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N. Oishi et al.
(Biocare Medical), developed with 3,3’-diaminobenzidine, and counterstained with hematoxylin. Stains were scored in a blind fashion by two hematopathologists (NO and ALF). For CA9, scor- ing was based on percentage of tumor cells with membranous staining.
BIA-ALCL xenograft models
Studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) under protocol A00002776. Five-week-old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from The Jackson Laboratory and main- tained under standard laboratory conditions. Cell lines TLBR-1, TLBR-2, and TLBR-3 were established from BIA-ALCL by one of the authors (ALE).15,20 and were maintained in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (Clontech), 1% peni- cillin/streptomycin (Gibco), and 100 U/mL interleukin-2 (R&D 202-IL-050). TLBR-1, -2, and -3 cells were cultured at a concentra- tion of 0.5 x 106/mL for 72 h and resuspended in phosphate- buffered saline. Mice were injected subcutaneously in the right flank with TLBR-1, -2, or -3 cells. Tumor volumes were calculated in mm3 using the formula l2×L/2, where l and L represent the short- est and longest dimensions, respectively.
Additional methods are described in the Online Supplementary Material.
Results
Increased expression of hypoxia signaling pathway genes is a hallmark of BIA-ALCL
We performed RNA sequencing to identify gene expres- sion signatures that might distinguish BIA-ALCL from other types of ALCL. Since BIA-ALCL are consistently of TN genetic subtype,9 we compared BIA-ALCL to other TN ALCL to avoid bias from the distinct expression pro- files of other genetic subtypes14 (Figure 1A). A distinct clus- ter of genes was upregulated in BIA-ALCL, which formed the basis for subsequent analyses. Two clusters of genes were expressed only in non-BIA cases: one was enriched for keratin genes and consisted of biopsies at epithelial sites (skin and tongue) and the other contained Y-linked genes and represented male patients.
We then performed GSEA to identify candidate molec- ular signatures for genes upregulated in BIA-ALCL (Figure 1B, Online Supplementary Table S1). We focused on the sec- ond highest ranking gene set, HALLMARK HYPOXIA (normalized enrichment score [NES], 2.727; false discov- ery rate q-value [FDR], 0.000), as a candidate molecular feature distinctive of BIA-ALCL. The highest ranking gene set, HALLMARK EPITHELIAL MESENCHYMAL TRAN- SITION (NES, 2.963; FDR, 0.000) and other subsequent pathways mostly related to collagen formation and extra- cellular matrix organization, likely reflecting stromal com- ponents in the fibrous capsule surrounding the breast implant and seroma in BIA-ALCL samples.21 Supporting these GSEA results, examination of differential expression of genes and absolute RPKM values between BIA-ALCL and non-BIA-ALCL revealed significantly higher expres- sion levels of downstream target genes of the hypoxia sig- naling pathway such as VEGFA, VEGFB, SLC2A3 (encod- ing GLUT3), and CA9 (carbonic anhydrase-9; RPKM, mean ± standard deviation: 16.5 ± 20.2 vs. 0.4 ± 0.7; P<0.001, t-test) (Online Supplementary Figure S1). Among genes associated with hypoxia, CA9 showed the highest fold-change between BIA-ALCL and non-BIA-ALCL
(FC=5.296, FDR, 3.07x10-8) (Figure 1C). Collectively, these results suggest that increased expression of hypoxia sig- naling pathway genes is a transcriptional hallmark of BIA- ALCL. We did not identify a significant difference in CA9 expression between in situ and tumor-type BIA-ALCL as described by Laurent et al.17 (Table 1), or distinct gene sig- natures associated with clinical stage; associations between gene expression and clinicopathological features should be evaluated in future, larger studies.
CA9 protein is consistently expressed in BIA-ALCL but not in other ALCL
CA9 is a well-established biomarker of hypoxia and tumoral expression of CA9 is widely used in the histopathological diagnosis of hypoxia-related cancers.22 Therefore, having identified a hypoxia-associated signa- ture and high CA9 mRNA expression in BIA-ALCL, we performed immunohistochemistry to investigate CA9 protein expression in BIA-ALCL and non-BIA-ALCL. CA9 was expressed on the cell membrane of BIA-ALCL cells (% positive staining, mean ± standard deviation, 91 ± 15%) but not in admixed inflammatory cells, validating the RNA sequencing data at the protein level (Figure 2A). A relatively narrow range of protein scores was observed by immunohistochemistry, compared to a wide range of CA9 gene expression values. The correlation between the two was not statistically significant, likely due to gene expression values reflecting contributions from non-neo- plastic cells whereas immunohistochemistry was scored only in the tumor cells. Conversely, CA9 was mostly neg- ative in non-BIA-ALCL (ALK-positive, 2 ± 6%; ALK-nega- tive, 5 ± 11%; cutaneous, 3 ± 5%; P<0.0001, Dunn multi- ple comparison test) (Figure 2B). We also stratified CA9 protein expression by genetic subtype of ALCL (Figure 2C). BIA-ALCL (all TN) showed significantly more CA9 expression than ALK-positive ALCL, DUSP22-rearranged ALCL, and TN non-BIA-ALCL, suggesting that high CA9 expression in BIA-ALCL is attributable to BIA presenta- tion rather than TN genetic subtype. Taken together, these data indicate that CA9 is specifically expressed in BIA- ALCL at the mRNA and protein levels.
Hypoxia-induced CA9 expression drives growth of BIA-ALCL cells
We next examined CA9 expression in BIA-ALCL cell lines under normoxic and hypoxic conditions. Expression of HIF-1α was evaluated to confirm the response to hypoxia. Western blotting of TLBR-1, -2, and -3 cells cul- tured under normoxic or hypoxic conditions revealed dis- tinct patterns of CA9 expression in each cell line, provid- ing unique models for further study (Figure 3A). In TLBR- 1, CA9 was expressed under baseline normoxic condi- tions, suggesting constitutive expression of the hypoxic program. CA9 expression was further induced by hypox- ia. In TLBR-2, CA9 was absent under normoxic conditions but was induced under hypoxic conditions, consistent with a canonical hypoxia response. In contrast, CA9 expression in TLBR-3 was absent under normoxic condi- tions and only minimally induced by hypoxia.
To explore the functional significance of these distinct patterns of CA9 expression, we evaluated the effects of hypoxia with or without siRNA-mediated silencing of CA9 on BIA-ALCL cell growth (Figure 3B). In TLBR-1, which showed evidence of a constitutive hypoxia pro- gram under normoxic conditions, hypoxia induced only a
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haematologica | 2021; 106(6)