Plasma Cells in Primary Melanoma. Prognostic Significance and Possible Role of IgA

Francesca M Bosisio; James S Wilmott; Nathalie Volders; Marjorie Mercier; Jasper Wouters; Marguerite Stas; Willeke AM Blokx; Daniela Massi; John F Thompson; Richard A Scolyer; Nicolas van Baren; Joost J van den Oord

Disclosures

Mod Pathol. 2016;29(4):347-358. 

In This Article

Results

Clinico-pathological Features of PC+ Melanomas

Of the 710 primary cutaneous melanomas from the University Hospital Leuven, 41 (3.7%) showed a PC component in the inflammatory infiltrate associated with the melanoma. According to the PC score, 7 (18%) showed scattered PCs (score PC1), 24 (58%) had discrete clusters of PCs (score PC2) and 10 (24%) contained sheets of PCs (score PC3). As PC1 melanomas represent a condition in which the quantity of PC is low and could be not biologically relevant, we focused on PC2 and PC3 cases to better characterize melanomas in which the amount of PC is important and labeled these 34 cases (82%) as 'PC-rich' melanomas in statistical analysis. From score PC1 to score PC3, an increase in the number of intra-tumoral PCs that were in direct contact with the melanoma cells was observed. In fact, while in 6 out of the 7 PC1 melanomas the PCs were only in peritumoral location, PC2 and PC3 melanomas showed the PCs to be located within the tumor mass (15/24 (62.5%) and 8/10 (80%) cases with intratumoral PCs, respectively, P=0.006). Clinical and histological features of PC-rich melanomas are shown in Supplementary Table S1 http://www.nature.com/modpathol/journal/v29/n4/suppinfo/modpathol201628s1.html?url=/modpathol/journal/v29/n4/full/modpathol201628a.html, and the statistical analysis is resumed in Figure 2. The average age at the diagnosis for PC-rich melanomas was 63 years (range 20–86, median 63) and 55 years for melanomas without PC (range 11–97, median 55), suggesting that PC-rich melanomas occur in patients 10 years older than PCneg melanomas (P=0.005). In all, 19/34 (56%) patients with PC-rich melanomas were males, and 15/34 (44%) were females. PC-rich melanomas affected, in decreasing order, the trunk, limbs, head and neck areas and acral sites, while PCneg melanomas were mostly on the limbs and less frequently on the trunk, head and neck area and acral sites. PC-rich melanomas were more often of the nodular subtype than PCneg melanomas (P=0.00046). Three strongly evidence-based markers of bad prognosis in melanoma (high Breslow thickness, >6 mitoses/mm2 and the presence of ulceration) were found to be associated with the presence of PCs in melanoma (P<0.0001). The incidence of PC+ melanomas increased to 19.5% if only cases with a Breslow thickness >2 mm were taken into account. A correlation with the presence of lymphatic/vascular invasion was also found (P=0.015). Histological examination of the 15 cases from Nijmegen, The Netherlands and 6 cases from Firenze, Italy revealed similar unfavorable prognostic features in melanomas with sheets of PC.

Figure 2.

Statistical analysis of the clinical (ad) and histopathological (ej) features of PC-rich, PC1 and PC− melanomas. Parameters that reach significance are marked by asterisks. ALM, acral lentiginous melanoma; H&N, head and neck; LVI, lymph/vascular invasion; NM, nodular melanoma; SSM, superficial spreading melanoma.

No significantly different clinical features were found between score PC1, PC2 and PC3. The three PC scores differed significantly only for two histopathological parameters, ie, the PC distribution in the tumor microenvironment and the thickness of the melanoma according to Breslow. Regarding the distribution of PCs, the number of intratumoral PCs increased from score PC1 to score PC3, indicating that, with increasing numbers of PCs in the inflammatory infiltrate, these PC tended to be more in direct contact with melanoma cells (P=0.006). Moreover, PC3 melanomas were significantly thicker than PC2 and PC1 melanomas (P=0.008). All other histopathological parameters (subtype, mitotic rate, ulceration, regression, lymphatic/vascular invasion, microsatellites) were not significantly correlated with the number of PCs in the infiltrate.

Immunohistochemical Features of PC+ Melanomas

Sixteen cases of PC+ primary melanomas were available for immunohistochemical analysis. Immunohistochemistry for Ig light chains showed the PCs to be polyclonal (data not shown) and staining for different isotypes of immunoglobulins (IgA, IgG, IgM, IgD, IgE) revealed that the major part (usually >50%) of the PCs in the melanoma-associated inflammatory infiltrate was positive for IgG (data not shown). Interestingly, an equally important PC component of the infiltrate expressed IgA. Using double chromogenic immunohistochemistry for the membranous PC marker CD138 (brown) and cytoplasmic IgA (red), the quantity of IgA+ PCs as a fraction on the total of the CD138+ PCs was evaluated in PC+ melanomas. In 3/16 (18%) of the cases, >50% of PCs expressed IgA, in 7/16 (44%), 10–50% of PCs expressed IgA and 6/16 (38%) cases were devoid of a significant IgA+ PC component. In total, 56% of cases showed >10% of all PCs to express IgA, and all these cases had clusters (PC2 score) or sheets (PC3 score) of PCs. The distribution of the IgA+CD138+ score from PC1 to PC3 melanomas showed that IgA+ PCs increased with increasing numbers of PCs in the melanoma (Table 1). In order to rule out that ulceration by itself was the trigger for IgA switch, 20 randomly chosen non-neoplastic ulcers and two (positive control) genital and mucosal ulcers were analyzed histologically and immunohistochemically. Nine had PCs in the infiltrate, and these ulcers occurred more frequently in the head and neck areas (Supplementary Table S3 http://www.nature.com/modpathol/journal/v29/n4/suppinfo/modpathol201628s1.html?url=/modpathol/journal/v29/n4/full/modpathol201628a.html). On double chromogenic immunohistochemistry, the number of IgA+ PCs was generally low in cutaneous non-neoplastic ulcers as compared with those at mucosal sites where equal numbers of IgA+ and IgG+ PCs were found.

As they represent a site of PC formation, the occurrence of tertiary lymphoid structures (TLS) in the inflammatory stroma was investigated by immunostainings in consecutive serial sections of 16 cases for CD20 (B cells), CD3 (T cells), CD21 (follicular dendritic cells (FDC)) and PNAd, a high endothelial venule (HEV) marker. The presence of B and T cells, as well as FDC and HEV together in a lymphoid aggregate, was considered to be sufficient to define a TLS.[31] According to this criterium, only 2 out of the 16 cases of PC+ melanoma presented with TLS (Table 1).

Molecular Analysis of Ig Genes Expressed by PC+ Melanomas

We first analyzed the isotype of the immunoglobulin-expressing cells present in two PC-rich primary melanomas, for which frozen material was available. We used RT-PCR to detect the IGHD, IGHM, IGHG, IGHA and IGHE transcripts, which encode the constant regions of Ig heavy chains (Figure 3a). The secreted isoforms of IgG and IgA were more expressed than both the corresponding membranous isoforms and other isotypes, thus confirming that they are the main isotypes expressed by PCs in melanoma.

Figure 3.

Molecular analysis of Ig genes expressed in PC-rich melanomas. (a) Identification by RT-PCR of the membrane-bound (m) and secreted (s) isoforms of the five Ig isotype families expressed in two primary melanomas with PC infiltration. (b) Immunoscope analysis of IgA-s and IgG-s in one of the two primary melanomas. (c and d) Immunoscope analysis of IgA-s in nine LN draining PC-rich melanomas. FFPE, formalin fixed paraffin embedded; LN, lymph node; N−, uninvolved lymph nodes; N+, lymph nodes involved by the tumor.

To further characterize the clonality of the infiltrating PCs, we analyzed the diversity of the Ig gene repertoire with the immunoscope approach. To this aim, we used two rounds of nested RT-PCR, including fluorochrome-coupled primers for the last PCR round, to amplify all the VDJ sequences present in IgG-s and IgA-s transcripts. The amplified fluorescent fragments were then separated according to their length by high-resolution electrophoresis (Figure 3b). In both tumors, the IgG were found to have a polyclonal pattern, whereas the IgA repertoire was more restricted, indicating oligoclonality.

Analysis of Lymph Nodes Draining PC+ Melanomas

In the loco-regional lymph nodes, the same score used for primary melanomas was applied to quantify the amount of PC infiltrates. In lymph nodes with score 1, scattered PCs were predominantly localized in the medullary sinuses. In lymph nodes with score 2 and 3, PCneg clusters and sheets were found in the medullary sinuses as well as in the sub-capsular region. In 12/14 lymph nodes draining the site of a PC+ melanoma, the PC score in the lymph node was the same or higher than the score in the corresponding primary melanoma. Therefore, a good correlation existed between the PC score in draining lymph nodes and primary melanoma (Figure 4). Using double IgA/CD138 immunohistochemistry, the percentage of IgA+PC varied between 10 and 50% of the total number of nodal PCs in 11 out of the 14 cases; moreover, a high percentage of IgA PCs was also found in cases with scattered PCs (PC1 score).

Figure 4.

Correlation between the plasma cells' score in primary melanomas and in lymph nodes. After application of the same scoring system used in primary melanomas, a good correlation was found between the amount of plasma cells in the tumoral site and in the draining lymph nodes (pictures on the right, from top to bottom, sparse plasma cells, clusters and sheets in the lymph nodes).

Gene Scan analysis, focused on the secretory part of IgA, was performed on frozen material from nine lymph nodes previously studied by immunohistochemistry. Seven of these cases had 10–50% IgA+ PCs in the lymph node, whereas the other two had no relevant IgA component in the lymph node. In seven out of the nine cases, Gene Scan Analysis showed oligoclonality of the secretory IgA component, suggesting an antigen-driven IgA-immune response (Figures 3c and d). In two out of the nine cases, a lymph node with sheets of PCs (PC3) showed predominance of IgG PCs and a score 1 case with prominent IgA+ PCs had a polyclonal IgA secretory component. These two cases proved that, even in the presence of a minority of IgA-positive PCs in the lymph node, polyclonality could be detected. Moreover, in the latter case (score 1 with prominent IgA+ PC), a restriction of IgM and IgG was detected, suggesting an antigen-driven immune response involving IgG and IgM, but not IgA.

Outcome of Patients With PC+ Melanomas

As our data showed that PCs are found in a low percentage of primary melanomas and that they are associated with unfavorable prognostic factors, we analyzed their outcome in a series of 85 thick melanomas from Australia. In this series, PCs were found in 33 cases (38%), an important increase in comparison to the incidence of 19.5% of PC+ melanomas that were found among the thick melanomas subgroup from the KUL data set. This could be due to differences between melanomas arising in the Belgian population compared with the Australian one. PC1 melanomas were 12 (36%), PC2 11 (33%) and PC3 10 (31%). In this series of thick melanomas, the presence of PCs correlated again with Breslow thickness (P=0.0002), subtype of melanoma (P=0.0037) and ulceration (P=0.0002). On the other hand, mitotic count and lymphatic/vascular invasion, associated with the presence of PCs in the original Leuven data set, lost significance in the series from Australia.

Immunohistochemical staining revealed that 21 out of the 33 cases (70%) presented a relevant IgA+ PC component. The distribution of the IgA score was homogeneous among the PC scores: also cases with sparse PCs showed predominant expression of IgA. In particular, 9 out of the 12 PC1 melanomas presented between 10 and 50% of IgA+ PCs in the infiltrate; 6 out of the 11 PC2 melanomas had IgA+ PCs (which in 2 cases comprised >50% of the PCs) and 9 out of the 10 PC3 melanomas had in almost all cases >50% of IgA+ PCs.

Of the 52 cases without PCs in the inflammatory infiltrate, 26 patients were alive without disease, 3 alive with melanomas, 11 dead owing to melanoma and 12 dead owing to a cause unrelated to melanoma or by unknown cause. Of the 21 PC-rich melanomas (PC2 and PC3), 10 patients were alive without disease, 9 dead owing to melanoma, 1 dead owing to other causes and none alive with melanoma. Of the 12 PC1 melanoma patients, 8 were alive without disease, 2 were alive with melanomas, 1 dead owing to melanoma and 1 dead owing to unknown cause. The Kaplan–Meier curves of survival were not significant for the absolute presence or absence of PCs in the inflammatory infiltrate but were significant for the difference in survival of patients with PC-rich melanomas vs those with melanomas lacking PCs and PC1 melanomas (Figure 5). Hence, patients with PC-rich melanomas showed a worse survival compared with those with PCneg melanomas (P=0.002). The survival of patients with PC1 melanomas appeared to be better than that of patients with PCneg melanomas, but this finding did not reach statistical significance. The same difference in survival was found when comparing the survival patients with ulcerated melanomas at all sites with that of ulcerated melanomas on the limbs, thereby eliminating the bias of ulceration and site of occurrence (Supplementary Figure S1 http://www.nature.com/modpathol/journal/v29/n4/suppinfo/modpathol201628s1.html?url=/modpathol/journal/v29/n4/full/modpathol201628a.html).

Figure 5.

Survival analysis of melanomas with plasma cells. The overall survival does not significantly differ between PC+ and PC− melanomas (a), but among the different PC scores, the score 3 and score 2 melanomas ('PC-rich') show a worse prognosis than score 0 ('PCneg') and score 1 melanomas (P=0.002) (b), therefore these two last groups could be prognostically grouped toghether under the definition of 'PClow' melanomas (c).

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