Neoplasia, 2015;17(9):735-741.

Patient-derived xenograft tumors are susceptible to formation of human T- and B-cell lymphoid tumors.

 

Bondarenko G1, Ugolkov A1, Rohan S2, Kulesza P2, Dubrovskyi O1, Gursel D3, Mathews J3, O’Halloran TV4, Wei JJ2, Mazar AP5.

  • 1Center for Developmental Therapeutics, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 2170 Campus Drive, Evanston, IL, USA; Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL, USA.
  • 2Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, 60611, IL, USA; Pathology Core Facility, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 710 North Fairbanks Court, Chicago, IL, USA.
  • 3Pathology Core Facility, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 710 North Fairbanks Court, Chicago, IL, USA.
  • 4Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL, USA.
  • 5Center for Developmental Therapeutics, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 2170 Campus Drive, Evanston, IL, USA; Department of Pharmacology, Feinberg School of Medicine, Northwestern University, 320 East Superior Street, Chicago, 60611, IL, USA; Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL, USA. Electronic address: a-mazar@northwestern.edu.

 

Abstract

Patient-derived xenograft (PDX) tumor models provide clinicians, researchers and drug developers a platform to potentially personalize cancer treatment as well as evaluate the efficacy of new cancer drugs in development through the use of patient tumor grafts.  By testing patients’ individual tumor grafts, researchers can determine the most effective treatment for cancer patients.  In addition, repositories of these models can be used to evaluate new cancer drugs under development and recent data has shown that these models have high predictive value for clinical activity (1).  Our study emphasizes the potential for artifact when creating PDX models by demonstrating that lymphocytic PDX tumors can preferentially grow in immunodeficient NSG mice after initial engraftment of human tumor samples.  We found that a portion of PDX tumors that were originally generated from patients’ samples of breast, colon, pancreatic, bladder and renal cancer, were histologically similar to lymphocytic neoplasms.  We also discovered that the initial passage of breast and pancreatic cancer PDX tumors had the potential to propagate into a lymphocytic tumor in one mouse and an adenocarcinoma in another mouse, despite arising from the same human tumor sample.  Additionally, we found that subcutaneous PDX tumors that resembled human adenocarcinoma histology grew slowly and were non-metastatic, while subcutaneous PDX lymphocytic tumors proved to grow quickly, and formed sizable metastatic lesions in mouse lymph nodes, liver, lungs and spleen.  The lymphocytic tumors were composed of Epstein-Barr (EB) virus positive B-cells, which expressed CD45 and CD20.  As B-cells are often found in malignant  tumors including the patient tumor samples that we engrafted, there is a reasonable probability that lymphocytic tumors can evolve in a variety of PDX tumor models. Although the use of PDX tumor models in the development of individualized therapy for cancer patients is an exciting opportunity, quality control standards that rely on cautious screening of lymphocytic markers in PDX tumors should be applied during model generation.

PMID: 26476081

 

Background

The human tumor xenograft has been a mainstay in the study of human cancer for many decades (2, 3).  Recent development of PDX models, where the tumors are passaged only in mice through transplantation of a primary or metastatic human tumor into an immunodeficient, non-obese diabetic/severe combined immunodeficient (NOD/SCID) gamma (NSG) mouse, recapitulate human cancer more faithfully than tumor cell line xenografts and can be continuously propagated to test the effectiveness of various cancer therapies. These models have also become a valuable tool for oncology drug research and development (3). Despite these potentials for advancement, certain disadvantages remain when using mouse xenograft models.  Immunodeficient mice which lack a T-cell immune response are vulnerable to T-cell controlled infections, particularly viral infections, and to the development of lymphoproliferative lesions (4, 5).  Recently, several studies discovered that human tumors engrafted in NSG and NOG mice are prone to Epstein-Barr virus-associated (EBV-associated) lymphomagenesis (4, 5).  The Epstein–Barr virus (EBV) is prevalent in over 90% of humans, with the infection found in human B-cells, T-cells, NK-cells, and epithelial cells. Since B-cells are normally present in malignant solid tumors, development of EBV-associated B-cell PDX tumor may evolve in a wide range of PDX tumor models.

 

Key Findings

We used freshly resected tumors acquired from patients with breast, colon, pancreatic, bladder and renal cancer to create PDX tumor models with NSG mice via subcutaneous (SC) transplantation. A fraction of PDX tumors generated from tumor tissue of the breast (1 out 3 cases), colon (2 out of 7 cases), pancreas (1 out of 5 cases), bladder (1 out of 2 cases) and kidney (1 out of 2 cases) were histologically similar to lymphocytic neoplasm (Fig. 1). Of the PDX tumors established from these patient samples, 15 out of 22 (68%) recapitulated histopathological characteristics of original human carcinoma, whereas 7 out of 22 (32%) consisted of lymphocyte-like cells.  Additionally, some SC lymphocytic PDX tumors possessed rare inclusions of carcinoma cells. Histologically, we found an increase in a mixed population of mononuclear cells in lymphocytic PDX tumors.  These, ranged from small lymphocytes, or plasma cells, to large abnormal lymphoid cells with pleomorphic nuclei and abundant basophilic cytoplasm, which resembled lymphoblastoid cells.

 

We observed that SC lymphocytic PDX tumors grew rapidly and formed large metastatic lesions in mouse spleen, lymph nodes, liver and lungs. In contrast to lymphocytic PDX tumors, SC PDX tumors that resembled primary human carcinoma histology grew slowly and remained non-metastatic.  These lesions were found in mouse tissue of the spleen, lymph nodes, liver and lung. We differentiated between carcinoma and lymphocytic-like PDX tumors using immunohistochemical staining for detection of cytokeratin, CD45, CD20 and CD3 expression and by in-situ hybridization (ISH) for detection of the Epstein-Bar virus (EBER). As EBV does not infect rodent cells, EBER positive staining was employed as a marker for infected human cells in the mouse tissue. Immunohistochemical staining confirmed that both the human leukocyte common antigen CD45 and the B-cell antigen CD20 were expressed in EBER-positive lymphocytic cells in 6 out of 7 lymphocytic PDX tumors and in the corresponding metastatic lesions. This indicated that EBV-associated lymphomagenesis might lead to the development of human B-cell PDX tumors from xenografted human carcinomas due to the high prevalence of EBV in humans and the presence of EBV-infected B-cells in solid human tumors.

 

In one case, we observed that the initial passage of a breast PDX tumor (PCF #373342) developed as an EBER-positive lymphocytic neoplasm in one mouse, and as a poorly differentiated adenocarcinoma in another mouse. In the aforementioned scenario, IHC staining confirmed a B-cell origin of lymphocytic PDX tumor (PCF #373342) by showing lymphocytic cells as cytokeratin-negative, CD45-positive and CD20-positive. SC lymphocytic PDX tumor (PCF# 373342) growth led to the formation of metastasis in mouse lymph node, lung and liver, while the adenocarcinoma PDX (from this same patient’s pleural effusion) did not metastasize. Further retransplantations of lymphocytic SC PDX tumor (PCF #373342) and latter passages in NSG mice demonstrated comparable aggressive growth of lymphocytic PDX tumor and the development of distant metastasis.

 

In a second case, initial subcutaneous transplantation of a human primary pancreatic tumor (PCF #379419) into NSG mice resulted in formation of an EBER-positive B-cell PDX tumor in one mouse and EBER-negative T-cell PDX tumor in another mouse. The T-cell origin of the lymphocytic PDX tumor (PCF #379419) was determined by IHC staining which demonstrated that lymphoid cells were cytokeratin-negative, CD45-positive, CD20-negative, and CD3-positive. T- and B-cell PDX tumors (PCF #379419) displayed distant metastases in the following mouse tissues: lung (T-cell), liver (B-cell) and spleen (B-cell). Due to the minor inclusions of pancreatic carcinoma cells in the B-cell SC PDX tumor (PCF #379419), further SC re-transplantation of this tumor in nude mice led to the ultimate growth of a pancreatic PDX tumor, which resembled a moderately differentiated adenocarcinoma. Therefore, re-transplantation of SC B-cell PDX tumors from NSG to nude mice could be a useful approach to clear SC PDX tumor from the EBV-positive B-cells, subsequently leading to the establishment of a carcinoma. Moreover, we determined that lymphocytic PDX tumors did not propagate in nude mice after the re-transplantation from NSG mice. In nude mice, we saw that PDX tumors could develop only as carcinomas. This phenomenon might be explained by the fact that nude mice maintain NK cells (that partake in the immune response to EBV) which are not present in highly immunodeficient NSG mice.

 

Conclusions

Our results suggest that human tumors engrafted in NSG mice are vulnerable to the development of lymphocytic PDX tumors. The potential development of lymphocytic PDX tumors instead of an expected carcinoma may significantly affect or mislead the efforts of clinicians to evaluate the effects of cancer drugs on a particular patient’s tumor grafts, leading to the selection of inappropriate clinical treatments for that patient. These results demonstrate the fast growing nature of SC lymphocytic PDX tumors which form large metastatic lesions in the spleen, lymph nodes, liver and lung of NSG mice.  In comparison to SC lymphocytic PDX tumors, we determined that SC PDX tumors that resembled primary human carcinoma histology remained slow growing and non-metastatic. Metastatic lesions found in the lymph nodes, spleen, lung or liver may serve as potential markers of growing lymphocytic, but not carcinoma PDX tumors. Our results also demonstrate that transplantation of human or PDX tumor to nude mice can clear EBV-positive B-cells in xenograft tumor resulting in the re-emergence of a carcinoma. The results of our study emphasize the necessity of thorough testing for lymphocytic markers to confirm that the appropriate (non-lymphocytic) PDX tumor models are used in the development of individualized therapy for cancer patients.

 

References

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