Development And Histopathological Characterization Of Tumorgraft Models Of Pancreatic Ductal Adenocarcinoma

Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer. Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types. Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing. Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models. We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation. Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained \u3c10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies. © 2013 Garcia et al

Pediatric Anaplastic Embryonal Rhabdomyosarcoma: Targeted Therapy Guided by Genetic Analysis and a Patient-Derived Xenograft Study

Therapy for rhabdomyosarcoma (RMS) has generally been limited to combinations of conventional cytotoxic agents similar to regimens originally developed in the late 1960s. Recently, identification of molecular alterations through next-generation sequencing of individual tumor specimens has facilitated the use of more targeted therapeutic approaches for various malignancies. Such targeted therapies have revolutionized treatment for some cancer types. However, malignancies common in children, thus far, have been less amenable to such targeted therapies. This report describes the clinical course of an 8-year-old female with embryonal RMS having anaplastic features. This patient experienced multiple relapses after receiving various established and experimental therapies. Genomic testing of this RMS subtype revealed mutations in BCOR, ARID1A, and SETD2 genes, each of which contributes to epigenetic regulation and interacts with or modifies the activity of histone deacetylases (HDAC). Based on these findings, the patient was treated with the HDAC inhibitor vorinostat as a single agent. The tumor responded transiently followed by subsequent disease progression. We also examined the efficacy of vorinostat in a patient-derived xenograft (PDX) model developed using tumor tissue obtained from the patient’s most recent tumor resection. The antitumor activity of vorinostat observed with the PDX model reflected clinical observations in that obvious areas of tumor necrosis were evident following exposure to vorinostat. Histologic sections of tumors harvested from PDX tumor-bearing mice treated with vorinostat demonstrated induction of necrosis by this agent. We propose that the evaluation of clinical efficacy in this type of preclinical model merits further evaluation to determine if PDX models predict tumor sensitivity to specific agents and/or combination therapies

Development and Histopathological Characterization of Tumorgraft Models of Pancreatic Ductal Adenocarcinoma

<div><p>Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer.  Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types.  Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing.  Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models.  We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation.  Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained <10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies.  </p> </div

Sister tumorgrafts originating from the same F0 or F1 tumor retained KRAS codon 12 mutational status.

<p>Electropherograms demonstrate that sister tumorgrafts (from mouse 1 [m1], mouse 2 [m2], mouse 3 [m3], and mouse 4 [m4] of each set) from all three models (UAB-PA2; UAB-PA4; UAB-PA10) conserved KRAS codon 12 status in F0, F1 and F2 tumors. Experimental details are reported in Materials and Methods. </p

Three generations of tumorgraft UAB-PA2 retain morphology similar to that of the primary tumor.

<p>UAB-PA2 tumor was serially passaged to the third generation (F3), and the morphology of H&E stained sections of tumors compared. The Results section contains details of histological analyses. </p

Cryopreservation of tumorgrafts does not alter the histology or growth kinetics in mice.

<p>(<b>a</b>) Photomicrographs of H&E stained sections of tumorgrafts produced from implantation of a primary tumor specimen within one hour of resection (UAB-PA2-F2)  compared to implantation of a specimen from the same tumor cryopreserved in liquid nitrogen for >30 days (UAB-PA2-F2-FV) show no histological differences. (<b>b</b>) Cryopreserved and fresh F0 tumor specimens produced tumorgrafts with similar growth kinetics in mice.  The apparent lag time of ~2 weeks prior to exponential growth of UAB-PA2-F2-FV tumorgrafts was not statistically significant (P = 0.0809). </p

Mutations in codon 12 of the KRAS gene of all twenty primary PDAC tumors (F0) were conserved in the F1 and F2 tumorgrafts derived from each tumor.

<p>Electropherograms for eight tumors are shown in this Figure; results for an additional six tumors are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078183#pone.0078183.s001" target="_blank">Figure S1</a>; results from all twenty F0, F1 and F2 tumors are summarized in Table 3. (<b>a</b>) The normal sequence of codon 12 from normal human pancreas DNA (wild type; WT) is GGT (encoding glycine [G]) as shown in the box. (<b>b</b>) Representative PCR results using a primer set that anneals to human, but not murine, KRAS sequences. UAB-PA3: NP (normal pancreas), F0 (primary tumor), and F1 and F2 tumorgrafts show readily detectable bands (214 base pairs). Mouse NP (normal pancreas) and negative control (cont [-]) lanes showed no bands. Experimental details are in Methods section. (<b>c</b>) Electropherograms show mutations in codon 12 of the KRAS gene in eight primary PDAC tumors (F0) and in the F1 and F2 tumorgrafts derived from each tumor. </p