| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Letters to the Editor |
Department of Pathology, Anatomy and Cell Biology Jefferson Medical College Philadel phia, Pennsylvania 19107
Institute of Normal and Pathological Cytomorphology Consiglio Nazionale delle Ricerc he 40136 Bologna, Italy
Laboratory of Cell Metabolism and Pharmacokinetics Center for Experimental Research Regina Elena Cancer Institute 00158 Rome, Italy
Department of Pathology, Anatomy and Cell Biology Jefferson Medical College Philadelphia, Pennsylvania 19107
In their letter, Gray et al. (1) question the results presented in our report regarding RB2/p130 mutations in primary NPC,1 published this year in Cancer Research (2) . In that study, we considered a group of 10 cases recruited from North Africa and found mutations in 30% of the primary tumors examined. Extending our investigation, we have since collected 120 specimens of NPCs derived from different areas of the world. In fact, 110 are African in origin and 10 are from a Chinese population that immigrated to the United States. Our screening for mutations in the RB2/p130 gene of this collection has yielded the following results: screening of cases of African origin have demonstrated a similar percentage of genetic alterations in the RB2/p130 gene to that published in Cancer Research (2) , whereas cases of Chinese origin have shown a wild-type RB2/p130 gene. Therefore, we are not at all surprised by the results described by Gray et al. (1) . Additionally, we have sequenced the DNA extracted from the HONE-1 cell line as well as the reverse transcriptase of its RNA messenger and have also found no mutations in this particular cell line, which is of Chinese origin.
Because DNA extracted from NPCs also contains normal genomic DNA derived from reactive lymphocytes that surround the cancer cells, there are, from our point of view, three basic strategies that should be followed. The first would be to screen the samples by immunohistochemistry using two different antibodies against pRb2/p130, one raised against the NH2 terminal region and the other against the COOH terminal region of the protein. This is because, in the presence of mutations that disrupt the amino acid sequence of the protein, only one of the two antibodies will be able to recognize the protein and to localize it in either compartment of the cell. Another strategy would be to microdissect and separate carcinoma cells from normal lymphocytes and extract the genomic DNA from these fractions. This procedure would collect very little DNA and allow for few but clean sequences. We are in the process of comparing the reproducibility of these two techniques for future studies. Another strategy is of course that suggested by Gray et al. (1) for subcloning the PCR products in plasmidic DNA, allowing for its amplification and direct sequencing. The idea is that if there is a heterozygous mutation, statistically this should be detected in some of the plasmid-transformed colonies. In our experience, with this technique, the rate of mutation does not necessarily reflect that of a direct sequence of the total genomic DNA amplification. This is because many attempts are needed before finding the bacterial colony transformed by the mutated DNA versus those transformed by a wild-type DNA. In essence, the odds of transforming a bacterial cell with a plasmid containing a mutated DNA are lower than those of transforming another bacterial cell with a wild-type DNA. In particular, this is true when screening for mutations in NPC, a cancer in which normal lymphocytes surround cancer cells. The copy number of mutated DNA is therefore lower than that of the wild-type one. Consequently, a fast way to screen for mutations was applied in the 10 samples from Northern Africa that we examined. DNA was amplified and subjected to single strand conformational polymorphism analysis. Samples were then sequenced, and mutations were detected. As a matter of fact, because the genomic DNA we used contained a mixture of normal genomic DNA derived from lymphocytes, plus that from cancer cells, it is understandable that the chromatogram might appear confusing. In fact, the chromatogram showed the presence of mutations in the cancerous DNA along with the normal sequence. Additionally, the presence of an insertion will cause a shift of the peaks that will be read by the automatic machine in a place that is further along the chromatogram. We had had the sequences examined by different experts in the field who participated in the human genome sequencing project. Because we found the presence of peaks in both the forward and reverse sequences, they affirmed that these were true mutations. This, evidently, was also the feeling of the peer reviewers that accepted our manuscript for publication in this journal after considering the sequences.
Additionally, regarding the use of primers that amplify exon 21, we are aware of possible difficulties encountered when using this particular set of primers. Unfortunately, in our opinion, the particular intron-exon boundary of exon 21 does not allow for designing other and better primers. This region harbors a stretch of thymidines that limit primer choices. Therefore, more attempts are needed before setting the technique for this particular exon screening.
Finally, two recent publications revealed the presence of loss of heterozygosity (LOH) in nasopharyngeal carcinoma in the chromosomal region where RB2/p130 maps. In the report published in Cancer Research by Lo et al. (3) , the authors described a newly found hot spot of LOH in NPC. The chromosomal zone reflects that in which the RB2/p130 gene is allocated, i.e., the chromosomal locus 16q12.2. The authors detected "high frequencies of allelic imbalance on 3p (96.3%), 9p (85.2%), 9q (88.9%), 11q (74.1%), 12q (70.4%), 13q (55.6%), 14q (85.2%), and 16q (55.6%). Nonrandom allelic changes of 12q and 16q were revealed for the first time." Also, the authors stated that "frequent deletions of these minimally deleted regions implied the presence of tumor suppressor genes that may be involved in the development of NPC. Frequent LOH of 16q on NPC is a novel finding in this allelotyping study. We have mapped a 21.4-cM (5.7-Mb) MDR to 16q22.3–23.1. The region is adjacent to the E-cadherin gene, which encoded a cell adhesion molecule and is associated with tumor invasiveness and metastasis. Zheng et al. showed that loss of E-cadherin expression was common in this cancer and significantly associated with advanced stages of this disease. The other candidate tumor suppressor at 16q, RB2/p130, is located at 16q12 and mapped between D16S415 and D16S503. This region was also deleted in 48.1% NPC tumors."
Another report published in Genes Chromosomes & Cancer by Fang et al. (4) showed that "several recurrent chromosomal abnormalities were identified in nasopharyngeal carcinoma. Fifty-seven tumors were analyzed by comparative genomic hybridization (CGH). In 47 cases, chromosomal imbalances were found. The most frequently detected loss of chromosomal materials involved chromosome arms 16q (26 cases, 55%), 14q (21 cases, 45%), 1p (20 cases, 43%), 3p (20 cases, 43%), 16p (19 cases, 40%), 11q (17 cases, 36%), and 19p (16 cases, 34%). Genomic alterations detected by CGH were compared and found to be largely consistent with those identified in banding analysis and loss of heterozygosity studies." The authors continue by saying: "However, several previously unrecognized recurrent alterations were also identified in the present study, including gain of 4q and 18q, and loss of 16q, 14q, and 19p. Identification of recurrent sites of chromosomal gain and loss identify regions of the genome that may contain oncogenes or tumor suppressor genes, respectively, which may be involved in the tumorigenesis of NPC."
The foregoing data demonstrate that the 16q chromosomal region containing the RB2/p130 gene is involved in NPC tumorigenesis and that RB2/p130 could be the tumor suppressor gene candidate in that process. We hope that our data taken together with those presented by Lo et al. (3) and Fang et al. (4) , as well as other studies examining the involvement of RB2/p130 in tumorigenesis (5) , provide the support necessary to resolve the issues raised by Gray et al. (1) .
FOOTNOTES
1 The abbreviation used is: NPC, nasopharyngeal carcinoma. 
Received 4/24/01. Accepted 6/ 1/01.
REFERENCES
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
