This page presents interesting papers and about single-cell, single-molecule qRT-PCR, or nano-liter volume range applieing real-time qRT-PCR:
Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte.
Sindelka R, Jonák J, Hands R, Bustin SA, Kubista M.
Nucleic Acids Res. 2008 36(2):387-92.
Laboratory of Gene Expression, Institute of Molecular Genetics, Academy of
Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic.
Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression profiles within single cells. We demonstrate its usefulness by dissecting an oocyte from Xenopus laevis into slices along its animal-vegetal axis, extracting its RNA and measuring the levels of 18 selected mRNAs by real-time RT-PCR. This identified two classes of mRNA, one preferentially located towards the animal, the other towards the vegetal pole. mRNAs within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. The polarization is substantial, though not extreme, with around 5% of vegetal gene mRNA molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. Most animal pole mRNAs were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mRNA expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mRNA gradients.
Quantification of mRNA in single cells and modelling of RT-qPCR induced noise.
Bengtsson M, Hemberg M, Rorsman P, Stahlberg A.
BMC Mol Biol. 2008 9: 63.
Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford,
The Churchill Hospital, Oxford, OX3 7LJ, UK.
BACKGROUND: Gene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells.
RESULTS: Low concentration of guanidine thiocyanate was used to fully lyse single pancreatic beta-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined.
CONCLUSION: Noise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene.
Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR.
Warren L, Bryder D, Weissman IL, Quake SR.
Proc Natl Acad Sci U S A. 2006 103(47): 17807-12.
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

We report here a systematic, quantitative population analysis of transcription factor expression within developmental progenitors, made possible by a microfluidic chip-based "digital RT-PCR" assay that can count template molecules in cDNA samples prepared from single cells. In a survey encompassing five classes of early hematopoietic precursor, we found markedly heterogeneous expression of the transcription factor PU.1 in hematopoietic stem cells and divergent patterns of PU.1 expression within flk2- and flk2+ common myeloid progenitors. The survey also revealed significant differences in the level of the housekeeping transcript GAPDH across the surveyed populations, which demonstrates caveats of normalizing expression data to endogenous controls and underscores the need to put gene measurement on an absolute, copy-per-cell basis.
220-plex microRNA expression profile of a single cell.
Tang F, Hajkova P, Barton SC, O'Carroll D, Lee C, Lao K, Surani MA.
Nat Protoc. 2006 1(3): 1154-1159.
Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental
Biology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.
Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.
MicroRNA quantitation from a single cell by PCR using SYBR Green detection and LNA-based primers.
NATURE METHODS FEBRUARY 2008   sponsored by Exiqon
We describe a new, highly sensitive and specific PCR approach for quantitation of microRNAs (miRNAs): the miRCURY™ LNA microRNA PCR system. The method, which allows detection of 10 copies of miRNA, is enabled by the use of Locked Nucleic Acids (LNA™). The LNA-conferred sensitivity facilitates accurate detection of miRNA expression in a single cell.
Split single-cell RT-PCR analysis of Purkinje cells.
Shigeyuki Esumi, Ryosuke Kaneko, Yoshimi Kawamura & Takeshi Yagi
Nature Protocols 1, - 2143 - 2151 (2006)
This protocol details a method for analyzing the expression of multiple genes from a single Purkinje neuron, including the determination of whether the gene expression is monoallelic or biallelic. The protocol describes how to extract a single, living Purkinje cell for reverse transcription, divide the cDNAs into three equal samples and subject those to triplicate amplification of multiple targets by two rounds of PCR (first a multiplex PCR then a gene-specific nested PCR) and finally discriminate the allelic expression of the transcript by direct sequencing of the PCR products. In optimal conditions, this method permits the analysis of the expression of 18 genes in a single Purkinje cell. This protocol can be completed in 5–6 d.
Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis.
Kurimoto K, Yabuta Y, Ohinata Y, Saitou M.
Nat Protoc. 2007;2(3): 739-52.
Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology,
RIKEN Kobe Institute, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (approximately 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5-6 days.
An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.
Kurimoto K, Yabuta Y, Ohinata Y, Ono Y, Uno KD, Yamada RG, Ueda HR, Saitou M.
Nucleic Acids Res. 2006 34(5): e42.
Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology,
RIKEN Kobe Institute, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.

Single-cell gene expression profiling.
Levsky JM, Shenoy SM, Pezo RC, Singer RH.
Science. 2002 297(5582): 836-840.
Department of Anatomy and Structural Biology, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
A key goal of biology is to relate the expression of specific genes to a particular cellular phenotype. However, current assays for gene expression destroy the structural context. By combining advances in computational fluorescence microscopy with multiplex probe design, we devised technology in which the expression of many genes can be visualized simultaneously inside
single cells with high spatial and temporal resolution. Analysis of 11 genes in serum-stimulated cultured cells revealed unique patterns of gene expression within individual cells. Using the nucleus as the substrate for parallel gene analysis, we provide a platform for the fusion of genomics and cell biology: "cellular genomics."

Gene expression and the myth of the average cell.
Levsky JM, Singer RH.
Trends Cell Biol. 2003 13(1): 4-6.
Department of Anatomy and Structural Biology, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
We all know that gene expression occurs within cells, yet we do not think of expression in terms of its fundamental unit - a single cell. Instead, we understand the expression of genes in terms of a cell population as all of our information comes from samples containing millions of cells. From a complex mixture of cells, we attempt to infer the probable state of an average cell in the population. In truth, what we obtain is an averaged cell, a contrivance for representing biological knowledge beyond the limits of detection. We never know the variation among the members of the population that our methods average into a mean. Recent technological advances allow the precise measurement of single-cell transcriptional states to study this variability more rigorously. How genes are expressed in the population is strikingly different to what we have assumed from extrapolating to an average cell. Does the average cell actually exist? As we discuss, it is becoming increasingly clear that it doesn't.

Combining laser capture microdissection with quantitative real-time PCR:
Effects of tissue manipulation on RNA quality and gene expression.
Kerman IA, Buck BJ, Evans SJ, Akil H, Watson SJ.
J Neurosci Methods. 2006 153(1): 71-85.
Molecular and Behavioral Neuroscience Institute, Department of Psychiatry,
University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109, USA.
Laser capture microdissection (LCM) is increasingly being used in quantitative gene expression studies of the nervous system. The current study aimed at determining the impact of various tissue manipulations on the integrity of extracted RNA in LCM studies. Our data indicate that various tissue preparation strategies prior to microdissection may decrease RNA quality by as much as 25%, thus affecting expression profiles of some genes. To circumvent this problem, we developed a strategy for reverse transcriptase real-time PCR that has considerable sensitivity and can be used to calculate relative changes in gene expression. This approach was validated in subregions of the rat cerebellum. Accordingly, expression of glial gene markers - myelin-associated glycoprotein and proteolipid protein 1 - was found 70-160-fold higher in the white matter layer of the cerebellar cortex as compared to the neuron-enriched granular layer. In contrast, expression of a specific neuronal maker, neuron-specific enolase, was found seven-fold higher in the granular layer, as compared to the white matter layer. Furthermore, this approach had high sensitivity and specificity as we were able to detect a 38% decrease in the expression of neuron-specific enolase without a change in the expression of glial markers following administration of the neurotoxin, ibotenic acid. These results demonstrate feasibility of performing accurate semi-quantitative gene expression analyses in LCM samples.
RNA expression profiling at the single molecule level.
Hesse J, Jacak J, Kasper M, Regl G, Eichberger T, Winklmayr M, Aberger F,
Sonnleitner M, Schlapak R, Howorka S, Muresan L, Frischauf AM, Schutz GJ.
Biophysics Institute, Johannes Kepler University Linz, A-4040 Linz, Austria;
Genome Res. 2006 Jun 29



We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm(2) size. A detection limit of 1.3 fM target oligonucleotide concentration-corresponding to only 39,000 molecules in the sample solution-and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

Quantitative single-cell RT-PCR and Ca(2+) imaging in brain slices.
Durand GM, Marandi N, Herberger SD, Blum R, Konnerth A.
Pflugers Arch. 2006 451(6): 716-726.
Institut fur Physiologie, Ludwig-Maximilians-Universitat, Pettenkofer Strasse 12, 80336, Munchen, Germany
We have established a quantitative reverse transcriptase-PCR (RT-PCR) approach for the analysis of RNA transcript levels in individual cells of living brain slices. Quantification is achieved by using rapid-cycle, real-time PCR protocols and high-resolution external cDNA standard curves for the gene of interest. The method consists of several procedures, including cell soma harvest, reverse transcription, and an optimized cDNA purification step, which allowed us to quantify transcripts in small types of neurons, like cerebellar granule cells. Thus, we detected in single granule cells an average of 20 transcript copies of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase. We combined two-photon calcium imaging and quantitative RT-PCR in single Purkinje and granule cells, respectively, and identified distinct glutamate receptor-dependent Ca(2+) responses in these two cell types. The approach was further tested by profiling the expression of the ionotropic glutamate receptor subunits NR2B and NR2C in the cerebellum. Our study revealed a developmental switch from an average of 15 NR2B copies/cell at postnatal day 8 (P8) to about five NR2C copies/cell after P26. Taken together, our results demonstrate that the new method is rapid, highly sensitive, provides reliable results in neurons of various sizes, and can be used in combination with Ca(2+) imaging.
RNA amplification strategies for small sample populations.
Ginsberg SD.
Methods. 2005 37(3): 229-237.
Center for Dementia Research, Nathan Kline Institute, Department of Psychiatry
and Physiology and Neuroscience, New York University School of Medicine,
Orangeburg, NY 10962, USA.
Advances in high throughput cloning strategies have led to sequencing of the human genome as well as progress in the sequencing of the genome of several other species. Consequently, the field of molecular genetics is blossoming into a multidisciplinary entity that is revolutionizing the way researchers evaluate a myriad of critical concepts such as development, homeostasis, and disease pathogenesis. There is tremendous interest in the quantitative assessment of tissue-specific expression of both newly identified and well characterized specific genes and proteins. At present, an ideal approach is to assess gene expression in single elements recorded physiologically in living preparations or by immunocytochemical or histochemical methods in fixed cells in vitro or in vivo. The quantity of RNA harvested from individual cells is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction based analyses, and linear RNA amplification including amplified antisense RNA amplification and a newly developed terminal continuation RNA amplification methodology have been developed for use in combination with microdissection procedures and cDNA/oligonucleotide microarray platforms. RNA amplification is a series of intricate procedures to amplify genetic signals from minute quantities of starting materials for microarray analysis and other downstream genetic methodologies. RNA amplification procedures effectively generate quantities of RNA through in vitro transcription. The present report illustrates practical usage of RNA amplification technologies within the context of regional, population cell, and single cell analyses in the brain.

GENE EXPRESSION OF SINGLE CHONDROCYTES
Eleswarapu, SV; Shieh, AC; Athanasiou, KA
Rice University, Houston, TX

POSTER


"Per cell" normalization method for mRNA measurement by quantitative PCR and microarrays.
Jun Kanno, Ken-ichi Aisaki, Katsuhide Igarashi, Noriyuki Nakatsu, Atsushi Ono, Yukio Kodama and Taku Nagao
BMC Genomics2006, 7:64
Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan and President, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Background: Transcriptome data from quantitative PCR (qPCR) and DNA microarrays are typically obtained from a fixed amount of RNA collected per sample. Therefore, variations in tissue cellularity and RNA yield across samples in an experimental series compromise accurate determination of the absolute level of each mRNA species per cell in any sample. Since mRNAs are copied from genomic DNA, the simplest way to express mRNA level would be as copy number per template DNA, or more practically, as copy number per cell.
Results: Here we report a method (designated the "Percellome" method) for normalizing the expression of mRNA values in biological samples. It provides a "per cell" readout in mRNA copy number and is applicable to both quantitative PCR (qPCR) and DNA microarray studies. The genomic DNA content of each sample homogenate was measured from a small aliquot to derive the number of cells in the sample. A cocktail of five external spike RNAs admixed in a dose-graded manner (dose-graded spike cocktail; GSC) was prepared and added to each homogenate in proportion to its DNA content. In this way, the spike mRNAs represented absolute copy numbers per cell in the sample. The signals from the five spike mRNAs were used as a dose-response standard curve for each sample, enabling us to convert all the signals measured to copy numbers per cell in an expression profile-independent manner. A series of samples was measured by qPCR and Affymetrix GeneChip microarrays using this Percellome method, and the results showed up to 90% concordance.
Conclusion: Percellome data can be compared directly among samples and among different studies, and between different platforms, without further normalization. Therefore, "percellome" normalization can serve as a standard method for exchanging and comparing data across different platforms and among different laboratories.

Multiplexing RT-PCR for the detection of multiple miRNA species in small samples.
Lao K, Xu NL, Yeung V, Chen C, Livak KJ, Straus NA.
Biochem Biophys Res Commun. 2006 343(1): 85-89.
Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404, USA.
MicroRNAs are short (approximately 22 nucleotides), non-coding RNAs that play critical roles in gene regulation and may be used as rapid precise diagnostic indicators of early stages of cancer. The small size of these RNAs makes detection of multiple microRNA species in very small samples problematic. Here we investigate the parameters associated with multiplexing RT-PCR to obtain relative abundance profiles of multiple microRNAs in small sample sizes down to the amount of RNA found in a single cell.

Gene expression profiling of individual bovine nuclear transfer blastocysts.
Joanna Somers, Craig Smith, Martyn Donnison, David N Wells, Harold Henderson, Lance McLeay and P L Pfeffer
Reproduction (2006) 131 1073–1084

During somatic cell nuclear transfer the gene expression profile of the donor cell has to be changed or reprogrammed extensively to reflect that of a normal embryo. In this study we focused on the switching on of embryonic genes by screening with a microarray consisting of 5,000 independent cDNA isolates derived from a bovine blastocyst library which we constructed for this purpose. Expression profiling was performed using linearly amplified RNA from individual day 7 nuclear transfer (NT) and genetically half-identical in vitro produced (IVP) blastocysts. We identified 92 genes expressed at lower levels in NT embryos whereas transcripts of 43 genes were more abundant in NT embryos (P 0.05,  1.5-fold change). A range of functional categories was represented among the identified genes, with a preponderance of constitutively expressed genes required for the maintenance of basal cellular function. Using a stringent quantitative SYBR-green real time RT-PCR based approach we found, when comparing the means of the expression levels of a larger set of individual embryos, that differences were small (2-fold) and only significant for two of the seven analysed genes (KRT18, SLC16A1). Notably, examination of transcript levels of a single gene in individual embryos could not distinguish an NT from a control embryo. This unpredictability of individual gene expression on a global background of multiple gene expression changes argues for a predominantly stochastic nature of reprogramming errors.

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.
Zheng Z, Luo Y, McMaster GK.
Clin Chem. 2006 52(7): 1294-1302.
Panomics, Inc., 6519 Dumbarton Circle, Fremont, CA 94555, USA.
BACKGROUND: Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variationsand biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format.
METHODS: We performed single- and
multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection.
RESULTS: The single- and multiplex assays could quantitatively measure as few as 6,000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood.
CONCLUSIONS: Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Expression profiling of small cellular samples in cancer: less is more.
Glanzer JG, Eberwine JH.
Br J Cancer. 2004 90(6): 1111-1114.
Department of Pharmacology, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6058, USA.
Expression profiling of tumours from cancer patients has uncovered several genes that are critically important in the progression of a normal cell to an oncogenic phenotype. Leading the way in these discoveries is the use of microarrays, a technology that is currently in transition from basic science applications to use in the clinic. Microarrays can determine the global gene regulation of an individual cancer, which may be useful in formulating an individualised therapy for the patient. Currently, cells used in breast cancer microarray studies often come from either homogenous cultures or heterogeneous biopsy samples. Both cell sources are at a disadvantage in determining the most accurate gene profile of cancer, which often consists of multiple subspecies of cancerous cells within a background of normal cells. Therefore, acquisition of small, but highly specific biopsies for analysis may be required for an accurate expression analysis of the disease. Amplification methods, such as polymerase chain reaction (PCR) and amplified antisense RNA (aRNA) amplification, have been used to amplify the mRNA signal from very small samples, which can then be used for microarray analysis. In this study, we describe the acquisition, amplification, and analysis of very small samples (<10000 cells) for expression analysis and demonstrate that the ultimate resolution of cancer expression analysis, one cell, is both feasible and practical.

Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer.
Klein CA, Blankenstein TJ, Schmidt-Kittler O, Petronio M, Polzer B, Stoecklein, NH, Riethmuller G.
Lancet. 2002 360(9334):  683-689.
Institut fur Immunologie, Ludwig-Maximilians-Universitat Munchen, D-80336 Muenchen, Germany.

BACKGROUND: Because cancer patients with small tumours often relapse despite local and systemic treatment, we investigated the genetic variation of the precursors of distant metastasis at the stage of minimal residual disease. Disseminated tumour cells can be detected by epithelial markers in mesenchymal tissues and represent targets for adjuvant therapies.
METHODS: We screened 525 bone-marrow, blood, and lymph-node samples from 474 patients with breast, prostate, and gastrointestinal cancers for single disseminated cancer cells by immunocytochemistry with epithelial-specific markers. 71 (14%) of the samples contained two or more tumour cells whose genomic organisation we studied by single cell genomic hybridisation. In addition, we tested whether TP53 was mutated. Hierarchical clustering algorithms were used to determine the degree of clonal relatedness of sister cells that were isolated from individual patients.
FINDINGS: Irrespective of cancer type, we saw an unexpectedly high genetic divergence in minimal residual cancer, particularly at the level of chromosomal imbalances. Although few disseminated cells harboured TP53 mutations at this stage of disease, we also saw microheterogeneity of the TP53 genotype. The genetic heterogeneity was strikingly reduced with the emergence of clinically evident metastasis.
INTERPRETATION: Although the heterogeneity of primary tumours has long been known, we show here that early disseminated cancer cells are genomically very unstable as well. Selection of clonally expanding cells leading to metastasis seems to occur after dissemination has taken place. Therefore, adjuvant therapies are confronted with an extremely large reservoir of variant cells from which resistant tumour cells can be selected.
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