Jeffrey Sklar

The t(14;18)(q32;21) chromosomal translocation characteristic of follicular lymphomas is the most common cytogenetic abnormality known to be associated with any specific type of hematolymphoid malignancy. A fragment of DNA containing the crossover point between chromosomes 14 and 18 was cloned from the tumor cells of a patient with a follicular lymphoma carrying this translocation. Nucleotide sequence analysis of the breakpoint DNA revealed that the break in chromosome 14 occurred in joining region 4(J4) of the nonfunctional immunoglobulin heavy chain allele. This finding and other structural similarities of the breakpoint with the functional diversity region-joining region (D-J) joint in this lymphoma suggest that D-J recombination enzymes played a role in the mechanism of the t(14;18) translocation. Hybridization analysis of DNA from 40 follicular lymphomas showed that the majority of t(14;18) translocations occur on chromosome 18 DNA within 4.2 kilobases of the cloned breakpoint. A DNA probe from this breakpoint-cluster region detects transcription products in the tumor cells from which it was cloned and in a B-lymphoma cell line containing a t(14;18) translocation.

We present statistical methods for analyzing replicated cDNA microarray expression data and report the results of a controlled experiment. The study was conducted to investigate inherent variability in gene expression data and the extent to which replication in an experiment produces more consistent and reliable findings. We introduce a statistical model to describe the probability that mRNA is contained in the target sample tissue, converted to probe, and ultimately detected on the slide. We also introduce a method to analyze the combined data from all replicates. Of the 288 genes considered in this controlled experiment, 32 would be expected to produce strong hybridization signals because of the known presence of repetitive sequences within them. Results based on individual replicates, however, show that there are 55, 36, and 58 highly expressed genes in replicates 1, 2, and 3, respectively. On the other hand, an analysis by using the combined data from all 3 replicates reveals that only 2 of the 288 genes are incorrectly classified as expressed. Our experiment shows that any single microarray output is subject to substantial variability. By pooling data from replicates, we can provide a more reliable analysis of gene expression data. Therefore, we conclude that designing experiments with replications will greatly reduce misclassification rates. We recommend that at least three replicates be used in designing experiments by using cDNA microarrays, particularly when gene expression data from single specimens are being analyzed.

One of the most common karyotypic abnormalities is the t(14;18) translocation, which is found in many lymphomas that have a characteristic follicular morphology. Recent molecular studies have shown that this chromosomal translocation results in the juxtaposition of the candidate proto-oncogene bcl-2 (B-cell leukemia-lymphoma) on chromosome 18 with the immunoglobulin heavy-chain locus on chromosome 14. However, because performing accurate cytogenetic studies in solid hematolymphoid neoplasms is difficult, knowledge of the prevalence of the t(14;18) translocation and, by association, the extent of bcl-2 involvement in human lymphomas is limited. We used a number of chromosome-18 DNA probes to analyze various subtypes of Hodgkin's and non-Hodgkin's lymphomas and test for structural abnormalities near or within the bcl-2 gene. Molecular features of the t(14;18) translocation were found in virtually all follicular neoplasms and about 28 percent of diffuse large-cell lymphomas. No changes in bcl-2 were found in several other subtypes of Hodgkin's and non-Hodgkin's lymphomas, including those previously suggested to originate from follicular-center cells and those about which cytogenetic data have been difficult to obtain. Our findings suggest a close pathogenetic relation between bcl-2 and a large group of non-Hodgkin's lymphomas, both with and without a follicular morphology. The methods employed in this study may be useful in improving the accuracy of diagnosis and subclassification of malignant lymphomas.