Monib Zirvi

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.

Detection of the BRAF V600E mutation in melanocytic lesions using the ligase detection reaction.

Background:  BRAF is a member of the RAF kinase family, and it promotes signaling through the RAS‐MAP kinase signal transduction cascade. Research has shown that a majority of melanomas and nevi exhibit an activating V600E (T1799A) mutation in BRAF exon 15. Additional studies of BRAF have demonstrated that the T1799A mutation is absent in uveal melanomas and Spitz nevi.

Acquired perforating calcific collagenosis after topical calcium chloride exposure.

A 24‐year‐old healthy man presented with a 6‐week history of numerous umbilicated coalescing erythematous papules with some scale and crust on his anterior medial thighs. The eruption began 1 to 2 weeks after he spilled calcium chloride rock salts on his pants while salting the sidewalk during a snow storm. The salts dissolved and remained in contact with his skin for at least 4 hours until he was able to change clothes. A skin biopsy shows thick and thin collagen fibers with partial calcification in the papillary and upper reticular dermis associated with a sparse infiltrate of neutrophils, lymphocytes and mononuclear histiocytes. There are foci of transepidermal elimination of calcified fibers with adjacent epidermal hyperplasia and ortho‐ and parakeratosis. Von Kossa stain highlights calcification of the fibers, and trichrome stain confirms the fibers are collagen. A Verhoeff‐van Gieson stain shows no abnormality of elastic fibers. The patient was treated with topical betametasone diproprionate cream twice daily for 3 weeks, as well as a short course of oral levofloxacin and topical gentamicin cream. The lesions resolved over 3 weeks with residual scarring. We report a unique case of acquired perforating calcific collagenosis secondary to topical calcium chloride exposure.