Additional reverse transcription-polymerase chain reaction of peripheral slices is not superior to analysis of the central slice in sentinel lymph nodes from melanoma patients.

BACKGROUND: The status of the sentinel lymph node (SLN) is an important prognostic factor in patients with cutaneous melanoma. Reverse transcription-polymerase chain reaction (RT-PCR) has been used as a sensitive means of detecting tumour cells in SLNs. OBJECTIVES: To determine whether RT-PCR analysis of the SLN using both the central and the peripheral slices is more sensitive than molecular analysis of the central slice only. METHODS: Eighty-three SLNs from 59 patients with primary cutaneous melanoma were identified by SLN mapping. All SLNs were bisected along their longitudinal axis to produce two equal halves. One half was used for histology and immunohistochemistry, and the other was analysed by RT-PCR for tyrosinase and MelanA. Parallel to the longitudinal axis, one central slice (approximately 2 mm in thickness) was cut manually. This central slice was used for our standard RT-PCR protocol. In the current study, up to eight additional peripheral slices (each approximately 2 mm in thickness) were cut parallel to the existing cut surface. These peripheral slices were analysed by additional RT-PCR. RESULTS: Standard RT-PCR of the central slice yielded positive results in 34 of 59 patients (57%). Additional RT-PCR of peripheral slices demonstrated positive findings in six additional patients (10%) who were initially negative by standard RT-PCR of the central slice. In detail, seven of those 34 patients positive by standard RT-PCR of the central slice had positive histological findings. In each of these seven patients, RT-PCR was positive both in the central slice as well as in the peripheral slices. The remaining 27 patients with positive RT-PCR results of the central slice showed negative histological findings. Only nine (33%) of these 27 patients had a positive RT-PCR also in the peripheral slices. Finally, all 25 patients with negative RT-PCR results in the central slice showed negative histological findings. Six of these patients (24%) revealed positive RT-PCR results on the analysis of peripheral slices. However, three of these patients expressed only MelanA but not tyrosinase. Thirty lymph nodes from 24 nonmelanoma patients served as negative controls for RT-PCR. In three of these 24 patients (13%) expression of MelanA but not tyrosinase was detected by RT-PCR. CONCLUSIONS: Molecular analysis of peripheral slices yielded six additional patients (10%) positive by RT-PCR who were initially negative by standard RT-PCR of the central slice. However, three of these six patients were found to express only MelanA but not tyrosinase. As MelanA expression was also found in 13% of control lymph nodes, positive MelanA expression alone in SLNs of melanoma patients requires cautious interpretation in order to avoid false-positive findings. Thus, additional molecular processing of peripheral slices did not significantly increase the number of patients with RT-PCR-positive SLNs.

Detection of melanoma cells in sentinel lymph nodes, bone marrow and peripheral blood by a reverse transcription-polymerase chain reaction assay in patients with primary cutaneous melanoma: association with Breslow's tumour thickness.

BACKGROUND: Tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) has been shown to be highly sensitive in detecting tumour cells in melanoma patients. OBJECTIVE: To assess whether the detection of minimal residual disease by RT-PCR is improved by concomitant analysis of sentinel lymph nodes (SLNs), bone marrow (BM) and peripheral blood (PB) in patients with primary melanoma. METHODS: Thirty-five SLNs, 41 BM samples and 26 PB specimens from 26 patients with primary cutaneous melanoma (tumour thickness > or = 0.75 mm) were examined by nested RT-PCR for tyrosinase and Melan-A. SLNs and BM samples were also analysed by histopathology. RT-PCR findings were related to tumour thickness of the primary melanoma. RESULTS: Overall, melanoma cells were detected by RT-PCR in 13 of 26 patients (50%). Seven patients had positive RT-PCR results in their SLNs (27%), including all patients (n = 4) with histologically positive SLNs, two patients had positive findings in their BM exclusively detected by RT-PCR (8%) and six patients in PB (23%). The presence of tumour cells detected by RT-PCR in SLNs was not related to the presence of melanoma cells in BM and/or PB. The incidence of RT-PCR-positive SLNs was significantly associated with greater tumour thickness (P = 0.004). Both patients with positive RT-PCR findings in their BM had a large tumour thickness (> or = 2 mm). No association between positive RT-PCR findings in PB and greater tumour thickness was observed. CONCLUSIONS: RT-PCR-positive SLNs were strongly associated with greater tumour thickness, underlining the prognostic significance of SLN positivity. Similar to certain epithelial malignancies, molecular investigation of the BM might provide complementary prognostic information in the early stages of melanoma. In contrast, no association between positive RT-PCR results in PB and increasing tumour thickness was found, implying that RT-PCR findings in PB are of doubtful clinical relevance in primary melanoma.

Does intensive histopathological workup by serial sectioning increase the detection of lymph node micrometastasis in patients with primary cutaneous melanoma?

Various histopathological techniques have been developed in order to improve the detection of micrometastasis in the regional lymph nodes of patients with malignant melanoma. Our standard histopathological examination of lymph nodes included haematoxylin and eosin (H & E) staining and immunohistochemistry (IH) using antibodies to HMB-45 and S-100 proteins of three paraffin sections at one level. In addition, lymph nodes were examined by molecular biological methods using tyrosinase reverse transcription-polymerase chain reaction (RT-PCR). In this study, we investigated the use of step sections and IH in lymph nodes regarded as negative by standard histopathology but positive by tyrosinase RT-PCR, suggesting the presence of tumour cells. In a series of 76 consecutive patients with stage I and II cutaneous melanoma, a total of 156 regional lymph nodes were examined by H & E staining, IH and tyrosinase RT-PCR. All lymph nodes were bisected along their long axis for separate evaluation. In 21 patients, at least one lymph node in the regional nodal basin reported as tumour-negative by standard histopathology was demonstrated to express tyrosinase (total number of nodes = 33). These 33 lymph nodes were re-examined by H & E and IH at 10 additional levels of the paraffin block. Only one lymph node from one patient had occult melanoma cells in deeper levels detected exclusively by IH. Six out of 20 patients with positive findings exclusively on tyrosinase RT-PCR developed tumour recurrences during a median follow-up of 34 months. We therefore conclude that additional step sectioning with IH does not significantly increase the detection of tumour-positive lymph nodes. Patients with melanoma cells detected exclusively by RT-PCR, however, were shown to be at increased risk for tumour recurrence.

Examination of regional lymph nodes by sentinel node biopsy and molecular analysis provides new staging facilities in primary cutaneous melanoma.

Histopathologic parameters of the primary tumor, such as Breslow's tumor thickness and Clark's level of invasion are the current basis for prognostic classifications of primary cutaneous melanoma. Once patients develop regional node metastasis, histopathologic features of the primary melanoma no longer contribute significantly to survival prediction. In this tumor stage, the extent of lymph node involvement is the main prognostic factor. This study addresses the question whether application of a highly sensitive molecular biology assay for detection of submicroscopic melanoma cells in sentinel lymph nodes may be suitable to improve melanoma staging. One hundred and sixteen patients with primary cutaneous melanoma with a total of 214 sentinel lymph nodes were enrolled. Sentinel lymph nodes were analyzed by histopathology including immunohistochemistry and by reverse transcription-polymerase chain reaction for tyrosinase. Patients were examined for tumor recurrences during a follow-up period of 19 mo (median). Disease-free survival probabilities were calculated and independent prognostic factors were determined by multivariate analysis. Using histopathology, micrometastatic nodal involvement was detected in 15 patients (13%). Of the 101 patients with histopathologically negative sentinel lymph nodes, 36 were reclassified by positive tyrosinase reverse transcription-polymerase chain reaction and 65 patients were still negative by reverse transcription-polymerase chain reaction. Recurrences were observed in 23 (20%) of 116 patients. These tumor recurrences were demonstrated in 10 patients (67%) with histopathologically positive sentinel lymph nodes, in nine patients (25%) with submicroscopic tumor cells detected by reverse transcription-polymerase chain reaction, and in four patients (6%) negative by both methods. The differences in recurrence rates were statistically significant (p = 0.01). In a multivariate analysis, histopathologic and reverse transcription-polymerase chain reaction status of the sentinel lymph node were demonstrated to be the only significant prognostic factors for predicting disease-free survival. Tyrosinase reverse transcription-polymerase chain reaction for the detection of minimal residual melanoma in sentinel lymph nodes is a powerful tool to determine patients who are at increased risk for subsequent metastasis. Moreover, a group of patients with high tumor thickness was identified by negative reverse transcription-polymerase chain reaction to be at low risk for recurrent disease. These data may have an impact on future tumor classifications of primary cutaneous melanoma.

Detection of melanoma micrometastasis in sentinel nodes by reverse transcription-polymerase chain reaction correlates with tumor thickness and is predictive of micrometastatic disease in the lymph node basin.

The sentinel node has been reported to be representative for the presence or absence of metastatic melanoma in the draining lymph node basin. In this study, for the first time sentinel nodes and adjoining nonsentinel nodes were analyzed for micrometastatic disease using tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) in comparison with standard immunohistochemistry. Successful identification of the sentinel nodes using a gamma probe-guided surgery was achieved in 73 (92%) of 79 patients with cutaneous stage I and II melanoma (tumor thickness > or =0.75 mm). A total of 794 regional lymph nodes, 148 sentinel nodes, and 646 adjoining nonsentinel nodes were evaluated. Tyrosinase RT-PCR was shown to increase the sensitivity for melanoma cell detection in sentinel nodes significantly (49% positivity) as compared with immunohistochemistry using antibodies against HMB-45 antigen and S-100 protein (18% positivity). Examination of sentinel nodes was highly predictive in determining the presence of regional lymph node micrometastasis by immunohistochemistry (99%) and RT-PCR (89%). Interestingly, detection of nodal micrometastasis by RT-PCR showed a strong positive correlation with tumor thickness of primary cutaneous melanoma. These results suggest the clinical significance and emphasize the importance of tyrosinase RT-PCR for detection of melanoma micrometastasis in sentinel nodes.

Staging, grading and related histopathological techniques in local therapy of rectal tumours.

Today, local therapy is an established alternative to radical resection for the treatment of rectal tumours. Selection of the operative technique requires an exact perioperative estimation of risks, with both clinical and histopathological examination. Of crucial importance in making a decision to perform a local resection is exact and meticulous histopathological preparation of the tissue. The most important criterion is the estimation of risk of lymph node metastases. This risk is assessed on the basis of the depth of invasion of the tumour, the histological grade of differentiation and the presence or absence of invasion of lymphatic vessels.

Fungal infections in cancer patients: an international autopsy survey.

In an attempt to estimate the frequency of fungal infections among cancer patients, a survey of autopsy examinations was conducted in multiple institutions in Europe, Japan and Canada. Fungal infections were identified most often in leukemic patients and transplant recipients (25% each). Fifty-eight percent of fungal infections were caused by Candida spp. and 30% by Aspergillus spp. There was considerable variability in the frequency of fungal infections in different countries. Nevertheless, this study clearly demonstrates that fungal infections represent a common complication in cancer patients, especially in patients with leukemia.