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Quantitative measurement of melanoma spread in sentinel lymph nodes and survival.

Ulmer A, Dietz K, Hodak I, Polzer B, Scheitler S, Yildiz M, Czyz Z, Lehnert P, Fehm T, Hafner C, Schanz S, Röcken M, Garbe C, Breuninger H, Fierlbeck G, Klein CA - PLoS Med. (2014)

Bottom Line: The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up.If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Tübingen, Tübingen, Germany.

ABSTRACT

Background: Sentinel lymph node spread is a crucial factor in melanoma outcome. We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.

Methods and findings: We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology. For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100. None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells. We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations. At a median follow-up of 49 mo (range 3-123 mo), 138 patients (13.4%) had died from melanoma. Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61-2.01, for a 10-fold increase in DCCD + 1). Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02-2.58). Upon multivariable testing DCCD was a stronger predictor of death than histopathology. The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.

Conclusions: Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death. A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up. If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically.

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Sample preparation, melanoma cell detection, and distribution of disseminated cancer cell densities.(A) The sentinel nodes were split into halves for routine histopathology and immunocytology. (B–D) After mincing (B), the small pieces were subjected to mechanical disaggregation (C), and single-cell suspensions were added to adhesion slides (D). (E) The detection of gp100-positive cells among the unstained lymphocytes. (a) gp100 staining of Mel Ho cell line cells mixed among peripheral blood leukocytes used as positive control. (b) Isotype control of positive control. Note that the large melanoma cells can be identified by size among the peripheral blood leukocytes and are completely unstained. (c) gp100-positive cell from melanoma patient with DCCD = 1. (d) Isotype control of the melanoma patient in (c). All four samples are taken from the same experiment. (F) The distribution of DCCD (number of DCCs per 106 isolated cells) in the sentinel nodes of 525 gp100-positive patients. Note that some patients had DCCD <1. The 502 zero values for DCCD are not included.
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pmed-1001604-g001: Sample preparation, melanoma cell detection, and distribution of disseminated cancer cell densities.(A) The sentinel nodes were split into halves for routine histopathology and immunocytology. (B–D) After mincing (B), the small pieces were subjected to mechanical disaggregation (C), and single-cell suspensions were added to adhesion slides (D). (E) The detection of gp100-positive cells among the unstained lymphocytes. (a) gp100 staining of Mel Ho cell line cells mixed among peripheral blood leukocytes used as positive control. (b) Isotype control of positive control. Note that the large melanoma cells can be identified by size among the peripheral blood leukocytes and are completely unstained. (c) gp100-positive cell from melanoma patient with DCCD = 1. (d) Isotype control of the melanoma patient in (c). All four samples are taken from the same experiment. (F) The distribution of DCCD (number of DCCs per 106 isolated cells) in the sentinel nodes of 525 gp100-positive patients. Note that some patients had DCCD <1. The 502 zero values for DCCD are not included.

Mentions: The underlying rationale of our detection assay (Figure 1A–1E) comprises two aspects. First, the spatially inhomogeneous distribution of melanoma cells may be equalized by lymph node disaggregation and generation of a single-cell suspension, which facilitates melanoma cell detection when only parts of the sample are screened; second, the number of melanoma cells can be counted and the amount of analyzed tissue can be quantified by referencing the number of melanoma cells to a defined number of isolated lymph node cells. This allows determining the DCCD, i.e. the number of DCCs per 1 million isolated cells.


Quantitative measurement of melanoma spread in sentinel lymph nodes and survival.

Ulmer A, Dietz K, Hodak I, Polzer B, Scheitler S, Yildiz M, Czyz Z, Lehnert P, Fehm T, Hafner C, Schanz S, Röcken M, Garbe C, Breuninger H, Fierlbeck G, Klein CA - PLoS Med. (2014)

Sample preparation, melanoma cell detection, and distribution of disseminated cancer cell densities.(A) The sentinel nodes were split into halves for routine histopathology and immunocytology. (B–D) After mincing (B), the small pieces were subjected to mechanical disaggregation (C), and single-cell suspensions were added to adhesion slides (D). (E) The detection of gp100-positive cells among the unstained lymphocytes. (a) gp100 staining of Mel Ho cell line cells mixed among peripheral blood leukocytes used as positive control. (b) Isotype control of positive control. Note that the large melanoma cells can be identified by size among the peripheral blood leukocytes and are completely unstained. (c) gp100-positive cell from melanoma patient with DCCD = 1. (d) Isotype control of the melanoma patient in (c). All four samples are taken from the same experiment. (F) The distribution of DCCD (number of DCCs per 106 isolated cells) in the sentinel nodes of 525 gp100-positive patients. Note that some patients had DCCD <1. The 502 zero values for DCCD are not included.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928050&req=5

pmed-1001604-g001: Sample preparation, melanoma cell detection, and distribution of disseminated cancer cell densities.(A) The sentinel nodes were split into halves for routine histopathology and immunocytology. (B–D) After mincing (B), the small pieces were subjected to mechanical disaggregation (C), and single-cell suspensions were added to adhesion slides (D). (E) The detection of gp100-positive cells among the unstained lymphocytes. (a) gp100 staining of Mel Ho cell line cells mixed among peripheral blood leukocytes used as positive control. (b) Isotype control of positive control. Note that the large melanoma cells can be identified by size among the peripheral blood leukocytes and are completely unstained. (c) gp100-positive cell from melanoma patient with DCCD = 1. (d) Isotype control of the melanoma patient in (c). All four samples are taken from the same experiment. (F) The distribution of DCCD (number of DCCs per 106 isolated cells) in the sentinel nodes of 525 gp100-positive patients. Note that some patients had DCCD <1. The 502 zero values for DCCD are not included.
Mentions: The underlying rationale of our detection assay (Figure 1A–1E) comprises two aspects. First, the spatially inhomogeneous distribution of melanoma cells may be equalized by lymph node disaggregation and generation of a single-cell suspension, which facilitates melanoma cell detection when only parts of the sample are screened; second, the number of melanoma cells can be counted and the amount of analyzed tissue can be quantified by referencing the number of melanoma cells to a defined number of isolated lymph node cells. This allows determining the DCCD, i.e. the number of DCCs per 1 million isolated cells.

Bottom Line: The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up.If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Tübingen, Tübingen, Germany.

ABSTRACT

Background: Sentinel lymph node spread is a crucial factor in melanoma outcome. We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.

Methods and findings: We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology. For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100. None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells. We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations. At a median follow-up of 49 mo (range 3-123 mo), 138 patients (13.4%) had died from melanoma. Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61-2.01, for a 10-fold increase in DCCD + 1). Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02-2.58). Upon multivariable testing DCCD was a stronger predictor of death than histopathology. The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.

Conclusions: Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death. A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up. If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically.

Show MeSH
Related in: MedlinePlus