Sentinel node in melanoma patients: triple negativity with routine techniques and PCR as positive prognostic factor for survival.

Lymph node mapping and sentinel lymph node biopsy are currently used to stage patients with cutaneous malignant melanoma. Immunohistochemical stains contribute to the detection of micrometastases; however, molecular biology techniques are associated with better diagnostic sensitivity. Sixty sentinel lymph nodes were included in this study. The primary lesions were malignant melanoma stage I or II, with a follow-up of longer than 2 years. Sentinel lymph nodes were studied with hematoxylin-eosin, immunohistochemistry for S-100 and HMB-45, and molecular biology techniques (reverse transcription (RT)-PCR) for the detection of tyrosinase messenger RNA. In 15 of 60 cases (25%), tyrosinase was detected by RT-PCR; three of these cases were also positive by immunohistochemistry. The population was divided into three groups: (i) hematoxylin-eosin-/immunohistochemistry+/molecular biology techniques+ (3 cases); (ii) hematoxylin-eosin-/immunohistochemistry-/molecular biology techniques+ (12 cases); (iii) hematoxylin-eosin-/immunohistochemistry-/molecular biology techniques- (45 cases). Correlation of the groups with overall survival showed the following: (i) 2 of 3 patients died (67%); (ii) 5 of 12 died (42%), and (iii) all 45 patients are alive, with no lymphadenectomy and a median follow-up of 84 months. The inclusion of molecular biology techniques appears to be of great value for the detection of sentinel lymph node micrometastases in patients with cutaneous malignant melanoma. In our series, those patients who showed negativity with all the three methods had a null recurrence rate. Therefore, this triple negativity could be a positive prognostic factor for overall survival. Our findings suggest the possibility of molecular oncological staging, which would allow the selection of patients with submicroscopic metastases for a complete treatment.

Sentinel lymph node: detection of micrometastases of melanoma in a molecular study.

INTRODUCTION: Lymph node status in patients with cutaneous malignant melanoma is the most important prognostic factor. Patients with clinically positive nodes (stage III) should undergo therapeutic lymphadenectomy; however, the surgical approach to the regional disease in patients with negative clinical examination (stage I and II) is still controversial. Selective lymphadenectomy consists of the intraoperative identification of the first node in the nodal basin, the sentinel lymph node (SLN). Routine examination, serial sectioning, and immunohistochemistry may underestimate the presence of tumor cells. PCR is a molecular biology technique that may be useful for the detection of malignant melanoma nodal metastases in the SLN. AIM: The aim of this study was to use tyrosinase messenger RNA (mRNA) amplification for the detection of micrometastases in fresh frozen SLNs. METHODS: 46 hematoxylin-eosin (HE)-negative sentinel node samples from 42 patients with malignant melanoma were included in this study. Formalin-fixed paraffin-embedded sections were immunostained with S-100 protein and HMB-45. A central portion of the node was submitted for PCR. This method was accomplished with a combination of reverse transcription and amplification of the tyrosinase complementary DNA and double- round PCR (nested reverse transcriptase [RT]-PCR). RESULTS: In 1 of the 42 SLN-negative patients, immunohistochemistry stains allowed the detection of micrometastases. With molecular biology, 14 of the 42 SLN patients were positive (33%); in another 12 (29%), only the nested RT-PCR was positive. Of the 42 patients, 24 were put into 3 groups and followed for a 5-year period with 1, 7, and 16 patients, respectively, in the groups. The first group involved 1 patient who had provided 2 SLN samples that were found to be SLN-positive using both techniques, immunohistochemistry stains and nested RT-PCR (he had hepatic metastasis and died 24 months after diagnosis). The second group, with only nested RT-PCR positive SLN samples, included 7 of 12 patients who were followed and had a median survival of 37 months; 4 died of widespread metastatic disease, the other 3 patients had event-free survival, but 1 consented to undergo a therapeutic lymphadenectomy as a result of a positive test. The last group consisting of 16 of 32 patients, with complete 5-year survival, who were SLN-negative with both techniques, immunohistochemistry stains and nested RT-PCR. Fourteen of the 16 (88%) were event-free survival during the follow-up, and 2 had local relapse. CONCLUSION: Tyrosinase mRNA amplification may be a negative prognostic factor for the detection of micrometastases in fresh frozen SLNs using molecular biology techniques.

Análisis inmunohistoquímico de p53 y c-erbB-2 en el carcinoma de mama / Immunohistochemical analysis of p53 and c-erbB-2 in breast cancer

We have studied by immunohistochemistry the nuclear expression of p53 and the finding of oncoprotein c-erbB-2 in a series of 85 breast carcinomas. The tissue was fixed in buffered formalin and embedded in paraffin. The primary antisera were obtained from Biogenex (USA), Bp53-12, monoclonal, used in a dilution of 1:100 for p53 and from Triton Diagnostics (USA), pAB-1, polyclonal, used in a dilution of 1:200 for c-erbB2. The detection system was the Vectastain Elite kit obtained from Vector Laboratories (USA). The results were compared with several morphological features of the tumors such as size and type of the tumor, extension of the intraductal component, nuclear grade, axillary lymph node metastasis and estrogen receptor data as well as with total and disease free survival. The oncoprotein p53 was detected in the nuclei in 25 (29.4 percent) of our cases (Fig 1). Its presence was correlated with tumor size (p < 0.01), high nuclear grade (p < 0.0001) and estrogen receptor negative tumors (p < 0.0001). There was an inverse relationship between p53 expression and 5 year disease free survival (p < 0.005). In 21 (24,7 percent) of the cases we found amplification of c-erbB-2. The staining pattern was membranous (Fig. 2). It was correlated significantly with the ductal type of invasive carcinoma (p < 0.05), an associated extensive intraductal component (p < 0.001), estrogen receptor negative tumors (p < 0.0001) and shortening of disease free survival in the patients with positive axillary lymph nodes (p < 0.005). In 9 cases we found staining for both markers without statistically significant relationship with the other features studied. We conclude that the presence of these genetic alterations demonstrated by immunohistochemistry were, in our series, unfavourable prognostic factors in invasive breast cancer.

Análisis inmunohistoquímico de p53 y c-erbB-2 en el carcinoma de mama / Immunohistochemical analysis of p53 and c-erbB-2 in breast cancer

We have studied by immunohistochemistry the nuclear expression of p53 and the finding of oncoprotein c-erbB-2 in a series of 85 breast carcinomas. The tissue was fixed in buffered formalin and embedded in paraffin. The primary antisera were obtained from Biogenex (USA), Bp53-12, monoclonal, used in a dilution of 1:100 for p53 and from Triton Diagnostics (USA), pAB-1, polyclonal, used in a dilution of 1:200 for c-erbB2. The detection system was the Vectastain Elite kit obtained from Vector Laboratories (USA). The results were compared with several morphological features of the tumors such as size and type of the tumor, extension of the intraductal component, nuclear grade, axillary lymph node metastasis and estrogen receptor data as well as with total and disease free survival. The oncoprotein p53 was detected in the nuclei in 25 (29.4 percent) of our cases (Fig 1). Its presence was correlated with tumor size (p < 0.01), high nuclear grade (p < 0.0001) and estrogen receptor negative tumors (p < 0.0001). There was an inverse relationship between p53 expression and 5 year disease free survival (p < 0.005). In 21 (24,7 percent) of the cases we found amplification of c-erbB-2. The staining pattern was membranous (Fig. 2). It was correlated significantly with the ductal type of invasive carcinoma (p < 0.05), an associated extensive intraductal component (p < 0.001), estrogen receptor negative tumors (p < 0.0001) and shortening of disease free survival in the patients with positive axillary lymph nodes (p < 0.005). In 9 cases we found staining for both markers without statistically significant relationship with the other features studied. We conclude that the presence of these genetic alterations demonstrated by immunohistochemistry were, in our series, unfavourable prognostic factors in invasive breast cancer.(AU)