmed by re-mapping each probe in combination with D3S6 as a marker for 3p14.

Multicolour fluorescence in situ hybridisation to order small, single-copy probes on metaphase chromosomes.

In constructing complete human chromosome maps, the relative order of markers and their precise chromosomal location will be combined. Multicolour in situ hybridisation, in which two probes are simultaneously hybridised to chromosomes and subsequently distinguished, potentially will provide both types of information. Using this technique, we have produced an ordered map of eight human chromosome 3 DNA markers, using small, single-copy probes that can detect target sequences ranging in size from 4 kb to as little as 500 bp.