Surface antigen profiles of leukocytes and melanoma cells in lymph node metastases are associated with survival in AJCC stage III melanoma patients

Melanoma lymph node metastases

Melanoma LN metastases (n = 38) were obtained from the Melanoma Institute Australia (MIA) Bio-specimen Bank with written informed patient consent and IRB approval (Sydney South West Area Health Service institutional ethics review committee (RPAH Zone) Protocol Nos., X06-0140, X08-0155/HREC 08/RPAH/262, X11-0023/HREC 11/RPAH/32 and X07-0202/HREC/07/RPAH/30). Fresh tumor samples were examined by a specialist melanoma pathologist (RAS) and carefully macro-dissected from non-tumor tissues avoiding necrotic areas and normal lymph node tissue. A high-percentage melanoma tumor content (>80 %) and low necrosis (<20 %) were verified by examining hematoxylin and eosin-stained sections and immunohistochemistry for melanoma antigens S-100 protein, HMB45 and MelanA/MART-1.

The fresh metastatic melanoma LN tumors were collected prospectively over a 13-month period from February 2009 and subsequent clinical follow-up data spaning 24.3–43.1 months (median 35.4 months) post LN resection was obtained. Nine patients were excluded from the study due to a high level of necrosis and poor cell viability (n = 3), a non-melanoma diagnosis following subsequent histopathology (n = 3) or evidence of distant metastases at diagnosis of the culprit primary melanoma (n = 3; stage IV at initial diagnosis). The remaining 29 cases were analyzed for demographic, primary tumor and metastasis pathologic and survival data as detailed in Table 1. As of May 2013, 13 patients were alive [11 with no sign of recurrence (NSR) and 2 with melanoma], 15 patients had died of melanoma and 1 patient had died of unrelated causes. One patient had synchronous AJCC stage IV disease at the time of LN recurrence and was excluded from the survival analyses. Patients with occult primary disease or with previous LN involvement were excluded from the DFI correlation analysis. Despite good cell viability, two patient samples had poor binding of cells to the microarray and were used for Western blot analysis.

In summary, we investigated the associations of CD45⁻ enriched melanoma cell (n = 25) and CD45⁺ leukocyte (n = 23) immuno-profiles in melanoma patients with distant metastasis-free survival (DMFS) and OS post LN resection of stage IIIb/c disease. Immuno-profiles of CD45⁻ enriched melanoma cells (n = 20) and CD45⁺ leukocytes (n = 19) were also correlated with DFI (time between diagnosis of culprit primary melanoma and LN resection; see Supplementary Table 1).

Preparation of viable, single cell suspensions

Fresh tumor samples were placed in Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, St. Louis. MO, USA) at 4 °C and processed within 16 h to ensure cell viability. Samples were finely diced with a scalpel blade and incubated in a dissociation buffer containing 1 % (w/v) collagenase type IV (Worthington, Lakewood, NJ, USA) and 0.2 % (w/v) DNase I (bovine pancreas; Sigma-Aldrich) in HBSS at 37 °C for 1 h, with gentle vortexing every 15 min. The semi-digested tissue was then passed through a fine wire mesh strainer and the resulting suspension centrifuged (450 × g, 4 °C, 5 min). Small aggregates were removed using 200 μm and then 50 μm Filcon cup filters (BD Biosciences, San Jose, CA, USA), resulting in a single-cell suspension with 70–90 % viability, that was frozen at −80 °C in fetal calf serum (FCS) with 10 % DMSO until required.

Separation of leukocytes from melanoma cells

Prior to surface profiling, samples were thawed and pelleted (400×g, 4 °C, 5 min) and the cells resuspended in RPMI growth medium with 0.2 % (w/v) DNase I for 10 min at room temperature. Leukocytes were bound to CD45 antibody microbeads (Miltenyi Biotec, Auburn, CA, USA) and separated from cell suspensions using an AutoMACS™ Pro Separator (Miltenyi Biotec) as per the manufacturer’s instructions. Briefly, 2 × 10⁷ cells were incubated with 40 μL anti-CD45 microbeads in a buffer containing PBS with 0.1 % BSA, 2 mM EDTA and 2 % AB serum at 4 °C for 10 min. Cells were diluted, pelleted (300×g, 4 °C, 10 min), resuspended in 500 μL of the above buffer and passed through a 50 μm filter before removal of magnetic beads with attached CD45⁺ cells using the ‘deplete’ setting on the AutoMACS™ Pro Separator. To confirm depletion of the CD45⁺ cells, a melanoma LN cell suspension was labelled with mouse monoclonal anti-CD45-PE or anti-IgG2a-PE (Miltenyi Biotec) and analyzed on a FACScalibur™ (BD Biosciences) before and after immuno-magnetic separation (Supplementary Fig 1).

Capture of cells on DotScan™ microarrays

The microarrays were constructed as duplicate dots (10 nl) on nitrocellulose-coated slides (Grace Bio-labs Inc., Bend, OR, USA) as previously described [25]. All array antibodies were specific to extracellular domain sequences where possible, if not the full-length protein. However, antibody avidity might be reduced following its immobilization to the nitrocellulose slide. CD45⁺ and CD45⁻ live cell suspensions were suspended at a density of 1.3 × 10⁷/ml in growth medium (RPMI with 10 % FCS and 2 % heat-inactivated human AB serum) and incubated on pre-moistened microarrays in a humidified chamber at 37 °C for 30 min. Captured cells were fixed in 3.7 % formaldehyde for 30 min at room temperature and washed in PBS. Arrays were imaged directly with an optical scanner (Medsaic Pty Ltd) without staining or labelling, and analyzed using DotScan™ software (Medsaic Pty Ltd) [23]. This software quantifies the density of cell binding on each antibody dot above background levels, on an 8-bit scale from 1–256. Dot intensities on an array reflect the proportion of cells expressing each antigen and/or the level of expression of a particular antigen per cell. The average numbers of cells bound to each dot, determined microscopically, correlate well with average binding density values. The number of cells captured on an antibody dot in the microarray also depends on the affinity of the antibody-antigen interaction.

Statistical analysis of antibody microarray results

Cell binding densities were corrected for background and isotype-control binding and the duplicate array information was averaged and log₂-transformed. Microarray data, consisting of 52 antigens for melanoma (CD45⁻) samples and 78 antigens for leukocytes (CD45⁺), were median normalized separately. DMFS was calculated from the time of LN resection to appearance of distant metastasis or last clinical follow-up. OS was calculated from the time of LN resection, to last clinical follow-up or death. Univariate (log-rank test) and multivariate (Wald’s test) survival models were used to assess associations between antigen levels and patient survival (DMFS and OS). Due to the small patient numbers (enriched melanoma cells, n = 25 and leukocytes, n = 23), we were unable to include all clinical variables in a single survival model. Instead, we restricted our attention to the patient’s age, gender and AJCC stage at LN resection (stage IIIb or IIIc) because these are the strongest known predictors of outcome in AJCC stage III melanoma patients. For patients with multiple primary melanomas (n = 3), the culprit primary was designated on the basis of the presence of the most adverse prognostic factors and its anatomic site, due to a greater likelihood that it was the source of the metastatic disease, as previously described [30, 31]. We censored patients who died of other causes, and for the DMFS analysis, patients who had no distant metastasis. For the DFI correlation analysis, Pearson’s r² values were calculated to determine the relationship between surface antigen levels and the time (in months) between the culprit primary melanoma diagnosis and LN resection of stage III metastatic disease.

Western blotting

Total protein extracts were made from CD45⁺ cell populations from 13 LN samples (see Supplementary Table 1) by lysing cells in a denaturing urea buffer containing 7 M urea, 2 M thiourea, 40 mM Tris, 65 mM DTT supplemented with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged (16,000×g, 15 min, 4 °C). Due to limited sample availability, the level of one surface antigen (CD55) was confirmed by Western blotting. Proteins were separated by 10 % SDS-PAGE and transferred to a PVDF membrane at 400 mA for 1 h using a Criterion™ Blotter (BioRad, Hercules, CA, USA). The membrane was blocked in 5 % (w/v) skim milk and incubated overnight at 4 °C with a 1:5,000 dilution of rabbit monoclonal antibody against CD55 (EPR6689, Epitomics, Inc., Burlingame, CA, USA) and 1:10,000 dilution of mouse monoclonal antibody against succinate dehydrogenase subunit A (SDHA; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Membranes were then incubated with donkey, anti-rabbit (Abcam, Waterloo, NSW, Australia) and goat, anti-mouse (Santa Cruz) secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature. Bands were visualized with ECL detection reagent and imaged using chemiluminescence film (GE Healthcare, Piscataway, NJ, USA). Bands were quantified using ImageQuantTL density analysis software (GE Healthcare). An average of the density of SDHA and the total protein stain was used as an internal loading control. To confirm the association with DMFS, CD55 level ratios were tested using the log-rank test described above.

Surface profiles of melanoma LN metastases

Single cell suspensions of 29 metastatic melanoma LN specimens were immuno-magnetically separated to isolate CD45⁺ leukocytes and enrich for melanoma cells (CD45⁻). Cell separation was confirmed by flow cytometry (Supplementary Fig. 1). This together with a failure of the CD45⁻ cells to bind CD45, CD45RA and CD45RO or other leukocyte specific antigens on the arrays confirmed the effectiveness of the cell separation. Adequate cell numbers (4 × 10⁶ cells), viability (>70 %) and binding were achieved for CD45⁻ and CD45⁺ cell populations from 26 to 24 patient samples, respectively (See Fig. 1 for optical scan of cell binding patterns). Cell binding to 52 and 78 antibodies was observed for CD45⁻ enriched melanoma cells and CD45⁺ leukocytes, respectively. Normalization and subsequent analyses were performed using this these data. Some antigens were expressed at variable levels on all CD45⁻ fractions, e.g., CD29, CD44, CD51, CD63, CD71, CD146 and CD151. Although the leukocyte (CD45⁺) antigen profiles were more diverse, perhaps reflecting the heterogeneity of the cell populations, as expected, there was consistent binding to the antibodies for CD45, and CD45RA and/or CD45RO for all patient samples.

DMFS and OS analyses

In leukocyte (CD45⁺) fractions, high levels of CD9 were associated with decreased DMFS on both univariate and multivariate analyses (HR = 2.67, p = 0.039 and HR = 3.73, p = 0.036, respectively; Fig. 2). In contrast, increased levels of CD39 and CD55 were associated with increased DMFS on univariate (HR = 0.29 and 0.28, p = 0.019 and 0.006, respectively) and multivariate analyses (HR = 0.01 and 0.10, p = 0.004 and 0.005, respectively). A significant association between increased levels of CD55 and DMFS was confirmed by Western blotting (HR = 2.8; p = 0.008; see Fig. 4). The CD45⁻ enriched melanoma cells displayed differential binding to CD117 (c-KIT), with higher levels associated with decreased DMFS on univariate analysis with borderline significance (HR = 1.56; p = 0.07).

Higher levels of CD39 on CD45⁺ leukocytes were also significantly associated with increased OS using both univariate and multivariate models (HR = 0.29, p = 0.045 and HR = 0.10, p = 0.016, respectively; Fig. 3). There is a documented synergy between CD39 and CD73 activities [23]. We found increased CD73 levels associated with DMFS and OS, with marginal significance (p = 0.08 and 0.13, respectively). When CD73 and CD39 levels were modelled together, CD39 remained significantly associated with DMFS (p = 0.027). Increased levels of CD11b, CD49d and CD79b on CD45⁺ leukocytes were associated with reduced OS (HR = 2.72, 2.46, 1.76, p = 0.025, 0.043, 0.044, respectively). However these associations were not statistically significant on multivariate analysis. Leukocyte results are summarised in Table 2 and survival plots for DMFS and OS are depicted in Figs. 2 and 3. No statistically significant association with OS was found for the 58 antigens tested on CD45⁻ enriched melanoma cells.

DFI correlation analyses

A proportion of patients with primary melanoma carry a risk for recurrence and distant metastases after a symptom-free period that can span decades, despite favourable prognostic determinants from their primary tumor. The relationship between this DFI, defined as the time between primary melanoma resection and resection of nodal metastases (spanning 3.6–247.2 months in this study) and antigen levels was also investigated. Enriched melanoma (CD45⁻) cells showed higher levels of 11 surface antigens in resected LNs from metastatic melanoma patients with a prolonged DFI (p < 0.05; average r² of 0.484; see Supplementary Fig. 2a and Table 3). Five of these antigens are cell adhesion molecules, i.e., integrins (CD29, CD49c), tetraspanin CD63, CD56 (neural adhesion molecule, NCAM) and CD107a (lysosome-associated membrane protein 1). For CD45⁺ leukocyte fractions, nine antigens were correlated with shorter DFI (p < 0.05; average r² of −0.590; Supplementary Fig. 2b and Table 3). Of these, eight antigens are related to T cell immunity, i.e., CD7, CD8, CD43, CD54, CD56, CD103, CD134 and CD166.

Survival analyses

Surface antigens associated with a prolonged disease free interval

The time interval between removal of the primary melanoma and detection of metastatic disease is very variable and can span many years, or even decades. The mechanisms by which metastatic disease may be present but remain clinically undetected for such prolonged periods, presumably influenced by interactions between the melanoma phenotype and the patient’s immune system, have not received sufficient attention to date [59]. A better understanding of the molecular cross-talk between tumor cells, immune cells and the extracellular matrix in secondary sites, and how these regulate metastatic disease progression, may lead to improved therapeutic strategies to eradicate them or at least to induce or maintain disseminated tumor cells in a clinically-silent chronic disease (“dormant”) state [60]. Adjuvant therapies, such as antibodies that target metastatic melanoma cells are of considerable interest [61, 62]. We performed correlation analyses of antigen levels and the DFI (time from primary diagnosis and detection of LN metastasis) to identify surface antigen signatures associated with melanoma progression following prolonged disease-free (latent) intervals.

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