Prognostic value of RT-PCR tyrosinase detection in peripheral blood of melanoma patients

Malignant melanoma (MM) prognosis has been related to tumour thickness and clinical stage and metastasis risk has been associated with presence of tumour cells in peripheral blood. The aim of this study was to determine the relationship between presence of tyrosinase in peripheral blood of MM patients and their clinical prognosis. Blood samples from 58 MM patients (stage I-IV) were analysed, using RT-PCR assay to detect tyrosinase mRNA. The results showed that positive RT-PCR assay for tyrosinase were significantly associated with clinical status and tumour thickness. After a median follow-up of 24 months, RT-PCR results were found to be significant correlated with recurrence (p<0.05) and clinical stage III (p<0.05). Separate analysis of stage III tumours to determine the prognostic value of tyrosinase presence in peripheral blood showed an overall 24-month survival rate of 70% in the RT-PCR negative group versus 10% in the positive group (p<0.02). These results suggest that detection of circulating melanoma cells may be especially relevant in stage III patients, in whom RT-PCR positivity defines a subpopulation at high risk of recurrence.     

Inducible nitric oxide synthase expression in benign and malignant cutaneous melanocytic lesions

Nitric oxide (NO) is synthesized by nitric oxide synthases (NOS) and plays an important role in tumour growth. In this study, inducible NOS (iNOS) expression was evaluated by immunohistochemistry in 34 melanocytic naevi (13 common melanocytic naevi, six Spitz naevi, and 15 so-called 'dysplastic naevi'), ten cutaneous melanomas in situ, 50 stage I invasive melanomas, and eight subcutaneous metastases of melanoma. In addition, four samples of melanocytic naevi and four samples of invasive melanomas were collected in order to perform western blot and northern blot analysis. By immunohistochemistry, melanocytic naevi never expressed iNOS. Among cases of melanoma in situ, two were negative, seven displayed staining in less than 20% of melanoma cells, and positivity was observed in 21-50% of melanoma cells in only one case. iNOS expression was detected in 46 out of 50 invasive melanomas (92%). Among these cases, 18 showed positivity in less than 20% of melanoma cells, 18 showed positivity in 21-50% of melanoma cells, and ten showed iNOS expression in more than 50% of cells. Statistical analysis revealed a significant difference in iNOS expression between melanocytic naevi and cutaneous melanomas (p<0.001). In addition, iNOS expression was significantly higher in invasive melanomas than in melanomas in situ (p=0.01). Among primary cutaneous melanomas, no significant correlation was found between iNOS expression and histopathological parameters (histotype, level, thickness and presence of regression/inflammatory infiltrate) and disease-specific survival. In subcutaneous melanoma metastases, iNOS expression was diffuse in more than 50% of cells. Statistical analysis revealed that subcutaneous melanoma metastases showed greater iNOS immunoreactivity than invasive melanomas (p=0.02). Molecular analyses confirmed that iNOS mRNA and protein were highly expressed in melanoma samples. In conclusion, iNOS was constantly absent in melanocytic naevi, whereas it was frequently expressed in melanomas, with up-regulation of the enzyme paralleling tumour progression. These data suggest that iNOS may play a role in the malignant transformation of melanocytes and in tumour growth. In addition, iNOS may be useful as an immunohistochemical marker for malignant melanocytic lesions.     

Autonomous histopathological regression of primary tumours associated with specific immune responses to cancer antigens

Spontaneous histopathological regression of cancer has been reported. The involvement of the immune system in such regression has been advocated, leading to the theory of immunological surveillance against cancer. A prediction of this theory is that common tumour antigens can be recognized upon repeated exposure by cell-mediated immunity, which leads to tumour regression and the subsequent appearance of tumour antigen-loss variants. However, no direct evidence has been provided in non-viral-induced experimental animal models of primary malignancy or in human primary cancer. This study examined two groups of melanoma patients where histopathological regression of the primary tumour was observed. Many of the 23 patients with multiple (> or =3) primary melanomas showed significant regression of their last melanoma (median 33%, mean 40) compared with matched melanomas from patients with a single primary melanoma (median 0%, mean 12) (p=0.0080), or compared with their first primary melanoma (p=0.0013). Regression was consistent with an 'immunization effect' seen in murine tumour transplantation studies, where inoculation with > or =3 asynchronous tumours induces transplantation rejection on subsequent challenge. A significant decrease in the expression of the melanoma common tumour antigen MART-1 in the last primary tumour from multiple melanoma patients (median 8%, mean 24) versus matched single melanoma patients (median 79%, mean 68) (p=0.0041) and in the last versus first tumour in multiple primary patients was found (p=0.0083). Metastases from 17 patients whose primary skin melanomas had completely regressed (occult primary melanoma) also showed significant MART-1 loss (median 0%, mean 11) compared with matched metastases from patients with non-regressing primary melanoma (median 51%, mean 50) (p=0.0013). MART-1 antigen-loss variants observed in the multiple primary and occult primary patients correlated with the presence of peripheral blood MART-1-specific cytotoxic T lymphocytes (CTLs) (p=0.03). No similar effects were observed with two other melanoma antigens, gp100 and CD63. Thus, in two groups of human melanoma patients, evidence is provided for histopathological tumour regression associated with cancer immune surveillance.     

False-positive cells in sentinel lymph nodes

Melanoma sentinel lymph nodes (SLN) are carefully evaluated to maximize sensitivity. Examination includes hematoxylin and eosin (H+E) stained sections at multiple levels through the node, with subsequent immunohistochemical (IHC) stains for melanocytic markers if H+E sections are negative for melanoma. However, not all IHC-positive cells in SLN are metastatic melanoma, as evidenced by the presence of MART-1 positive cells in SLN from breast cancer patients with no history of melanoma (so-called 'false-positive' cells). These 'false-positive cells' could be nodal nevus, non-melanocytic cells with cross-reacting antigenic determinants, phagocytic cells containing melanocyte antigens, or possibly melanocytes or melanocyte stem cells liberated at the time of biopsy of the cutaneous melanoma. Examination of SLN requires careful correlation of H+E and IHC findings.     

Fluorescence In Situ Hybridization (FISH) Analysis of Melanocytic Nevi and Melanomas: Sensitivity, Specificity, and Lack of Association With Sentinel Node Status

A 4-color fluorescence in situ hybridization (FISH) assay, using probes to chromosomes 11q, 6p, 6q, and 6 cent, has recently been proposed as an ancillary tool for the diagnosis of melanoma. The authors report herein their experience with this assay. To determine the sensitivity and specificity of the assay for histopathologically unequivocal cases, they analyzed 50 melanocytic nevi, 50 primary melanomas, and 15 metastatic melanomas. Of 50 melanocytic nevi, 47 were FISH negative on initial readout (test sensitivity, 94%); 49 were FISH negative after correction for tetraploidy (test specificity, 98%). Of 50 primary melanomas, 41 were FISH positive (test sensitivity, 82%). Of 15 metastatic lesions, 13 were FISH positive (test sensitivity, 85%). Of the 9 FISH-negative melanomas, 6 metastasized. The tumors of the 5 patients who had survived thick primary melanoma for more than 5 years without recurrence were all FISH positive. Half of the patients whose primary melanoma was tested by FISH had undergone sentinel lymph node (SLN) biopsy. When the authors compared the FISH results of those 25 melanomas with the SLN status, no statistically significant correlation was found. These findings document limitations of the current FISH assay. A rare nevus may be FISH positive. Some primary metastasizing melanomas are FISH negative. Even metastatic melanomas can be FISH negative. Awareness of the limitations in test sensitivity and specificity of the FISH assay is important to avoid an erroneous diagnosis by overreliance on cytogenetic findings. Correlation with clinical and histopathological findings is paramount for accurate diagnosis.     

Combined application of RT-PCR and immunohistochemistry on paraffin embedded sentinel lymph nodes of prostate cancer patients

The detection of tumor cells in the sentinel lymph node (SLN) is of great importance for the prognosis of cancer patients. At present, immunohistochemistry and RT-PCR for tumor marker expression are the most sensitive techniques available for this analysis. However, so far, most RT-PCR-based analyses of SLNs have been performed on fresh material, excluding a direct comparison with the (immuno)histologic results. In our view, this does not entirely aid routine diagnosis. We established an efficient method for RNA extraction and RT-PCR from paraffin sections of SLNs from prostate cancer patients and compared the results with the (immuno)histologic data of adjacent sections. Amplifiable RNA was obtained from 133 SLNs of 68 prostate cancer patients. Correlation of PSA-specific RT-PCR with (immuno)histologic findings showed a positive and negative predictive value of 83% and 100%, respectively, for the prostate cancer patients investigated. Four of 12 patients with biochemical relapse, but without (immuno)histologically detectable tumor cells were RT-PCR-positive for PSA. We found that single sections of paraffin-embedded SLNs are suitable for routinely performed RT-PCR. Combined with (immuno)histology, PSA-specific RT-PCR is a revealing supplementary technique for the detection of tumor cells in SLNs of prostate cancer patients.     

Metastatic melanoma volume in sentinel nodes: objective stereology-based measurement predicts disease recurrence and survival

Aims:  Sentinel lymph node (SLN) status is the most important prognostic factor in intermediate thickness melanoma. The amount of metastatic disease in positive SLNs varies greatly between patients, and this tumour burden appears to influence the prognosis of node-positive patients. The aim was to use objective stereological techniques to correlate accurately total SLN tumour burden with recurrence and patient survival.
Methods and results:  SLNs from 327 patients were examined by complete step sectioning and immunohistochemistry. The total metastasis volume (TMV) of 156 positive SLNs from 99 patients (30.3%) was measured using stereological methods based on the 2D-nucleator and Cavalieri's principle. The maximum metastasis diameter was also measured. These two measurements were correlated with disease recurrence and patient survival. The mean TMV for SLN+ patients was 10.5 mm³ (median 0.05 mm³; range 0.0001–623.7 mm³). Median follow-up was 26.3 months. On multivariate analysis, TMV was an independent predictor of recurrence when corrected for primary tumour thickness ( P  = 0.001) and was a stronger prognosticator compared with the maximum metastasis diameter ( P  < 0.0001 versus P  = 0.01).
Conclusions:  Combining total step sectioning of SLNs with stereological assessment of metastases, we found metastasis volume to be a highly significant predictor of disease recurrence and survival.     

The development of optimal pathological assessment of sentinel lymph nodes for melanoma

1158 sentinel lymph nodes (SLNs), excised from patients with primary cutaneous melanoma, were assessed pathologically using histology with immunohistochemistry (IHC) on all nodes, and RT-PCR for Mart-1 and tyrosinase on 55 nodes. RT-PCR was compared with the histology and IHC assessed on the same nodes. The evaluation of progressively more detailed protocols for histology and IHC modulated by the RT-PCR results led to a procedure that consistently detects metastases in 34% of patients submitted to SLN biopsy for cutaneous melanomas with a vertical growth phase and a mean thickness of 2.02 mm (range 0.25, with regression, to 19 mm). As this technique is virtually free of false positives and produces only a marginally lower detection rate than RT-PCR, which was subject to false positives of 7% in our study, it is suggested that this extended protocol should be the basis on which further evaluation of the place of RT-PCR in SLN assessment takes place. The evolved protocol described here has been adopted by the EORTC as the standard procedure for pathological handling of sentinel lymph nodes for melanoma when SLN status is a criterion in their clinical trials or studies.     

Automated digital volume measurement of melanoma metastases in sentinel nodes predicts disease recurrence and survival

Riber‐Hansen R, Nyengaard J R, Hamilton‐Dutoit S J, Sjoegren P & Steiniche T
(2011) Histopathology 59 , 433–440 Automated digital volume measurement of melanoma metastases in sentinel nodes predicts disease recurrence and survival Aims: Total metastatic volume (TMV) is an important prognostic factor in melanoma sentinel lymph nodes (SLNs) that avoids both the interobserver variation and unidirectional upstaging seen when using semi‐quantitative size estimates. However, it is somewhat laborious for routine application. Our aim was to investigate whether digital image analysis can estimate TMV accurately in melanoma SLNs. Methods and results: TMV was measured in 147 SLNs from 95 patients both manually and by automated digital image analysis. The results were compared by Bland–Altman plots (numerical data) and kappa statistics (categorical data). In addition, disease‐free and melanoma‐specific survivals were calculated. Mean metastatic volume per patient was 10.6 mm³ (median 0.05 mm³; range 0.0001–621.3 mm³) and 9.62 mm³ (median 0.05 mm³; range 0.00001–564.3 mm³) with manual and digital measurement, respectively. The Bland–Altman plot showed an even distribution of the differences, and the kappa statistic was 0.84. In multivariate analysis, both manual and digital metastasis volume measurements were independent progression markers when corrected for primary tumour thickness [manual: hazard ratio (HR): 1.21, 95% confidence interval (CI): 1.07–1.36, P = 0.002; digital: HR: 1.21, 95% CI: 1.06–1.37, P = 0.004]. Conclusions: Stereology‐based, automated digital metastasis volume measurement in melanoma SLNs predicts disease recurrence and survival.     

Pathologic evaluation of sentinel lymph nodes in colorectal carcinoma

BACKGROUND: The identification of lymph node metastases in colorectal resection specimens is necessary for accurate tumor staging. However, routine lymph node dissection by the pathologist yields only a subset of nodes removed surgically and may not include those nodes most directly in the path of lymphatic drainage from the tumor. Intraoperative mapping of such sentinel lymph nodes (SLNs) has been reported in cases of melanoma and breast cancer. We applied a similar method to cases of colorectal carcinoma, with emphasis on the pathology of the SLNs. METHODS: Eighty-three consecutive patients with colorectal carcinoma were evaluated after intraoperative injection of 1 to 2 mL of 1% isosulfan blue dye (Lymphazurin) into the peritumoral subserosa. Blue-stained lymph nodes were suture-tagged by the surgeon within minutes of the injection for identification by the pathologist, and a standard resection was performed. Designated SLNs were sectioned at 10 levels through the block; a cytokeratin immunostain (AE1) was also obtained. To evaluate the possibility that increased detection of metastases in the SLN might be solely due to increased histologic sampling, all initially negative non-SLNs in the first 25 cases were sectioned also at 10 levels. RESULTS: Sentinel lymph nodes were identified intraoperatively in 82 (99%) of 83 patients and accounted for 152 (11.9%) of 1275 lymph nodes recovered, with an average of 1.9 SLNs per patient. A total of 99 positive lymph nodes (38 positive SLNs and 61 positive non-SLNs) were identified in 34 node-positive patients. The SLNs were the only site of metastasis in 17 patients (50%), while 14 patients (41%) had both positive SLNs and non-SLNs. Three patients (9%) had positive non-SLNs with negative SLNs, representing skip metastases. In patients with positive SLNs, 91 (19%) of 474 total lymph nodes and 53 (12%) of 436 non-SLNs were positive for metastasis. In patients with negative SLNs, 8 (1%) of 801 total lymph nodes and 8 (1.2%) of 687 non-SLNs were positive for metastasis. Multilevel sections of 330 initially negative non-SLNs in the first 25 patients yielded only 2 additional positive nodes (0. 6%). All patients with positive SLNs were correctly staged by a combination of 4 representative levels through the SLN(s) together with a single cytokeratin immunostain. CONCLUSIONS: Intraoperative mapping of SLNs in colorectal carcinoma identifies lymph nodes likely to contain metastases. Focused pathologic evaluation of the 1 to 4 SLNs so identified can improve the accuracy of pathologic staging.