Biomarker concordance between primary colorectal cancer and ovarian metastases: a Dutch cohort study.

PURPOSE: The genetic characteristics and mismatch repair (MMR) status of the primary tumor and corresponding metastases in colorectal cancer (CRC) are generally considered to be highly concordant. This implies that either the primary or metastatic tumor can be used for testing gene mutation and MMR status. However, whether this is also true for CRC and their ovarian metastases is currently unknown. Ovarian metastases generally show a poorer response to systemic therapy compared to other metastatic sites. Differences in biomarker status between primary CRC and ovarian metastases could possibly explain this difference in therapy response. METHODS: The study cohort was selected from CRC patients treated in two Dutch hospitals. Eligible patients with CRC and ovarian metastasis who were surgically treated between 2011 and 2018 were included. CRC and corresponding ovarian metastatic tissues were paired. Gene mutation status was established using next-generation sequencing, while the MMR status was established using either immunohistochemistry or microsatellite instability analysis. RESULTS: Matched samples of CRC and ovarian metastasis from 26 patients were available for analysis. A biomarker concordance of 100% was detected. CONCLUSION: Complete biomarker concordance was found between MMR proficient CRC and their matching ovarian metastasis. Biomarker testing of MMR proficient CRC tissue appears to be sufficient, and additional testing of metastatic ovarian tissue is not necessary. Differences in therapy response between ovarian metastases and other metastases from CRC are thus unlikely to be caused by differences in the genetic status.

Recommendations for the clinical interpretation and reporting of copy number gains using gene panel NGS analysis in routine diagnostics.

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity.

The combination of IDH1 mutations and MGMT methylation status predicts survival in glioblastoma better than either IDH1 or MGMT alone.

BACKGROUND: Genetic and epigenetic profiling of glioblastomas has provided a comprehensive list of altered cancer genes of which only O(6)-methylguanine-methyltransferase (MGMT) methylation is used thus far as a predictive marker in a clinical setting. We investigated the prognostic significance of genetic and epigenetic alterations in glioblastoma patients. METHODS: We screened 98 human glioblastoma samples for genetic and epigenetic alterations in 10 genes and chromosomal loci by PCR and multiplex ligation-dependent probe amplification (MLPA). We tested the association between these genetic and epigenetic alterations and glioblastoma patient survival. Subsequently, we developed a 2-gene survival predictor. RESULTS: Multivariate analyses revealed that mutations in isocitrate dehydrogenase 1 (IDH1), promoter methylation of MGMT, irradiation dosage, and Karnofsky Performance Status (KFS) were independent prognostic factors. A 2-gene predictor for glioblastoma survival was generated. Based on the genetic and epigenetic status of IDH1 and MGMT, glioblastoma patients were stratified into 3 clinically different genotypes: glioblastoma patients with IDH1mt/MGMTmet had the longest survival, followed by patients with IDH1mt/MGMTunmet or IDH1wt/MGMTmet, and patients with IDH1wt/MGMTunmet had the shortest survival. This 2-gene predictor was an independent prognostic factor and performed significantly better in predicting survival than either IDH1 mutations or MGMT methylation alone. The predictor was validated in 3 external datasets. DISCUSSION: The combination of IDH1 mutations and MGMT methylation outperforms either IDH1 mutations or MGMT methylation alone in predicting survival of glioblastoma patients. This information will help to increase our understanding of glioblastoma biology, and it may be helpful for baseline comparisons in future clinical trials.

Multiplex blood reporters for simultaneous monitoring of cellular processes.

Reporters secreted into the conditioned medium of cells in culture or into blood in vivo have shown to be useful tools for simple and noninvasive monitoring of biological processes in real-time. Here, we characterize the naturally secreted Vargula luciferase as a secreted blood reporter and show that this reporter can be multiplexed with the secreted Gaussia luciferase and alkaline phosphatase for simultaneous monitoring of three different cellular processes in the same biological system. We applied this system to monitor the response of three different subsets of glioma cells to a clinically relevant chemotherapeutic agent in the same well in culture or animal in vivo. This system could be extended to any field to detect multiple processes in the same biological system and is amenable for high-throughput screening to find drugs that affect multiple cellular populations/phenomena simultaneously.

Significance of complete 1p/19q co-deletion, IDH1 mutation and MGMT promoter methylation in gliomas: use with caution.

The histopathological diagnosis of diffuse gliomas often lacks the precision that is needed for tailored treatment of individual patients. Assessment of the molecular aberrations will probably allow more robust and prognostically relevant classification of these tumors. Markers that have gained a lot of interest in this respect are co-deletion of complete chromosome arms 1p and 19q, (hyper)methylation of the MGMT promoter and IDH1 mutations. The aim of this study was to assess the prognostic significance of complete 1p/19q co-deletion, MGMT promoter methylation and IDH1 mutations in patients suffering from diffuse gliomas. The presence of these molecular aberrations was investigated in a series of 561 diffuse astrocytic and oligodendroglial tumors (low grade n=110, anaplastic n=118 and glioblastoma n=333) and correlated with age at diagnosis and overall survival. Complete 1p/19q co-deletion, MGMT promoter methylation and/or IDH1 mutation generally signified a better prognosis for patients with a diffuse glioma including glioblastoma. However, in all 10 patients with a histopathological diagnosis of glioblastoma included in this study complete 1p/19q co-deletion was not associated with improved survival. Furthermore, in glioblastoma patients >50 years of age the favorable prognostic significance of IDH1 mutation and MGMT promoter methylation was absent. In conclusion, molecular diagnostics is a powerful tool to obtain prognostically relevant information for glioma patients. However, for individual patients the molecular information should be interpreted with caution and weighed in the context of parameters such as age and histopathological diagnosis.

Effects of targeting the VEGF and PDGF pathways in diffuse orthotopic glioma models.

Currently available compounds that interfere with VEGF-A signalling effectively inhibit angiogenesis in gliomas, but influence diffuse infiltrative growth to a much lesser extent. Development of a functional tumour vascular bed not only involves VEGF-A but also requires platelet-derived growth factor receptor-ß (PDGFRß), which induces maturation of tumour blood vessels. Therefore, we tested whether combined inhibition of VEGFR and PDGFRß increases therapeutic benefit in the orthotopic glioma xenograft models E98 and E473, both displaying the diffuse infiltrative growth that is characteristically observed in most human gliomas. We used bevacizumab and vandetanib as VEGF(R) inhibitors, and sunitinib to additionally target PDGFRß. We show that combination therapy of sunitinib and vandetanib does not improve therapeutic efficacy compared to treatment with sunitinib, vandetanib or bevacizumab alone. Furthermore, all compounds induced reduction of vessel leakage in compact E98 tumour areas, resulting in decreased detectability of these mostly infiltrative xenografts in Gd-DTPA-enhanced MRI scans. These data show that inhibition of VEGF signalling cannot be optimized by additional PDGFR inhibition and support the concept that diffuse infiltrative areas in gliomas are resistant to anti-angiogenic therapy.

Taqman genotyping assays can be used on decalcified and paraffin-embedded tissue from patients with osteosarcoma.

BACKGROUND: In cancers like osteosarcoma with a 5-year overall survival of 50-60%, archived histological specimens can be a useful source of biological material. However, this material generally has been decalcified and formalin-fixed for many years. In our study, we investigated whether DNA obtained from these tissues can be used for reliable single nucleotide polymorphism (SNP) genotyping. PROCEDURE: We studied two SNPs in the drug transporter MDR1 using Taqman® SNP genotyping assays. Genotypes of the germ line DNA derived from freshly isolated DNA of 20 surviving patients with osteosarcoma were compared with genotypes obtained from archived material from decalcified formalin-fixed, paraffin-embedded (FFPE) blocks of the same patients. RESULTS: Decalcified FFPE-derived DNA yielded smaller PCR fragments compared to DNA extracted from peripheral blood cells, with a reliable size of ∼200 bp. However, we were able to evaluate each SNP in 19 of 20 cases included in this study. All successfully genotyped samples showed 100% concordance between genotypes obtained from DNA of FFPE tissue and the genotypes obtained from DNA of blood from the same patients. CONCLUSIONS: In conclusion, we have demonstrated that decalcified FFPE tissue can be used for genetic polymorphism analysis using Taqman® allelic discrimination assays. This forms a unique opportunity to combine new insights in genetic research with historical patient cohorts.

The nature and timing of specific copy number changes in the course of molecular progression in diffuse gliomas: further elucidation of their genetic "life story".

Up till now, typing and grading of diffuse gliomas is based on histopathological features. However, more objective tools are needed to improve reliable assessment of their biological behavior. We evaluated 331 diffuse gliomas for copy number changes involving 1p, 19q, CDKN2A, PTEN and EGFR(vIII) by Multiplex Ligation-dependent Probe Amplification (MLPA®, Amsterdam, The Netherlands). Specifically based on the co-occurrence of these aberrations we built a model for the timing of the different events and their exact nature (hemi- → homozygous loss; low-level gain → (high-copy) amplification) in the course of molecular progression. The mutation status of IDH1 and TP53 was also evaluated and shown to correlate with the level of molecular progression. The relevance of the proposed model was confirmed by analysis of 36 sets of gliomas and their 39 recurrence(s) whereas survival analysis for anaplastic gliomas confirmed the actual prognostic relevance of detecting molecular malignancy. Moreover, based on our results, molecular diagnostic analysis of 1p/19q can be further improved as different aberrations were identified, some of them being indicative for advanced molecular malignancy rather than for favorable tumor behavior. In conclusion, identification of molecular malignancy as proposed will aid in establishing a risk profile for individual patients and thereby in therapeutic decision making.

MAPK pathway activation through BRAF gene fusion in pilocytic astrocytomas; a novel oncogenic fusion gene with diagnostic, prognostic, and therapeutic potential.

Recently, a new mechanism for activation of B-RAF was identified resulting from a tandem duplication, generating a fusion protein with constitutive BRAF activity and thereby activating the MAPK pathway. Different fusion variants involving BRAF and KIAA1549 were demonstrated, present in 80% of pilocytic astrocytomas in children. As the KIAA1549-BRAF fusion gene is detected at a much lower frequency in diffuse low-grade astrocytomas and survival was much longer than expected in the patients with a 'non-pilocytic' astrocytoma carrying the fusion gene, identification of this fusion gene can be of diagnostic and prognostic value. In the near future, interference with the (fusion gene causing) activation of the MAPK signalling cascade may open new therapeutic avenues for children with pilocytic astrocytomas, as a first line of defence against tumour growth or in situations where the tumour has become refractory to other therapeutic modalities.

Molecular diagnostics of gliomas: state of the art.

Modern neuropathology serves a key function in the multidisciplinary management of brain tumor patients. Owing to the recent advancements in molecular neurooncology, the neuropathological assessment of brain tumors is no longer restricted to provide information on a tumor's histological type and malignancy grade, but may be complemented by a growing number of molecular tests for clinically relevant tissue-based biomarkers. This article provides an overview and critical appraisal of the types of genetic and epigenetic aberrations that have gained significance in the molecular diagnostics of gliomas, namely deletions of chromosome arms 1p and 19q, promoter hypermethylation of the O6-methylguanine-methyl-transferase (MGMT) gene, and the mutation status of the IDH1 and IDH2 genes. In addition, the frequent oncogenic aberration of BRAF in pilocytic astrocytomas may serve as a novel diagnostic marker and therapeutic target. Finally, this review will summarize recent mechanistic insights into the molecular alterations underlying treatment resistance in malignant gliomas and outline the potential of genome-wide profiling approaches for increasing our repertoire of clinically useful glioma markers.

Six year survival after prolonged temozolomide treatment in a 30-year-old patient with glioblastoma.

Glioblastoma (GBM) is the most malignant primary brain tumour in adults. Since 2005 surgery followed by radiotherapy with concomitant Temozolomide (TMZ) is the standard care for patients with a GBM. Despite these improved treatment strategies, survival of GBM-patients remains poor; and there are very few patients who survive for a long time. Also there is no standard therapeutic strategy after six cycles of TMZ, and further treatment is at the physician's discretion. We report a case of a young patient with a glioblastoma who, not only showed dramatic clinical and radiological improvement after TMZ treatment but who now also (under continued TMZ therapy) survives over 6 years, with complete remission clinically and radiologically. Up till now there are no studies describing TMZ treatment in GBM patients for as long as 6 years.

MGMT promoter methylation is prognostic but not predictive for outcome to adjuvant PCV chemotherapy in anaplastic oligodendroglial tumors: a report from EORTC Brain Tumor Group Study 26951.

PURPOSE: O6-methylguanine-methyltransferase (MGMT) promoter methylation has been shown to predict survival of patients with glioblastomas if temozolomide is added to radiotherapy (RT). It is unknown if MGMT promoter methylation is also predictive to outcome to RT followed by adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in patients with anaplastic oligodendroglial tumors (AOT). PATIENTS AND METHODS: In the European Organisation for the Research and Treatment of Cancer study 26951, 368 patients with AOT were randomly assigned to either RT alone or to RT followed by adjuvant PCV. From 165 patients of this study, formalin-fixed, paraffin-embedded tumor tissue was available for MGMT promoter methylation analysis. This was investigated with methylation specific multiplex ligation-dependent probe amplification. RESULTS: In 152 cases, an MGMT result was obtained, in 121 (80%) cases MGMT promoter methylation was observed. Methylation strongly correlated with combined loss of chromosome 1p and 19q loss (P = .00043). In multivariate analysis, MGMT promoter methylation, 1p/19q codeletion, tumor necrosis, and extent of resection were independent prognostic factors. The prognostic significance of MGMT promoter methylation was equally strong in the RT arm and the RT/PCV arm for both progression-free survival and overall survival. In tumors diagnosed at central pathology review as glioblastoma, no prognostic effect of MGMT promoter methylation was observed. CONCLUSION: In this study, on patients with AOT MGMT promoter methylation was of prognostic significance and did not have predictive significance for outcome to adjuvant PCV chemotherapy. The biologic effect of MGMT promoter methylation or pathogenetic features associated with MGMT promoter methylation may be different for AOT compared with glioblastoma.

Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas.

Somatic mutations in the IDH1 gene encoding cytosolic NADP+-dependent isocitrate dehydrogenase have been shown in the majority of astrocytomas, oligodendrogliomas and oligoastrocytomas of WHO grades II and III. IDH2 encoding mitochondrial NADP+-dependent isocitrate dehydrogenase is also mutated in these tumors, albeit at much lower frequencies. Preliminary data suggest an importance of IDH1 mutation for prognosis showing that patients with anaplastic astrocytomas, oligodendrogliomas and oligoastrocytomas harboring IDH1 mutations seem to fare much better than patients without this mutation in their tumors. To determine mutation types and their frequencies, we examined 1,010 diffuse gliomas. We detected 716 IDH1 mutations and 31 IDH2 mutations. We found 165 IDH1 (72.7%) and 2 IDH2 mutations (0.9%) in 227 diffuse astrocytomas WHO grade II, 146 IDH1 (64.0%) and 2 IDH2 mutations (0.9%) in 228 anaplastic astrocytomas WHO grade III, 105 IDH1 (82.0%) and 6 IDH2 mutations (4.7%) in 128 oligodendrogliomas WHO grade II, 121 IDH1 (69.5%) and 9 IDH2 mutations (5.2%) in 174 anaplastic oligodendrogliomas WHO grade III, 62 IDH1 (81.6%) and 1 IDH2 mutations (1.3%) in 76 oligoastrocytomas WHO grade II and 117 IDH1 (66.1%) and 11 IDH2 mutations (6.2%) in 177 anaplastic oligoastrocytomas WHO grade III. We report on an inverse association of IDH1 and IDH2 mutations in these gliomas and a non-random distribution of the mutation types within the tumor entities. IDH1 mutations of the R132C type are strongly associated with astrocytoma, while IDH2 mutations predominantly occur in oligodendroglial tumors. In addition, patients with anaplastic glioma harboring IDH1 mutations were on average 6 years younger than those without these alterations.

Chromosomal and methylation alterations in sporadic and familial adenomatous polyposis-related duodenal carcinomas.

Primary carcinomas of the small intestine are rare and the mechanism of their pathogenesis is poorly understood. Patients with familial adenomatous polyposis (FAP) have a high risk of developing duodenal carcinomas. The aim of this study is to gain more insight into the development of duodenal carcinomas. Therefore, five FAP-related duodenal carcinomas were characterized for chromosomal and methylation alterations, which were compared to those observed in sporadic duodenal carcinomas. Comparative genomic hybridization (CGH) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed in 10 primary sporadic and five primary FAP-related duodenal carcinomas. In the FAP-related carcinomas, frequent gains were observed on chromosomes 8, 17 and 19, whereas in sporadic carcinomas they occurred on chromosomes 8, 12, 13 and 20. In 60% of the sporadic carcinomas, gains in the regions of chromosome 12 were observed which were absent in the FAP-related carcinomas (P=0.04). Hypermethylation was observed in the immunoglobulin superfamily genes member 4 (IGSF4), TIMP metallopeptidase inhibitor 3 (TIMP3), Estrogen receptor 1 (ESR1), adenomatous polyposis coli (APC), H-cadherin (CDH13) and paired box gene 6 (PAX6) genes. Hypermethylation of PAX6 was only observed in FAP-related carcinomas (3/5) and not in sporadic carcinomas (P=0.02). In conclusion, in contrast to sporadic duodenal carcinomas, gains on chromosome 12 were not observed in duodenal carcinomas of patients with FAP. Identification of the genes in these regions of chromosome 12 could lead to a better understanding of the carcinogenesis pathways leading to sporadic and FAP-related duodenal carcinomas. Furthermore, hypermethylation seems to be a general feature of both FAP-related duodenal carcinomas as well as sporadic duodenal carcinomas with the exception of the PAX6 gene, which is methylated only in FAP-related carcinomas.

MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas.

Expression of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), encoded by the O6-methylguanine (O6-mG) -DNA-methyltransferase (MGMT) DNA repair gene, results in resistance to alkylating agents, and hypermethylation of the MGMT promoter is associated with chemosensitivity as it prevents AGT expression. As the interpretation of the results of immunohistochemistry to evaluate AGT expression proved to be difficult, the aim of our present study is to establish a feasible, reliable, and robust method for MGMT promoter hypermethylation testing that can be easily implemented in a diagnostic setting and is applicable to routinely processed tissue. MGMT hypermethylation analysis using methylation-specific (MS-) multiplex ligation-dependent probe amplification (MLPA) was performed on 62 glioma samples of 55 individual tumors (including 12 cell lines) and compared to the more conventionally used, but improved, MS-polymerase chain reaction (PCR). In contrast to MS-PCR, MS-MLPA (i) is not based on bisulfite conversion of unmethylated cytosines (a somewhat troublesome step in MS-PCR), (ii) provided methylation status of all samples, (iii) proved to be semiquantitative, (iv) can be used to evaluate methylation status of multiple sequences (CpG dinucleotides) simultaneously, and (v) allows for a combined copy number detection and methylation specific analysis. The potential therapeutic value of MGMT hypermethylation evaluation using MS-MLPA was shown in a group of 20 glioblastoma patients receiving temozolomide chemotherapy. We conclude that MS-MLPA is a robust and reliable method that can be easily applied to differently processed tissues, including those fixed in formalin and embedded in paraffin. The semiquantitative aspect of MS-MLPA may prove to be of great value, especially in predicting response to alkylating agents, not only for gliomas as evaluated in this study but also for tumors in general.

Molecular diagnostics as a tool to personalize treatment in adult glioma patients.

Gliomas, the most frequent primary brain tumors in humans, form a heterogeneous group, encompassing many different histological types and malignancy grades. Within this group, the diffuse infiltrative gliomas are by far the most common in adults. The major representatives in this subgroup are the diffuse astrocytic, oligodendroglial, and mixed oligo-astrocytic tumors. Especially in these diffuse gliomas, the role of molecular diagnostics is rapidly increasing. After summarizing the most relevant genetic aberrations and pathways in these tumors detected up till now, this review will discuss the clinical relevance of this information. Several molecular markers have been identified in diffuse gliomas that carry diagnostic and prognostic information. In addition, some of these and other markers predict the response of these gliomas to particular (chemo)therapeutic approaches. The techniques used to obtain this molecular information, as well as the advantages and disadvantages of the different techniques will be discussed. Finally, future perspectives will be presented with regard to the contribution of molecular diagnostics to tailor-made therapy in glioma patients.

Cyclin D1 genotype and expression in sporadic hemangioblastomas.

Central nervous system (CNS) hemangioblastomas are highly-vascularized tumors occurring in sporadic form or as a manifestation of von Hippel-Lindau disease (VHL). The VHL protein (pVHL) regulates various target genes, one of which is the CCND1 gene, encoding cyclin D1, a protein that plays a critical role in the control of the cell cycle. Overexpression of cyclin D1 is found in many cancers. The CCND1 gene contains a common G --> A polymorphism (870G > A) that enhances alternative splicing of the gene. CCND1 genotype is associated with clinical outcome in a number of cancers although prognostic significance varies with tumor type. In VHL disease, CCND1 genotype has been suggested as a genetic modifier that influences susceptibility to hemangioblastomas. In order to analyze whether CCND1 genotype plays a role in sporadic CNS hemangioblastomas, we investigated CCND1 genotype in tumor tissue of 17 sporadic and also in five VHL-related CNS hemangioblastomas. In addition, in these tumors the extent and localization of cyclin D1 expression was investigated by immunohistochemistry. We found no deviation in CCND1 genotype distribution and allele frequencies from expected values. Also, there was no correlation between age at onset and CCND1 genotype. The expression of cyclin D1 as detected by immunohistochemistry was highly variable within and between tumors, without a clear correlation with CCND1 genotype. We conclude that, whereas variable but sometimes high cyclin D1 expression is a feature of sporadic hemangioblastomas, CCND1 genotype is unlikely to be an important genetic modifier in the oncogenesis of these tumors.

Molecular pathogenesis of oligodendroglial tumors.

Based on their histopathological appearances, most diffusely infiltrative gliomas can be classified either as astrocytic tumors (As), pure oligodendroglial tumors (Os) or mixed oligoastrocytic tumors (OAs). The latter two may be grouped together as oligodendroglial tumors (OTs). The distinction between As and OTs is important because of the more favorable clinical behavior of OTs. Unfortunately, the histopathological delineation of OAs, Os and As can be difficult because of vague and subjective histopathological criteria. Over the last decade, the knowledge on the molecular genetic background of OTs has drastically increased. This review provides an overview of molecular genetic aberrations in OTs and discusses the pathobiological and clinical significance of these aberrations. In contrast to As, OTs frequently show frequent loss of heterozygosity on chromosome arms 1p and 19q. Since these aberrations are significantly correlated with clinically relevant parameters, such as prognosis and chemosensitivity, and given the difficulties in histopathological typing and grading of glial tumors, genetic testing should be included in routine glioma diagnostics. It is to be expected that the identification of the relevant tumor suppressor genes located on 1p and 19q will lead to more refined genetic tests for OTs. Furthermore, as microarray technology is rapidly increasing, it is likely that clinically relevant markers for OTs will be identified on other chromosomes and need to be included into routine glioma diagnostics as well.

Comparative genomic hybridization: practical guidelines.

Comparative genomic hybridization (CGH) is a technique used to identify copy number changes throughout a genome. Until now, hundreds of CGH studies have been published reporting chromosomal imbalances in a large variety of human neoplasms. Additionally, technical improvements of specific steps in a CGH experiment and reviews on the technique have appeared. However, full CGH protocols are only occasionally published. In this paper a review of CGH is presented, including technique, pitfalls, and difficulties. Our own protocol is completely described and discussed, including the different optimization experiments used to establish this protocol and points requiring special attention. Although this protocol results in reliable and sensitive CGH experiments in our hands, readers should keep in mind that other laboratories may prefer other protocols. Testing different options, among others, as discussed in the current paper generates the most appropriate protocol. This paper shows the complexity of the CGH technique and may serve as a guideline for starting CGH or as a troubleshooting guide for those who perform CGH.