CD1d- and PJA2-related immune microenvironment differs between invasive breast carcinomas with and without a micropapillary feature

Invasive micropapillary carcinoma (IMPC) of the breast is characterized by its unique morphology and frequent nodal metastasis. However, the mechanism for development of this unique subtype has not been clearly elucidated. The aim of this study was to obtain a better understanding of IMPC. Using representative cases of mixed IMPC, mRNA expression in the micropapillary area and usual invasive area was compared. Then, immunohistochemical analyses for 294 cases (76 invasive carcinomas with a micropapillary feature [ICMF] and 218 invasive carcinomas without a micropapillary feature [ICNMF]) were conducted. Clinicopathological analyses were also studied. DNA microarray analyses for mixed IMPC showed that BC-1514 (C21orf118) was commonly upregulated in the micropapillary area. CAMK2N1, CD1d, PJA2, RPL5, SAMD13, TCF4, and TXNIP were commonly downregulated in the micropapillary area. Immunohistochemically, we confirmed that BC-1514 was more upregulated in ICMF than in ICNMF. CD1d and PJA2 were more downregulated in ICMF than ICNMF. All patients with cases of PJA2 overexpression survived without cancer recurrence during the follow-up period, although the differences for disease-free (p = 0.153) or overall survival (p = 0.272) were not significant. The CD1d- and PJA2-related tumour microenvironment might be crucial for IMPC. Further study of the immune microenvironment and micropapillary features is warranted.


DNA microarray
After approval by the Institutional Review Board of Kawasaki Medical School ethics committee (approval number 909 and 2136), two representative cases of mixed IMPC were extracted from the database of the Department of Pathology, Kawasaki Medical School. Case 1 was ER-, PgR-, and HER2 3+, with histologic grade 2 and pT2N3aM0 ( Fig. 1a and c). Case 2 was ER+, PgR-, and HER2-, with histologic grade 2 and pT1cN0M0 ( Fig. 1b  and d). The micropapillary structure was confirmed with EMA and MUC1. Paraffin sections were cut from the blocks and deparaffinized. They were stained with toluidine blue. Under microscopy, the tissue was separated to the IMPC area and the ICNST area of each case by scalpal blade. RNA was extracted and hybridization on a 3D-Gene Human Oligochip (Toray, Tokyo, Japan). The microarray data from the IMPC and ICNST areas were compared.

Antibodies
Polyclonal antibodies were generated for SAMD13, TCF4 and TXNIP using synthetic peptides of their specific amino

Tissue microarray
The first tissue microarray was constructed with 231 consecutive surgical cases of invasive breast cancers from Kawasaki Medical School Hospital from September 2009 to December 2010. The first microarray contained 13 cases of invasive carcinoma with a micropapillary feature (ICMF: pure or mixed IMPC and ICNST with a focal micropapillary feature). The second tissue microarray was constructed with 63 cases of ICMF from January 2011 to December 2014. A KIN-2 system (Azumaya, Tokyo, Japan) with a 2-mm needle was used for the tissue microarray. For cases of ICMF, only the micropapillary area was sampled. ER and PgR were judged using 1% cutoff [14]. For HER2, HercepTest 3+ or HercepTest 2+ and FISH positive were regarded as positive [15].
Immunohistochemistry was analysed using a histoscore that was calculated by multiplying the positive area (%) and intensity (0-3: 0 for negative, 1 for weak, 2 for moderate, and 3 for strong staining). Immunohistochemical analyses were evaluated in blinded manner.

Statistical analyses
Statistical analyses were performed using IBM SPSS Statistics for Windows (version 25; IBM Corp., Armonk, NY). p < 0.05 was considered significant.

DNA microarray
The commonly up-and downregulated factors in IMPC compared with ICNST by DNA microarray are shown in Table 1 and Table 2. BC-1514 (C21orf118) is the only gene that showed over threefold increasing expression in the IMPC area compared with the ICNST area. SAMD13, CAMK2N1, TCF4, TXNIP, RPL5, PJA2, and CD1d showed over threefold decreasing expression in the IMPC area compared with the ICNST area.
The expression of CD1d by ICNMF is similar to that of normal breast tissue (p = 0.373). However, expression of CD1d by ICMF is lower than that of normal breast tissue (p = 0.008). The expression of PJA2 by ICNMF is higher than that of normal breast tissue (p < 0.001). The expression of PJA2 by ICMF is similar to that of normal breast tissue (p = 0.259).

Survival analyses
The median follow-up periods for disease-free survival was 87.8 months (range, 0.4 to 113.7 months) and for overall survival was 88.2 months (range, 0.4 to 119.27 months). The univariate Cox hazard survival analyses are shown in Table 5. pT, pN, and pathological stage were significant for Table 1 DNA microarray data of mixed IMPC. Factors more commonly upregulated in the IMPC than ICNST areas. The ratio of the expression in IMPC area to the expression in ICNST area was shown as heat map. Red column represents over 3.0, pink represents 2.5 to 3.0, yellow represents 2.1 to 2.4 disease free survival (p = 0.001, < 0.001 and 0.011). pT, pN, stage, histological grade, and PgR were significant for overall survival (p < 0.001, 0.004, < 0.001, 0.001, and 0.005, respectively). BC-1514 (C15, W12), CD1d, and PJA2 did not show any significant difference for disease-free survival or overall survival. All patients with cases having high PJA2 survived without cancer recurrence (19 of 19) using the third quadrant as a cut-off value, but there was no significant difference (p = 0.153 for disease-free survival [ Fig. 3] and p = 0.272 for overall survival).

Discussion
CD1d-presents lipid antigens to activate natural killer T (NKT) cells, through the interaction with the T-cell receptor present on NKT membranes [16]. NKT cells display antitumour immune responses when activated by the synthetic glycosphingolipid, α-galactosylceramide (αGalCer) [16]. CD1d expression was reported in the intestines, liver, pancreas, kidney, uterus, skin, conjunctiva, thymus, tonsil, and breast [17]. We demonstrated that CD1d expression is lower in ICMF than in ICNMF  [19], although we could not show a significant correlation between prognosis and CD1d expression. To improve the immune microenvironment, strategies such as αGalCer [20] administration may be a therapeutic option for breast cancer.
PJA2, also known as PRAJA2, regulates the protein kinase A signal strength and duration in response to cAMP [21]. PJA2 increases the accumulation of ubiquitylated malignant fibrous histiocytoma amplified sequence 1, which promotes M1 macrophage polarization and M2 to M1 macrophage transformation [22]. M1 macrophage promotes antitumour immunity, while M2 macrophage promotes tumour progression [23]. We showed lower expression of PJA2 in ICMF than ICNMF by both DNA microarray and immunohistochemistry. In IMPC, M2 macrophage polarization by a shortage of PJA2 might facilitate tumour progression.
CD1d, PJA2, and granzyme A precursor also showed lower expression in IMPC than in ICNST in our DNA microarray screening. Granzyme A, an enzyme present in cytotoxic T lymphocytes, has tumouricidal activity [24,25]. The potential of IMPC to evade the immune system may be supported by multiple mechanisms.   We demonstrated BC-1514 mRNA upregulation in IMPC and greater immunohistochemical expression in ICMF than ICNMF. However, the record of BC-1514 for Homo sapiens has been withdrawn by the National Center for Biotechnology Information because of insufficient evidence (https://www.ncbi.nlm.nih.gov/gene/378829, last accessed on June 6, 2018). Our immunohistochemistry for BC-1514 probably showed some cross reaction to an unknown substance.
One of the limitations of this study is that our DNA microarray data was obtained from only two mixed IMPC cases. We thought that the candidate markers for micropapillary feature were well narrowed down by just two cases. However, four (CAMK2N1, RPL5, SAMD13, and TCF4) of eight markers showed contradictory results, and TXNIP did not show a significant difference by immunohistochemistry. Additional DNA microarray would be promising to increase the accuracy, and should have improved the efficiency of our immunohistochemical study. Our DNA microarray data should be interpreted with caution.