Detection of occult melanoma cells in paraffin-embedded histologically negative sentinel lymph nodes using a reverse transcriptase polymerase chain reaction assay.
OBJECTIVE
Detection of occult metastasis before the development of clinical disease could allow more accurate staging, appropriate follow-up procedures, and adjuvant therapies in patients with malignant melanoma (MM). The sentinel lymph node (SLN) has been proposed as a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. In this study, we screened both paraffin-embedded SLNs and peripheral-blood (PB) samples from MM patients at various stage of disease using a multimarker reverse transcriptase polymerase chain reaction (RT-PCR) assay. The prognostic significance of the presence of PCR-positive markers was also evaluated.


METHODS
Total RNA was obtained from paraffin-embedded SLN sections and PB samples of 75 MM patients. RT-PCR was performed using tyrosinase and MelanA/MART1 as melanoma-associated markers. Radiolabeled PCR products were analyzed on denaturing polyacrylamide gels.


RESULTS
Good sensitivity of the RT-PCR assay on archival tissues was demonstrated after comparison of RT-PCR results on frozen and paraffin-embedded SLNs from 16 MM patients. Significant correlation between the disease stage and marker expression in both PB and SLN samples was observed; the highest value was for patients who were positive for both markers in SLN (P =.006). Progression of disease was significantly associated with the total number of PCR-positive markers in both PB (P =.034) and SLN (P =.001) samples.


CONCLUSIONS
Although sensitivity is lowered by the use of paraffin-embedded specimens, our data indicate that RT-PCR analysis of serial sections from archival SLNs may be helpful in improving detection of occult micrometastases, thus improving staging of patients with melanoma.
Palmieri
G
G
Institute of Molecular Genetics, National Research Council of Italy, Alghero (SS). gpalmieri@yahoo.com
Ascierto
P A
PA
Cossu
A
A
Mozzillo
N
N
Motti
M L
ML
Satriano
S M
SM
Botti
G
G
Caracò
C
C
Celentano
E
E
Satriano
R A
RA
Lissia
A
A
Tanda
F
F
Pirastu
M
M
Castello
G
G
D014408:Biomarkers, Tumor / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis; D005188:False Negative Reactions; D006801:Humans; D008207:Lymphatic Metastasis / Q000175:diagnosis; D008545:Melanoma / Q000175:diagnosis* / Q000473:pathology; D016612:Paraffin Embedding; D011379:Prognosis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012680:Sensitivity and Specificity; D021701:Sentinel Lymph Node Biopsy; D012878:Skin Neoplasms / Q000175:diagnosis* / Q000473:pathology; D013048:Specimen Handling
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm