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The RAV12 monoclonal antibody recognizes the N-linked glycotope RAAG12: expression in human normal and tumor tissues.

Aberrant glycosylation is one of the hallmarks of human cancer and is hypothesized to contribute to oncogenic transformation, tumor invasion, and metastasis. (1-3) The altered glycosylation profiles can result from changes in the expression levels of native glycans or from the generation of novel altered glycan structures. These altered glycans represent tumor-associated antigens that are tumor specific or overexpressed on tumors and therefore are candidates for antibody-based targeted cancer therapy. The Lewis blood group family of carbohydrate antigens represents a prototypical tumor-associated antigen that exhibits restricted expression, as well as differential subcellular localization, on normal tissues when compared with tumor tissues. In the case of Lewis Y (Ley), expression on normal tissues is restricted to granulocytes and epithelial surfaces, particularly in mucosa of the gastrointestinal tract, and expression is largely confined to the cytoplasm. (4-6) In contrast, Ley expression on tumor tissue is observed on the cell surface as well as in the cytoplasm. (7) Overexpression of Ley has been observed on colon, breast, ovary, lung, pancreas, and prostate carcinomas (7) and the expression correlated with increased invasiveness and metastasis and decreased patient survival rates. (8,9) Recently, Klinger et al (10) reported that the cell surface receptors ErbBl and ErbB2 are modified with Ley on human breast and epidermoid tumor cells, and the epidermal growth factor- and heregulin-stimulated receptor signaling cascades in these cells can be blocked by monoclonal anti-Ley antibodies. Thus, aberrant or differentially expressed carbohydrate antigens present on the surface of tumor cells may serve as important bioactive targets for monoclonal antibody antitumor therapies.

We previously reported the identification of RAAG12, a primate-restricted carbohydrate antigen recognized by the monoclonal antibody RAV12. (11) The RAV12 binding epitope on RAAG12 consists minimally of the trisaccharide Gal[beta]1-3GlcNAc[beta]1-3Gal, a precursor to the Lewis blood group antigens, suggesting that the RAAG12 glycotope may be the product of misprocessed or underprocessed carbohydrate moieties related to the Lewis blood group antigens. Coimmunoprecipitation studies showed that RAAG12 is present on multiple membrane-associated proteins, including growth factor receptors and adhesion-associated molecules. Importantly, RAV12 induced oncotic cell death in the uniformly RAAG12-positive COLO 205 colon adenocarcinoma cell line in vitro, and RAV12 exhibited potent antitumor activity toward multiple RAAG12positive gastrointestinal tumor xenografts in athymic mice. (11) As a step toward defining molecular mechanisms that may contribute to RAV12-mediated oncotic cell death, we have begun to investigate growth factor receptor-mediated signaling events that may be perturbed by RAV12. Recent evidence from our laboratory indicates that RAV12, through interaction with RAAG12-bearing insulin-like growth factor 1 receptors, can modulate receptor function and block insulin-like growth factor 1 stimulated growth of COLO 205 colon adenocarcinoma cells.12 Based on these promising preclinical data, we undertook an extensive immunohistochemical study to investigate the expression pattern of RAAG12 to (1) identify potential normal tissues that may be affected by RAV12 treatment and (2) aid in the selection of appropriate clinical patient populations. In this report we describe the expression pattern of RAAG12 in human normal and tumor tissues. The preclinical data, together with results presented in this study, have led to the initiation of a phase I/IIA clinical study of RAV12 in patients with metastatic or recurrent adenocarcinoma.

MATERIALS AND METHODS Cell and Tissue Sample Collection and Preparation

Nonmalignant biopsy or autopsy tissue (referred to as normal tissue herein) was obtained by Pathology Associates Division, Charles River Laboratories, Inc (Frederick, Maryland) for the good laboratory practice cross-reactivity study. Fresh tissue samples were embedded in Tissue-Tek OCT medium (Sakura Finetek USA, Inc, Torrance, California), frozen on dry ice, and stored in sealed plastic bags at less than -70[degrees]C Tissues were cut into approximately 5-|j,m sections. Just prior to staining, all slides, with the exception of positive control [[beta].sub.2]-macroglobulin slides, were fixed in 10% formalin for 10 seconds. The [[beta].sub.2]-macroglobulin slides were fixed for 10 minutes in acetone.

Frozen tumors and/or adjacent nonmalignant tissues were obtained from Ardais (Lexington, Massachusetts), Phenopath Laboratories (Seattle, Washington), Cureline (South San Francisco, California), Oncotech (Tustin, California), and C. Mangham, BSc, MB, ChB, FRCPath (University of Birmingham, Birmingham, England). Formalin-fixed, paraffin-embedded tumors and/or adjacent normal tissues were obtained from Cureline, Asterand (Detroit, Michigan), and Dr C. Mangham. Formalin-fixed, paraffin-embedded tumor and/or normal tissue microarrays were obtained from Biogenex (San Ramon, California), Imgenex (San Diego, California), Petagen (Seoul, South Korea), and Asterand. All tumor samples were cut into 4- to 6-| m sections for immunohistochemistry staining.

COLO 205 colon adenocarcinoma tumor cells were obtained from the American Type Culture Collection (Manassas, Virginia). The cell line was maintained in F12/DMEM (Invitrogen, Carlsbad, California), supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) in a humidified atmosphere of 95% air and 5% CO2 at 37[degrees]C.

Immunohistochemistry

For the good laboratory practice normal tissue survey (Pathology Associates Division, Charles River Laboratory), biotinylated RAV12 (2 and 50 | g/mL) was used to stain frozen human biopsy and autopsy tissue sections. Samples were stained using the VectaStain Elite ABC Kit (Vector Laboratories, Burlingame, California) according to the manufacturer's protocol.

Frozen or formalin-fixed, paraffin-embedded tumor and adjacent normal tissue sections were stained either using the VectaStain Elite ABC Kit with biotin-conjugated antibodies or the Dako Envision Plus Kit (Dako, Carpinteria, California) with unconjugated murine antibodies, according to the manufacturer's protocol, using 5 [micro]g/mL biotinylated RAV12 (RAV12-bio) or 3 [micro]g/mL biotinylated KID3 (KID3-bio; KID3 is the parental murine antibody of chimeric RAV12) or 3 [micro]g/mL unconjugated KID3. Antigen retrieval was necessary and was performed by incubating rehydrated slides in Dako Target Retrieval Solution (pH 6.0) in the DC2002 Decloaking Chamber Pro (Biocare Medical, Walnut Creek, California) at 125[degrees]C for 30 seconds, then cooled to room temperature. Frozen sections were fixed with a 50:50 mixture of acetone:ethanol or pure acetone prior to performing the previously described immunohistochemistry staining.

To limit nonspecific staining, necessary blocking steps with appropriate reagents were performed on both frozen and formalinfixed, paraffin-embedded sections prior to immunohistochemistry staining.

Subrenal Capsule Tissue Toxicology Model

Differential toxicity of RAV12 toward colon adenocarcinoma xenografts compared with normal colon tissue was assessed by the subrenal capsule tissue toxicology model as described by Mather and Young. (13) Intact human fetal colon tissue was isolated, and 1 X 1 X 1-mm pieces of tissue (1 piece per kidney) were surgically implanted under one kidney capsule of severe combined immunodeficient mice through a paralumbar surgical approach. (14) The tissue was then allowed to mature in the mouse for 18 to 22 weeks. Following maturation of the tissue, a subset of the tissue xenografts were harvested and assessed histologically for RAAG12 expression and tissue morphology as previously described. COLO 205 colon adenocarcinoma tumor cells were harvested with 10 mmol/L EDTA in phosphate-buffered saline and resuspended in F12/DMEM containing 10% fetal bovine serum. Then 5 X [10.sup.5] cells were resuspended in 50-[micro] L buttons of rat tail type I collagen and cultured in vitro overnight. The tumor cellcontaining collagen buttons were surgically placed under the contralateral kidney capsules of the remaining mice that bear the matured human fetal colon tissue, as was done with the fetal tissue. Following a 3-day recovery period, mice were treated with KID3 antibody (the murine parent of the chimeric RAV12 antibody) as follows: 100 mg/kg KID3 loading dose intraperitoneally (day 3), followed by 50 mg/kg KID3 intraperitoneally (days 6, 10, and 13). Mice were killed on day 17. Tissue xenografts were assessed histologically for cell and tissue morphology, and tumor xenografts were assessed histologically for tumor size, following staining with hematoxylin.

RESULTS

RAAG12 Expression in Human Tumor Tissues

Archival human tumor samples representing a broad range of tumor types were evaluated for the frequency, intensity, and pattern of expression of RAAG12. The major tumor types demonstrating the most frequent and uniform RAAG12 expression pattern were colorectal, gastric, and pancreatic adenocarcinomas and intrahepatic cholangiocarcinomas, as summarized in Table 1. More than 90% of each of these tumor types expressed RAAG12 (colorectal [107 of 119;90%], gastric [88 of 94;94%], and pancreatic [66 of 66; 100%]), and staining was uniform and encompassed the whole cell membrane in most samples. Representative examples of the RAAG12 expression pattern on human colorectal (Figure 1, A), gastric (Figure 1, B), and pancreatic (Figure 1, C) adenocarcinomas are shown.

RAAG12 was also expressed at moderate to high frequencies in additional types of adenocarcinomas and carcinomas. Overall, 56% of lung cancers expressed RAAG12, with 68% of the lung adenocarcinomas and 49% of squamous cell carcinomas of the lung expressing RAAG12. Additionally, endometrial carcinoma (2 of 2; 100%), ovarian carcinoma (13 of 20; 65%), liver adenocarcinoma (4 of 6; 67%), and gallbladder carcinoma (2 of 3; 67%) also exhibited moderate to high frequency of RAAG12 expression, although a lower proportion of tumors exhibited uniform staining. RAAG12 was also expressed at lower frequency and/or uniformity of expression in esophageal carcinomas, breast carcinomas, prostate adenocarcinomas, urinary tract carcinomas, and thyroid carcinomas.

[FIGURE 1 OMITTED]

Persistence of RAAG12 Expression in Metastatic Colorectal Cancer and Correlation With Primary Tumor Expression

To evaluate the persistence of RAAG12 expression in metastatic disease, colorectal cancer samples from 94 primary tumors, 1 recurrent tumor, and 24 metastatic tumors (11 from lymph notes and 13 from distant sites) were evaluated for RAAG12 expression. As summarized in Table 1, the frequency and uniformity of RAAG12 expression by metastatic colorectal tumors was similar to that of unmatched primary tumors. Matched primary and metastatic colorectal adenocarcinomas from the same patient also showed a high correlation of RAAG12 expression. A comparison of 10 primary tumors with matched metastatic tumor specimens from lymph nodes revealed that 9 of 11 metastatic tumor specimens (1 patient had 2 metastatic lymph node lesions available) showed a 1:1 correlation with the corresponding primary tumor in the pattern, frequency, and intensity of RAAG12 expression. Regarding the 2 noncorrelative cases, one patient's primary tumor exhibited RAAG12 reactivity, while the metastatic lesion was negative. In the second case, the patient had 2 distinct metastatic lesions--one of the metastatic lesions exhibited RAAG12 expression similar to the positive primary tumor, while the other metastatic lesion was negative. These data are consistent with the frequency and uniformity of expression of RAAG12 observed in the unmatched primary and metastatic colorectal adenocarcinomas.

RAAG12 Expression in Normal Human Tissues

RAAG12 expression on normal human tissues, as determined by immunohistochemistry, is summarized in Table 2. Representative photomicrographs of normal colon (Figure 2, A), stomach (Figure 2, B), pancreas (Figure 2, C), liver (Figure 2, D), skin (Figure 2, E), and lung (Figure 2, F) tissue specimens stained for RAAG12 expression are shown.

In addition to the expression summarized in Table 2, rare to occasional weak staining of macrophages was observed within small vessels (usually lymphatics) in multiple tissues including colon, lung, tonsil, pancreas, red pulp/cords of Billroth in spleen, adjacent to epithelium in thymus, and sinusoids in lymph node. In the epithelial tissues, the stained macrophages were located very close to prominently stained epithelial cells (eg, in colon, only lymphatics in close proximity to the muscularis mucosae were involved), consistent with possible physiologic uptake or processing of shed or secreted antigen by nearby macrophages or mononuclear cells. Peripheral blood mononuclear leukocytes were negative in the 3 blood smears examined; however, subcapsular or sinusoidal macrophages were stained in some lymph nodes, again consistent with the hypothesis that epithelial cell antigen may be shed in vivo and taken up by macrophages.

Subcellular Location of RAAG12 in Human Normal and Tumor Tissues

Distinct patterns of subcellular localization of RAAG12 were observed in the tumor tissues, particularly in gastric, colorectal, and pancreatic adenocarcinomas, as well as in normal tissues. In normal epithelia from individuals without malignancy, RAAG12 was expressed primarily in the cytoplasm; however, occasional membrane staining was observed in some epithelia. Whole membrane and/or desmosomal staining were seen only in a few epithelia (transitional and stratified squamous), while in normal polarized epithelia such as gastrointestinal tract epithelium and ductal epithelium, RAAG12 expression was restricted primarily to the cytoplasm and apical membrane surface. Normal colon epithelium (Figure 3, A) and gastric epithelium (Figure 3, B) exhibited prominent apical membrane staining, and gastric epithelium also exhibited strong cytoplasmic granule staining. Bile ducts exhibited cytoplasmic staining in addition to strong apical membrane staining (Figure 3, C). Additionally, goblet cell staining was observed in bronchial epithelium (Figure 3, D). Most normal tissue samples adjacent to tumors exhibited RAAG12 subcellular localization similar to that observed in normal tissues from individuals without malignancy (data not shown).

In contrast, in tumors derived from polarized epithelia such as gastrointestinal adenocarcinomas, RAAG12 was present on other membrane domains, including the basolateral surface. In well-differentiated tumors, RAAG12 expression was observed on apical membrane surfaces together with basolateral membrane expression. A representative example of RAAG12 expression in a well-differentiated colon (Figure 4, A), pancreas (Figure 4, C), and stomach (Figure 4, E) adenocarcinoma is shown. RAAG12 expression became progressively less restricted as the differentiation state of the tumors decreased. Moderately differentiated tumors showed increased basolateral staining relative to well-differentiated tumors, while poorly differentiated tumors exhibited whole membrane staining. A representative example of RAAG12 expression in a poorly differentiated colon (Figure 4, B), pancreas (Figure 4, D), and gastric (Figure 4, F) adenocarcinoma is shown. Similar patterns of expression were observed in adenocarcinomas derived from other polarized epithelia (breast, ovarian surface epithelium, and lung; data not shown). Thus, there were significant differences in the pattern of RAAG12 expression, particularly in the case of poorly differentiated tumors.

Differential Sensitivity to RAV12

As a step toward assessing whether RAV12 may be cytotoxic toward normal tissues that are reactive with RAV12, we used a murine subrenal capsule xenograft model (13) to determine whether RAV12 treatment was differentially cytotoxic toward RAAG12-positive colon tumor xenografts compared with normal colon tissue implanted under the contralateral kidney. Normal human 12-week fetal colon epithelial tissue exhibited an age-appropriate developmental phenotype with weak RAAG12 staining (Figure 5, A). Following implantation and maturation of 14-week fetal colon epithelial tissue under the mouse kidney capsule for 18 weeks in vivo, the tissue achieved a cellular architecture and RAAG12 expression pattern similar to the adult human colon (Figure 5, B). Following the 18-week maturation of the normal fetal colon tissue, COLO 205 colon adenocarcinoma cells were implanted under the contralateral kidney capsule and allowed to establish for 3 days. The mouse was then treated twice weekly during a 2-week period with KID3, the murine parent monoclonal antibody of RAV12. We have previously shown that the KID3 and RAV12 antibodies exhibit comparable antitumor activity in vivo. (11) Treatment with KID3 had no observable effect on the matured normal colon tissue (Figure 5, C), and the tissue appeared identical to tissue from control, phosphate-buffered saline-treated animals (Figure 5, D). In contrast, KID3 treatment eliminated the COLO 205 colon adenocarcinoma tumor xenograft (Figure 5, E) relative to tumor xenografts from control, phosphate-buffered saline-treated animals (Figure 5, F).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

COMMENT

We have developed a chimeric monoclonal antibody, RAV12, which recognizes the primate-restricted carbohydrate antigen RAAG12 expressed on a number of human carcinomas. RAV12 binds with high affinity to RAAG12 and exhibits cytotoxic activity in vitro, via an oncotic cell death mechanism, toward RAAG12-expressing human gastrointestinal tumor cell lines. The cytotoxic activity of RAV12 in vitro toward RAAG12-expressing tumor cells was reflected in the potent antitumor activity observed in multiple gastrointestinal xenografts in vivo, including colon, gastric, and pancreatic tumors. As a step toward defining appropriate cancer patient populations for treatment, as well as to aid in identifying potential normal tissues that may be reactive to RAV12, we undertook an extensive immunohistochemical study to investigate expression of RAAG12 in human normal and tumor tissues. In this report we described the expression of RAAG12 in human tumor and normal tissue, placing emphasis on the overall frequency, density, and subcellular location of the RAAG12 glycotope.

In cancer, the major tumor types exhibiting the highest frequency and most uniform RAAG12 expression were colorectal, gastric, and pancreatic adenocarcinomas, with more than 90% of specimens of these tumor types expressing RAAG12. In addition to these tumor types, RAAG12 was also expressed at lower frequency and / or uniformity of expression in a number of other carcinomas and adenocarcinomas, including esophageal, ovarian, liver, breast, and prostate. Thus, adenocarcinomas--especially adenocarcinomas of gastrointestinal origin--would be considered strong candidates for RAV12 treatment. In tissues from normal, non-tumor-bearing individuals, no RAAG12 expression was observed on tissues from cardiovascular, endocrine, hematopoietic, neuromuscular, and central nervous systems. RAAG12 expression was observed on polarized epithelium, with the most uniform expression in mucosal epithelia of the gastrointestinal tract, bile ducts, and pancreatic ducts.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Importantly, RAAG12 exhibited significant differences in subcellular localization in RAAG12-positive normal polarized epithelia when compared with tumors derived from those tissues. Differences in subcellular location of antigens between normal and malignant tissues has been observed for other tumor antigens, including MUC1 (CD277; epithelial membrane antigen), which has been extensively investigated as a potential target for cancer immunotherapy. (15) MUC1 is a high-molecular-weight membrane-associated glycoprotein, which is often aberrantly glycosylated and overexpressed in adenocarcinoma and plays a role in cell-cell and cell-matrix interactions. In renal cell carcinoma, the subcellular localization of MUC1 correlates with tumor stage and grade--low stage and grade tumors show predominantly apical membranous staining, while high stage and grade tumors exhibit predominantly circumferential membranous staining. (16) In contrast, normal kidney epithelium exhibits strong, diffuse cytoplasmic and apical membrane MUC1 expression. Similar observations of differential subcellular localization of MUC1 have been reported for transitional cell carcinomas of the bladder (17) and breast carcinomas. (18)

In the clinical setting, investigators have been targeting MUC1 using vaccine-based approaches to elicit MUC1-specific immune responses in patients with MUC1-positive cancers. (15) Several of these clinical studies have reported positive results with respect to the development or amplification of MUC1-specific immune responses (19-23) and evidence of therapeutic benefit, (24,25) with minimal adverse events. Administration of a murine anti-MUC1 monoclonal antibody to patients with MUC1-positive metastatic cancer was also shown to be well tolerated. (26) The lack of significant adverse events in these clinical studies is consistent with the notion that antigen accessibility may be limited in normal tissues due to localization to apical membrane surfaces and the cytoplasm.

Analogous to the observations reported for MUC1, RAAG12 appears to be an aberrantly glycosylated glycotope whose expression in normal polarized epithelia, as found in the gastrointestinal tract and various ducts and glands, was primarily confined to the cytoplasm and apical membrane. Conversely, adenocarcinomas derived from these tissues showed a progressive increase in the membrane area that expressed RAAG12 (apical and increasingly basolateral), and the increased membrane expression directly correlated with the differentiation state of the tumor. The cytoplasmic and apical membrane localization of RAAG12 on normal polarized secretory/absorptive epithelia may confer differential sensitivity by limiting antigen accessibility in normal tissues, as has been reported for membrane proteins including ErbB2. (27) Normal tight junctions and apical restriction of antigen has been shown to prevent antibodies from traversing from the basolateral surface to the antigen-bearing apical surface in vitro and in murine xenograft models. (28)

In the case of RAV12, transient, self-limiting elevations in liver function tests and pancreatic enzyme blood levels, as well as transient vomiting, were observed in cynomolgus monkeys treated with RAV12; however, no significant histopathologic gastrointestinal or hepatic abnormalities were observed in the long-term multiple dose intravenous infusion toxicity study with RAV12 (data not shown). Furthermore, we observed a differential sensitivity, between normal polarized colon epithelia and COLO 205 adenocarcinoma xenografts, toward the cytotoxic activity of the murine parent of RAV12, KID3, using an in vivo subrenal capsule tissue toxicity model. Thus, the differences in subcellular localization of RAAG12 may limit, or prevent, RAV12 reactivity toward RAAG12 present in the cytoplasm and apical membrane surfaces of normal polarized epithelium. We are currently investigating whether transcytosis of antibody to the RAAG12 positive apical surface, perhaps via Fc receptor-mediated transport, may have contributed to the limited toxicity observed in cynomolgus monkeys.

In summary, this study provides information regarding the frequency, pattern of expression, and subcellular location of the target of the monoclonal antibody RAV12, a novel glycotope RAAG12 expressed in human tumor and normal tissues. RAAG12 expression was strongest and most uniform in tumors of gastrointestinal origin, while expression on normal tissues was greatest on polarized epithelium and stratified squamous epithelium. There are significant differences in the subcellular localization of RAAG12 in normal polarized epithelia tissues relative to tumor tissues. These differences in RAAG12 subcellular location may limit accessibility to RAAG12 expressed by normal tissues of intravenously administered RAV12 antibody. Additional nonclinical studies, as well as the ongoing phase I/IIA clinical study of RAV12 in patients with metastatic or recurrent adenocarcinoma, will aid in defining the relationship between RAAG12 expression/subcellular location and the potential for a differential response to RAV12 treatment in tumor versus normal tissue in patients.

References

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(9.) Muramatsu T. Carbohydrate signals in metastasis and prognosis of human carcinomas. Glycobiology. 1993;3:291-296.

(10.) Klinger M, Farhan H, Just H, et al. Antibodies directed against Lewis-Y antigen inhibit signaling of Lewis-Y modified ErbB receptors. Cancer Res. 2004; 64:1087-1093.

(11.) Loo D, Pryer N, Young P, et al. The glycotope-specific RAV12 monoclonal antibody induces oncosis in vitro and has antitumor activity against gastrointestinal adenocarcinoma tumor xenografts in vivo. Mol Cancer Ther. 2007;6:856-865.

(12.) Li JC, Li R. RAV12 accelerates the desensitization of Akt/PKB pathway of insulin-like growth factor I receptor signaling in COLO205. Cancer Res. 2007; 67:8856-8864.

(13.) Mather JP, Young PF, inventors; Raven Biotechnologies, Inc, assignee. Animal model for toxicology and dose prediction. US patent 10,448,766. 2004.

(14.) Parmar H, Young P, Emerman JT, et al. A novel method for growing human breast epithelium in vivo using mouse and human mammary fibroblasts. Endocrinology. 2002;143:4886-4896.

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(17.) Walsh MD, Hohn BG, Thong W, et al. Mucin expression by transitional cell carcinomas of the bladder. Br J Urol. 1994;73:256-262.

(18.) Rahn JJ, Dabbagh L, Pasdar M, Hugh JC. The importance of MUC1 cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients and review of the literature. Cancer. 2001;91:1973-1982.

(19.) Gilewski T, Adluri S, Ragupathi G, et al. Vaccination of high-risk breast cancer patients with mucin-1 (MUC1) keyhole limpet hemocyanin conjugate plus QS-21. Clin Cancer Res. 2000;6:1693-1701.

(20.) Karanikas V, Thynne G, Mitchell P, et al. Mannan mucin-1 peptide immunization: influence of cyclophosphamide and the route of injection. J Immu nother. 2001;24:172-183.

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Suzanne K. Coberly, MD; Francine Z. Chen, MD; Mark P. Armanini, MS; Yan Chen, MS; Peter F. Young, MS; Jennie P. Mather, PhD; Deryk T. Loo, PhD

Accepted for publication November 7, 2008.

From the Departments of Pathology (Drs Coberly and F. Chen, Mr Armanini, and Mrs Y. Chen) and Cell Biology (Drs Mather and Loo and Mr Young), MacroGenics West, Inc, South San Francisco, California. Dr Coberly is now with the Department of Pathology, Amgen, South San Francisco, California.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Deryk T. Loo, PhD, MacroGenics West, Inc, One Corporate Dr, South San Francisco, CA 94080 (e-mail: dloo@ravenbio.com).
Table 1. Frequency of RAAG12 Expression on Human Tumor Types

                                        Frequency of RAAG12 Expression

                                                        % [greater
                                                         than or
                                            Total %       equal
Tumor Type                        No.       Positive     to] 75%

Gastrointestinal tumors
  Colorectal carcinoma (total)    119            90            64
    Primary                        94            91            69
    Metastatic                     24            83            54
    Reoccurrence (NOS)              1           100           100
  Stomach adenocarcinoma           94            94            67
  Gastric dysplasia                 2           100           100
  Gastric sarcoma                   1             0             0
  Gastric lymphoma                  1             0             0
  Esophageal carcinoma
    (squamous cell cancer only)     7            71             0
  Tongue carcinoma (SCC)            3             0             0
  Salivary gland tumors             5            60             0
  Pancreatic adenocarcinoma        66           100            68
  Liver cholangiocarcinoma          2           100           100
  Liver adenocarcinoma
    (metastatic adenocarcinoma
    or cholangiocarcinoma)          6            67            50
  Liver hepatocellular
    carcinoma                       2             0             0
  Gallbladder carcinoma             3            67            33
Respiratory tumors
  Lung (total)                     94            56            13
  Adenocarcinoma                   38            68            18
  SCC                              39            49             5
  Adenosquamous carcinoma           4           100            50
  Large cell carcinoma              6            33            17
  Small cell carcinoma              2            50             0
  Malignant carcinoid               1           100             0
Reproductive tumors
  Ovarian carcinoma                20            65            25
  Ovarian lymphoma                  1             0             0
  Breast adenocarcinoma            36            44            19
  Breast sarcoma                    1           100             0
  Uterine (endometrioid)
    carcinoma                       2           100           100
  Uterine (cervical) carcinoma     10            20             0
  Prostatic adenocarcinoma         24            33            29
  Testis tumors                     6            17            17
  Penile carcinoma (SCC)            2            50             0
Urinary tract tumors
  Kidney tumors                    21            24            14
  Bladder/urethral carcinoma
    (TCC)                           7            57             0
Endocrine tumors
  Adrenal carcinoma                 1             0             0
  Thyroid carcinoma (all)          13            46             0
Hematopoietic tumors
  Lymphomas (all)                  10             0             0
Skin tumors
  SCC                               1             0             0
Skeletal system tumors
  Bone tumors (all)                11             0             0
Soft tissue tumors
  Soft tissue sarcoma (all)        13             8             0
  Melanocytic tumors
    (including clear cell
    sarcoma)                        4             0             0
Brain tumors
  Brain tumors (all)                8            13            13
All metastatic tumors
    (unknown primary)
  Adenocarcinoma (NOS)              3            67            67
  Carcinoma (NOS)                   5            40            40
  SCC (NOS)                         1           100           100

                                      Frequency of RAAG12 Expression

                                  % [greater   % [greater
                                   than or      than or
                                    equal        equal
Tumor Type                         to] 50%      to] 10%     % = 1%-10%

Gastrointestinal tumors
  Colorectal carcinoma (total)         8            8           11
    Primary                            7            7           11
    Metastatic                         8            8           13
    Reoccurrence (NOS)                 0            0            0
  Stomach adenocarcinoma               3           14           10
  Gastric dysplasia                    0            0            0
  Gastric sarcoma                      0            0            0
  Gastric lymphoma                     0            0            0
  Esophageal carcinoma
    (squamous cell cancer only)        0           29           43
  Tongue carcinoma (SCC)               0            0            0
  Salivary gland tumors                0            0           60
  Pancreatic adenocarcinoma           12           15            5
  Liver cholangiocarcinoma             0            0            0
  Liver adenocarcinoma
    (metastatic adenocarcinoma
    or cholangiocarcinoma)             0           17            0
  Liver hepatocellular
    carcinoma                          0            0            0
  Gallbladder carcinoma                0           33            0
Respiratory tumors
  Lung (total)                         9           12           23
  Adenocarcinoma                      13           13           24
  SCC                                  8           13           23
  Adenosquamous carcinoma              0            0           50
  Large cell carcinoma                 0            0          1 7
  Small cell carcinoma                 0           50            0
  Malignant carcinoid                  0            0          100
Reproductive tumors
  Ovarian carcinoma                    0           10           30
  Ovarian lymphoma                     0            0            0
  Breast adenocarcinoma                3            8           14
  Breast sarcoma                       0          100            0
  Uterine (endometrioid)
    carcinoma                          0            0            0
  Uterine (cervical) carcinoma         0            0           20
  Prostatic adenocarcinoma             0            0            4
  Testis tumors                        0            0            0
  Penile carcinoma (SCC)               0           50            0
Urinary tract tumors
  Kidney tumors                        0            0           10
  Bladder/urethral carcinoma
    (TCC)                             29           14           14
Endocrine tumors
  Adrenal carcinoma                    0            0            0
  Thyroid carcinoma (all)              0            8           39
Hematopoietic tumors
  Lymphomas (all)                      0            0            0
Skin tumors
  SCC                                  0            0            0
Skeletal system tumors
  Bone tumors (all)                    0            0            0
Soft tissue tumors
  Soft tissue sarcoma (all)            0            0            8
  Melanocytic tumors
    (including clear cell
    sarcoma)                           0            0            0
Brain tumors
  Brain tumors (all)                   0            0            0
All metastatic tumors
    (unknown primary)
  Adenocarcinoma (NOS)                 0            0            0
  Carcinoma (NOS)                      0            0            0
  SCC (NOS)                            0            0            0

Abbreviations: NOS, not otherwise specified; SCC, squamous cell
carcinoma; TCC, transitional cell carcinoma.

Table 2. Frequency and Subcellular Localization of RAAG12 in Human
Normal Tissue From Nonmalignant Individuals

                                                     Localization
% Positive
  Cells                Tissue of Origin              WM   AM   C

>50%         Bladder (transitional cell)             X         X
             Esophagus (striated squamous
               epithelium)                           X         X
             Tonsil (mucosa)                         X         X
             Breast (glandular/ductal)                    X    X
             Colon (mucosal)                              X    X
             Liver (biliary duct)                         X    X
             Pancreas (centroacinar and ductal
               epithelium)                                X    X
             Skin (sweat glands and ducts)                X    X
             Placenta (amniotic membrane)                      X
             Uterus (endometrium)                              X
>25%-50%     Eye (corneal)                           X         X
             Ureter (transitional cell)              X         X
             Uterus (cervical epithelium)            X         X
             Duodenum (mucosal)                           X    X
             Fallopian tube (mucosal)                     X    X
             Kidney (collecting ducts and distal
               tubules)                                   X    X
             Lung (bronchiolar epithelium)                X    X
             Stomach (mucosal)                            X    X
             Ovary (surface, follicular)                       X
             Prostate (acinar)                                 X
             Spinal cord (central canal)                       X
             Testis (germinal epithelium,
               epididymal epithelium)                          X
             Thymus (Hassall corpuscles and other
               stratified squamous epithelia)                  X
[less than   Embryonic duct remnants (Rathke
or equal       pouch, thymic/parathyroid duct)                 X
to] 25%      Parathyroid (chief cells)                         X
             Pituitary (adenohypophysis)                       X
Negative     Adrenal gland
             Blood leukocytes
             Blood vessels (endothelium)
             Bone marrow
             Brain (cerebellum and cerebrum)
             Connective tissue
             Heart
             Lymph node
             Pancreas (p islets)
             Peripheral nerve
             Skeletal muscle
             Skin (epidermis)
             Smooth muscle
             Spinal cord (nervous tissue)
             Spleen
             Thyroid

                                                       Frequency of
% Positive                                             Observation
  Cells                Tissue of Origin              (No./No. Donors)

>50%         Bladder (transitional cell)                   3/3
             Esophagus (striated squamous
               epithelium)                                 3/3
             Tonsil (mucosa)                               3/3
             Breast (glandular/ductal)                     4/4
             Colon (mucosal)                               4/4
             Liver (biliary duct)                          3/3
             Pancreas (centroacinar and ductal
               epithelium)                                 3/3
             Skin (sweat glands and ducts)                 3/3
             Placenta (amniotic membrane)                  1/3
             Uterus (endometrium)                          2/2
>25%-50%     Eye (corneal)                                 3/3
             Ureter (transitional cell)                    3/3
             Uterus (cervical epithelium)                  1/3
             Duodenum (mucosal)                            3/3
             Fallopian tube (mucosal)                      3/3
             Kidney (collecting ducts and distal
               tubules)                                    3/3
             Lung (bronchiolar epithelium)                 3/3
             Stomach (mucosal)                             3/3
             Ovary (surface, follicular)                   2/3
             Prostate (acinar)                             3/3
             Spinal cord (central canal)                   3/3
             Testis (germinal epithelium,
               epididymal epithelium)                    1/3, 2/3
             Thymus (Hassall corpuscles and other
               stratified squamous epithelia)              2/3
[less than   Embryonic duct remnants (Rathke
or equal       pouch, thymic/parathyroid duct)             3/3
to] 25%      Parathyroid (chief cells)                     2/3
             Pituitary (adenohypophysis)                   3/3
Negative     Adrenal gland                                 0/4
             Blood leukocytes                              0/3
             Blood vessels (endothelium)                   0/3
             Bone marrow                                   0/3
             Brain (cerebellum and cerebrum)               0/3
             Connective tissue                             0/3
             Heart                                         0/3
             Lymph node                                    0/3
             Pancreas (p islets)                           0/3
             Peripheral nerve                              0/3
             Skeletal muscle                               0/3
             Skin (epidermis)                              0/3
             Smooth muscle                                 0/3
             Spinal cord (nervous tissue)                  0/3
             Spleen                                        0/3
             Thyroid                                       0/3

Abbreviations: AM, apical membrane; C, cytoplasm; WM, whole membrane.
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Author:Coberly, Suzanne K.; Chen, Francine Z.; Armanini, Mark P.; Chen, Yan; Young, Peter F.; Mather, Jenni
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Clinical report
Geographic Code:1USA
Date:Sep 1, 2009
Words:5430
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