Loss of heterozygosity analysis: Practically and conceptually flawed?

Abstract

The Knudson "two-hit" hypothesis has provided the rationale for studies that aim to identify tumor-suppressor genes by mapping regions of allelic loss (loss of heterozygosity, LOH). Although LOH has been found in practically all types of tumors, very few such projects have been successful in identifying their tumor-suppressor targets. The prime explanation for this failure is probably that researchers have, in general, been too credulous about the two-hit hypothesis, and too willing to ignore factors such as intratumor heterogeneity, contamination by normal cells, karyotypic complexity, homozygous deletions, gene dosage changes, and polymerase chain reaction artifacts. We suggest ways of minimizing these problems. Unfortunately, there is no guarantee that existing or newer methods, such as genomic microarrays and in situ single-nucleotide polymorphism analysis, will solve the difficulties of LOH analysis. The future prospects for LOH studies are, as ever, uncertain.

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma

“…This technique involves the detection of gene dosage imbalances by PCR-based microsatellite genotyping; however, there are a number of methodological difficulties (Tomlinson et al, 2002). Tumour samples not only contain contaminating normal cells, but are also genetically heterogeneous; therefore, a deletion would have to be present in a substantial number of cells to be detected by LOH analysis.…”

“…In addition, most tumours are aneuploid and considerable LOH frequency differences have been detected between flowsorted aneuploid and diploid cells (Bonsing et al, 2000). LOHbased studies therefore tend to be inconsistent, particularly as definitions for LOH thresholds are variable (Tomlinson et al, 2002). In contrast, FISH involves direct visualisation of the nuclear DNA content on a single-cell basis.…”

Genomic losses at 5q13.2 and 8p23.1 in dysplastic hepatocytes are common events in hepatitis B virus-related hepatocellular carcinoma

“…DNA from matched tumor and corresponding non-tumor liver tissue was analyzed for LOH at 8p23.1 by amplification of two published microsatellite markers, D8S503 (forward, GGT TAC GAG TTT TGT CCT TTG and reverse, GAA ACA AAC CAA TGT AGG AGTG) and D8S1130 (forward, GAA GAT TTG GCT CTG TTGGA and reverse, TGT CTT ACT GCT ATA GCTT), which were selected from the Genome Database (http://www.gdb.org), as described previously (16,17). PCR was performed in a total volume of 25 µl, consisting of 2 µl DNA solution (concentration, ~100 ng/µl), 5 µl 5X Colorless GoTaq ® Flexi Buffer (Applied Biosystems), 2 µl 25 mM MgCl 2 , 0.5 µl 200 µM dNTP, 0.15 µl 5 U/µl GoTaq ® Hot Start Polymerase, and 0.8 µl primer sets (1.25 µmol/l of each primer), in a Bio-Rad thermal cycler (Bio-Rad Laboratories, Inc.) using the following program: Initial denaturing step at 95˚C for 5 min, followed by 30 cycles of denaturation at 95˚C for 50 sec, annealing at 59˚C or 53˚C for 60 sec, extension at 72˚C for 60 sec and a final extension at 72˚C for 3 min.…”

Large‐scale genomic analysis of ovarian carcinomas

“…These difficulties made it difficult to pinpoint the TSGs targeted by microsatellite based LOH analyses. The lack of success of LOH analyses to identify specific gene targets led some investigators to question the validity of the whole approach (Tomlinson et al, 2002).…”