No abstract

Some studies have shown an inverse relationship between microsatellite instability in colon cancer and mutations in p53 and K-ras, whereas others have not. We therefore evaluated these features in a population-based sample of 496 individuals with colon cancer. Microsatellite instability was determined by a panel of 10 tetranucleotide repeats, the Bethesda consensus panel of mono- and dinucleotide repeats, and coding mononucleotide repeats in transforming growth factor-beta receptor type II, hMSH3, BAX, hMSH6, and insulin-like growth factor receptor type II. Mutations in codons 12 and 13 in K-ras were evaluated by sequencing. p53 overexpression (as detected by immunohistochemistry) was used as an indicator of p53 mutation; this was evaluated in 275 of the tumors. K-ras mutations were present in 33.2% of tumors, p53 overexpression in 51.5%, and microsatellite instability (as determined by the Bethesda consensus panel) in 12.5%. K-ras mutations were significantly less common in unstable tumors than stable tumors (11.8% versus 36.9%, P: < 0.001). p53 overexpression was significantly less common in unstable tumors than stable tumors (20.0% versus 55.7%, P: < 0.001). These inverse relationships between microsatellite instability and ras gene mutations and p53 overexpression were shown to be independent of tumor site in logistic regression analyses. All other measures of instability also showed statistically significant inverse relationships independent of tumor site with alterations in ras and p53, and instability results determined by the panel of 10 tetranucleotide repeats were highly significantly related to those determined by the Bethesda consensus panel. Coding mononucleotide repeat mutations were significantly more common in unstable tumors than stable tumors (85.7% versus 1.0%, P: < 0.001). We conclude that there is an inverse relationship between microsatellite instability and mutations in p53 and K-ras, and that the molecular profile of colon cancers with microsatellite instability is characterized by relatively infrequent mutations in K-ras and p53 and relatively frequent mutations in coding mononucleotide repeats.

Few human tumors are collected such that RNA is preserved for molecular analysis. Completion of the Human Genome Project will soon result in the identification of more than 100,000 new genes. Consequently, increasing attention is being diverted to identifying the function of these newly described genes. Here we describe a multidisciplinary tumor bank procurement protocol that preserves both the integrity of tissue for pathologic diagnosis, and the RNA for molecular analyses. Freshly excised normal skin was obtained from five patients undergoing wound reconstruction following Mohs micrographic surgery for cutaneous neoplasia. Tissues treated for 24 hours with RNAlater™ were compared histologically and immunohistochemically to tissues not treated with RNAlater. Immunohistochemical stains studied included: CD45, CEA, cytokeratin AE1/3, vimentin, S-100, and CD34 on formalin-fixed, paraffin embedded tissue and CD45 staining of frozen tissue. Slides were blinded and evaluated independently by three pathologists. The histologic and immunohistochemical parameters of tissue stored in RNAlater were indistinguishable from tissue processed in standard fashion with the exception of S-100 stain which failed to identify melanocytes or Langerhan's cells within the epidermis in any of the RNAlater -treated tissues. Interestingly, nerve trunks within the dermis stained appropriately for S-100. Multiple non-cutaneous autopsy tissues were treated with RNAlater, formalin, liquid nitrogen (LN2), and TRIzol Reagent . The pathologists were unable to distinguish between tissues treated with RNAlater, formalin, or frozen in LN2, but could easily distinguish tissues treated with TRIzol Reagent because of extensive cytolysis. RNA was isolated from a portion of the tissue treated with RNAlater and used for molecular studies including Northern blotting and microarray analysis. RNA was adequate for Northern blot analysis and mRNA purified from RNAlater -treated tissues consistently provided excellent templates for reverse transcription and subsequent microarray analysis. We conclude that tissues treated with RNAlater before routine processing are indistinguishable histologically and immunohistochemically from tissues processed in routine fashion and that the RNA isolated from these tissues is of high quality and can be used for molecular studies. Based on this study, we developed a multidisciplinary tumor bank procurement protocol in which fresh tissue from resection specimens are routinely stored in RNAlater at the time of preliminary dissection. Thus, precious human tissue can be utilized for functional genomic studies without compromising the tissue's diagnostic and prognostic qualities.

Fibroepithelial lesions with cellular stroma (FELCS) in breast core needle biopsy (CNB) specimens may result in either fibroadenoma or phyllodes tumor at excision. We evaluated histologic features, proliferation indices (by Ki-67 and topoisomerase II a immunostaining) and p53 expression in 29 cases of FELCS in CNB specimens and correlated these with excision findings in a blinded manner. On excision, 16 patients had fibroadenomas and 12 had phyllodes tumors. All CNB specimens with mildly increased stromal cellularity were fibroadenomas on excision (n=4), and all with markedly cellular stroma were phyllodes tumors (n=4). Among CNB specimens with moderate cellularity (12 fibroadenomas and 8 phyllodes tumors), only stromal mitoses were discriminatory histologically. Stromal proliferation indices were significantly higher in CNB that were phyllodes tumors vs fibroadenomas. Assessment of stromal cellularity, mitoses, and proliferation indices might help determine the probability of phyllodes tumor occurring and guide management of these cases.

DNA topoisomerases play important roles in basic cellular biology. Recently they have been identified as the molecular targets of a variety of pharmaceutical agents. Some of the drugs that target the topoisomerases are anticancer drugs. These anticancer drugs work by a novel mechanism of action. They inhibit the topoisomerase molecule from religating DNA strands after cleavage. This leaves a cell with DNA breaks, which if not repaired, become lethal. In other words, these drugs convert the topoisomerase molecule into a DNA damaging agent. This is a stoichiometric relationship. Each anticancer drug molecule has the potential of interacting with one topoisomerase molecule to cause one DNA lesion. The clinical implication of this mechanism of drug action is that sensitivity to topoisomerase targeting drugs should be dependent on high topoisomerase levels. This is clearly true in laboratory systems. With new developments in in situ immunohistochemistry, topoisomerase expression can now be easily estimated in human cancers. From this information, it may be possible to predict the sensitivity or resistance of human cancers to topoisomerase targeting anticancer drugs.