Introduction

Placental alkaline phosphatase (PLAP), also known as alkaline phosphatase, placental type (ALPP), is encoded by the ALPP gene at chromosome 2q37.1 [1]. PLAP is a dimer of 65 kDa consisting of 535 amino acids and is thought to play a role in guiding migratory cells and transport specific molecules over the plasma membrane [1, 2]. PLAP is expressed in the placenta from the ninth week of gestation and its concentration increases continually throughout pregnancy [2]. PLAP can be separated into three distinct isoenzymes corresponding to early, mid, and term placenta [3]. In normal human tissues, the expression of PLAP is largely restricted to the placenta but low-level RNA expression has also been reported for uterine cervix, fallopian tube, and – to a lower level – the lung [4, 5].

PLAP expression also occurs in tumors [4, 6-60]. This is particularly known for testicular germ cell tumors [6-8, 10-36]. PLAP expression, often at high levels, has been described to occur in up to 100% of testicular germ cell neoplasia in situ [20, 28, 30], up to 100% of seminoma [4, 6, 7, 9, 20-23, 30, 35, 36, 38, 41, 42], up to 100% of embryonal carcinoma [35, 42], up to 87% of yolk sac tumors [30], and up to 100% of choriocarcinomas [12, 16, 19]. Antibodies targeting PLAP are thus regularly used for the detection and classification of testicular tumors [61]. Various studies have demonstrated, however, that PLAP expression can also occur in non-germinal cell tumors, but immunohistochemical data are controversial. For example, positive PLAP immunostaining has been described to occur in 0–100% of adenocarcinomas of the ampulla of Vater [4, 35], 0–38% of gastric adenocarcinoma [4, 35], 0–100% of rhabdomyosarcomas [44, 56], 20–80% of high-grade serous carcinomas of the ovary [43, 59], and 0–18% of clear cell renal cell carcinomas [4, 35]. These conflicting data are probably caused by the use of different antibodies, immunostaining protocols, and criteria to determine PLAP positivity in these studies.

To better understand the prevalence of PLAP immunostaining in different tumor types, a comprehensive study analyzing a large number of neoplastic and non-neoplastic tissues under highly standardized conditions is needed. We thus analyzed PLAP expression in more than 16,000 tumor tissue samples from 131 different tumor types and subtypes as well as 76 non-neoplastic tissue types by immunohistochemistry (IHC) in a tissue microarray (TMA) format.

Materials and methods Tissue microarrays

TMAs composed of normal and tumor tissues were employed for this study. The normal TMA contained eight samples from eight different donors from each of 76 different normal tissue types. The cancer TMAs contained a total of 16,166 primary tumors from 131 tumor types and subtypes. Histopathological data including grade, pathological tumor (pT) stage, and pathological lymph node (pN) status were available from 583 ovarian cancers, 259 endometrial cancers, and 1,784 colorectal cancers. The dataset on colorectal cancer also included molecular information on mismatch repair protein deficiency. The composition of both normal and cancer TMAs is described in the Results section. All samples were from the archives of the Institutes of Pathology, University Hospital of Hamburg, Germany; the Institute of Pathology, Clinical Center Osnabrueck, Germany; and Department of Pathology, Academic Hospital Fuerth, Germany. Tissues were fixed in 4% buffered formalin and then embedded in paraffin. TMA tissue spot diameter was 0.6 mm. The use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12) and by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work was carried out in compliance with the Helsinki Declaration.

Immunohistochemistry

Freshly cut TMA sections were immunostained under the same experimental conditions. Two different primary antibodies were used for PLAP detection: MSVA-350R (rabbit recombinant; MS Validated Antibodies, Hamburg, Germany) and IR779 (mouse monoclonal 8A9, Agilent DAKO, Santa Clara, CA, USA). The normal tissue array was analyzed with both MSVA-350R and IR779, while the multitumor array was analyzed with MSVA-350R only. For MSVA-350R, slides were deparaffinized with xylol, rehydrated through a graded alcohol series, and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121°C in pH 9.0 Target Retrieval Solution (Agilent). Endogenous peroxidase activity was blocked with Peroxidase Blocking Solution (Agilent) for 10 min. The primary antibody was diluted 1:150 and applied for 60 min at 37°C. For IR779, the slides were deparaffinized and rehydrated as described previously, and exposed to heat-induced antigen retrieval for 15 min in Agilent's PT Link pretreatment module at 95°C in pH 9.0 retrieval buffer. Slides were transferred to an Autostainer Link 48 device (Agilent) for peroxidase blocking (5 min) and incubation of the primary antibody (ready to use prediluted for 20 min at room temperature). Both antibodies were visualized using the respective EnVision reagents (Agilent) for manual and automated staining according to the manufacturer's directions. One pathologist (NG) analyzed all immunostainings. For tumor tissues, the percentage of positive neoplastic cells was estimated, and the staining intensity was semiquantitatively recorded (0, 1+, 2+, and 3+). For statistical analyses, the staining results were categorized into four groups. Tumors without any staining were considered negative. Tumors with 1+ staining intensity in ≤70% of cells and 2+ intensity in ≤30% of cells were considered weakly positive. Tumors with 1+ staining intensity in >70% of cells, 2+ intensity in 31–70%, or 3+ intensity in ≤30% were considered moderately positive. Tumors with 2+ intensity in >70% or 3+ intensity in >30% of cells were considered strongly positive.

Statistics

Statistical calculations were performed with JMP 14 software (SAS Institute Inc., Cary, NC, USA). Contingency tables and the chi-square test were performed to search for associations between PLAP and tumor phenotype. Survival curves were calculated according to Kaplan–Meier. The log-rank test was applied to detect significant differences between groups. A P value of ≥0.05 was considered as statistically significant.

Results Technical issues

A total of 12,381 (76.6%) of 16,166 tumor samples were interpretable in our TMA analysis. Non-interpretable samples demonstrated lack of unequivocal tumor cells or loss of the tissue spot during technical procedures. A sufficient number of samples of each normal tissue type was evaluable.

PLAP in normal tissues

With two different antibodies (MSVA-350R and IR779), particularly strong PLAP immunostaining was found in the placenta. Here, strong PLAP positivity was seen in chorion cells as well as in cyto- and syncytiotrophoblast of mature placenta (Figure 1A,D). Staining was only moderate and limited to the surface cell membrane in the trophoblast of early placenta, and only weak in amnion cells. Also, for both antibodies, weak PLAP staining was seen at the apical membrane of epithelial cells in the endocervix (Figure 1B,E), endometrium, and the fallopian tube, although this did not occur in all samples analyzed. PLAP immunostaining was lacking for both antibodies in most other tissues including all epithelial cells of the gastrointestinal and the genitourinary tract, gallbladder, liver, pancreas, salivary and bronchial glands, breast glands, Brunner glands, thyroid, pituitary gland, adrenal gland, parathyroid gland, testis, epididymis, seminal vesicle, prostate, non-keratinizing and keratinizing squamous epithelium of various different sites, skin appendages, hematopoietic and immune cells, and the brain. Staining of muscular tissues revealed complete absence of staining by MSVA-350R (Figure 1C) while Agilent Dako IR779 showed moderate to strong staining of smooth muscle (Figure 1F) and weak to moderate staining of skeletal muscle. These latter findings were considered to be due to cross-reactivity.

PLAP immunostaining of normal tissues (comparison of antibodies). The panels show (A) strong PLAP positivity of trophoblastic cells in the placenta, (B) weak to moderate apical staining of endocervical glands, and (C) absence of staining in smooth muscle from the colon wall for the antibody MSVA-350R. Using the antibody IR779, identical findings are seen for (D) placenta and (E) endocervical glands but an additional strong staining occurred in (F) smooth muscle cells.

PLAP in cancer

By using MSVA-350R, positive PLAP immunostaining was detectable in 1,503 (12.1%) of the 12,381 analyzable tumors, including 761 (6.1%) with weak, 184 (1.5%) with moderate, and 558 (4.5%) with strong immunostaining. Overall, 48 (36.6%) of 131 tumor categories showed detectable PLAP expression with 22 (16.8%) tumor categories showing strong positivity in at least one case (Table 1). Representative images of PLAP-positive tumors are shown in Figure 2. The highest rate of positive staining was found in testicular tumors, followed by tumors of the female genital tract, gastroesophageal, and pancreaticobiliary cancers. It is of note that only weak PLAP immunostaining was occasionally found in 21 different tumor entities. In most of these tumors, PLAP immunostaining was limited to a small fraction of tumor cells (Figure 2D,E). None of the 48 leiomyomas, 84 leiomyosarcomas, 7 rhabdomyosarcomas, or 91 angiomyolipomas showed any PLAP staining. A graphical representation of the rank order of PLAP positive and strongly positive cancers is shown in Figure 3.

Table 1. PLAP immunostaining in human tumors. PLAP immunostaining Tumor entity On TMA (n) Analyzable (n) Negative (%) Weak (%) Moderate (%) Strong (%) Positive (%) Tumors of the skin Pilomatrixoma 35 32 100.0 0.0 0.0 0.0 0.0 Basal cell carcinoma 88 56 100.0 0.0 0.0 0.0 0.0 Benign nevus 29 26 100.0 0.0 0.0 0.0 0.0 Squamous cell carcinoma of the skin 90 82 100.0 0.0 0.0 0.0 0.0 Malignant melanoma 48 44 100.0 0.0 0.0 0.0 0.0 Merkel cell carcinoma 46 44 100.0 0.0 0.0 0.0 0.0 Tumors of the head and neck Squamous cell carcinoma of the larynx 110 96 93.8 5.2 1.0 0.0 6.3 Squamous cell carcinoma of the pharynx 60 46 97.8 2.2 0.0 0.0 2.2 Oral squamous cell carcinoma (floor of the mouth) 130 118 100.0 0.0 0.0 0.0 0.0 Pleomorphic adenoma of the parotid gland 50 44 100.0 0.0 0.0 0.0 0.0 Warthin tumor of the parotid gland 104 96 100.0 0.0 0.0 0.0 0.0 Adenocarcinoma, NOS (papillary cystadenocarcinoma) 14 12 91.7 8.3 0.0 0.0 8.3 Salivary duct carcinoma 15 12 100.0 0.0 0.0 0.0 0.0 Acinic cell carcinoma of the salivary gland 181 143 100.0 0.0 0.0 0.0 0.0 Adenocarcinoma NOS of the salivary gland 109 79 98.7 1.3 0.0 0.0 1.3 Adenoid cystic carcinoma of the salivary gland 180 119 100.0 0.0 0.0 0.0 0.0 Basal cell adenocarcinoma of the salivary gland 25 23 100.0 0.0 0.0 0.0 0.0 Basal cell adenoma of the salivary gland 101 91 100.0 0.0 0.0 0.0 0.0 Epithelial–myoepithelial carcinoma of the salivary gland 53 52 98.1 1.9 0.0 0.0 1.9 Mucoepidermoid carcinoma of the salivary gland 343 262 98.5 1.1 0.0 0.4 1.5 Myoepithelial carcinoma of the salivary gland 21 20 100.0 0.0 0.0 0.0 0.0 Myoepithelioma of the salivary gland 11 9 100.0 0.0 0.0 0.0 0.0 Oncocytic carcinoma of the salivary gland 12 12 100.0 0.0 0.0 0.0 0.0 Polymorphous adenocarcinoma, low grade, of the salivary gland 41 33 100.0 0.0 0.0 0.0 0.0 Polymorphous adenoma of the salivary gland 53 35 100.0 0.0 0.0 0.0 0.0 Tumors of the lung, pleura, and thymus Adenocarcinoma of the lung 246 160 79.4 19.4 0.6 0.6 20.6 Squamous cell carcinoma of the lung 130 65 98.5 1.5 0.0 0.0 1.5 Small cell carcinoma of the lung 20 16 100.0 0.0 0.0 0.0 0.0 Mesothelioma, epithelioid 39 32 100.0 0.0 0.0 0.0 0.0 Mesothelioma, other types 76 63 98.4 1.6 0.0 0.0 1.6 Thymoma 29 29 100.0 0.0 0.0 0.0 0.0 Tumors of the female genital tract Squamous cell carcinoma of the vagina 78 63 100.0 0.0 0.0 0.0 0.0 Squamous cell carcinoma of the vulva 130 116 94.8 5.2 0.0 0.0 5.2 Squamous cell carcinoma of the cervix 130 124 91.9 6.5 0.0 1.6 8.1 Endometrioid endometrial carcinoma 236 223 40.4 31.8 9.0 18.8 59.6 Endometrial serous carcinoma 82 72 56.9 31.9 4.2 6.9 43.1 Carcinosarcoma of the uterus 48 38 68.4 23.7 2.6 5.3 31.6 Endometrioid carcinoma, high grade, G3 13 13 84.6 15.4 0.0 0.0 15.4 Endometrial clear cell carcinoma 8 7 85.7 14.3 0.0 0.0 14.3 Endometrioid carcinoma of the ovary 110 91 41.8 38.5 6.6 13.2 58.2 Serous carcinoma of the ovary (NOS) 559 462 50.2 32.9 8.4 8.4 49.8 Mucinous carcinoma of the ovary 96 71 85.9 7.0 2.8 4.2 14.1 Clear cell carcinoma of the ovary 50 40 90.0 10.0 0.0 0.0 10.0 Carcinosarcoma of the ovary 47 38 60.5 28.9 5.3 5.3 39.5 Brenner tumor 9 9 77.8 22.2 0.0 0.0 22.2 Tumors of the breast Invasive breast carcinoma of no special type 1,391 1185 99.2 0.8 0.0 0.0 0.8 Lobular carcinoma of the breast 294 236 99.6 0.0 0.4 0.0 0.4 Medullary carcinoma of the breast 26 26 96.2 3.8 0.0 0.0 3.8 Tubular carcinoma of the breast 27 26 100.0 0.0 0.0 0.0 0.0 Mucinous carcinoma of the breast 58 44