Patient-derived xenografts effectively capture responses to oncology therapy in a heterogeneous cohort of patients with solid tumors.

BACKGROUND: While patient-derived xenografts (PDXs) offer a powerful modality for translational cancer research, a precise evaluation of how accurately patient responses correlate with matching PDXs in a large, heterogeneous population is needed for assessing the utility of this platform for preclinical drug-testing and personalized patient cancer treatment. PATIENTS AND METHODS: Tumors obtained from surgical or biopsy procedures from 237 cancer patients with a variety of solid tumors were implanted into immunodeficient mice and whole-exome sequencing was carried out. For 92 patients, responses to anticancer therapies were compared with that of their corresponding PDX models. RESULTS: We compared whole-exome sequencing of 237 PDX models with equivalent information in The Cancer Genome Atlas database, demonstrating that tumorgrafts faithfully conserve genetic patterns of the primary tumors. We next screened PDXs established for 92 patients with various solid cancers against the same 129 treatments that were administered clinically and correlated patient outcomes with the responses in corresponding models. Our analysis demonstrates that PDXs accurately replicate patients' clinical outcomes, even as patients undergo several additional cycles of therapy over time, indicating the capacity of these models to correctly guide an oncologist to treatments that are most likely to be of clinical benefit. CONCLUSIONS: Integration of PDX models as a preclinical platform for assessment of drug efficacy may allow a higher success-rate in critical end points of clinical benefit.

Patient-derived xenografts as tools in pharmaceutical development.

Successful drug development in oncology is grossly suboptimal, manifested by the very low percentage of new agents being developed that ultimately succeed in clinical approval. This poor success is in part due to the inability of standard cell-line xenograft models to accurately predict clinical success and to tailor chemotherapy specifically to a group of patients more likely to benefit from the therapy. Patient-derived xenografts (PDXs) maintain the histopathological architecture and molecular features of human tumors, and offer a potential solution to maximize drug development success and ultimately generate better outcomes for patients. Although imperfect in mimicking all aspects of human cancer, PDXs are a more predictable platform for preclinical evaluation of treatment effect and in selected cases can guide therapeutic decision making in the clinic. This article summarizes the current status of PDX models, challenges associated with modeling human cancer, and various approaches that have been applied to overcome these challenges and improve the clinical relevance of PDX cancer models.

DNA methylation profiles delineate epigenetic heterogeneity in seminoma and non-seminoma.

BACKGROUND: It remains important to understand the biology and identify biomarkers for less studied cancers like testicular cancer. The purpose of this study was to determine the methylation frequency of several cancer-related genes in different histological types of testicular cancer and normal testis tissues (NT). METHODS: DNA was isolated from 43 seminomas (SEs), 14 non-SEs (NSEs) and 23 NT, and was assayed for promoter methylation status of 15 genes by quantitative methylation-specific PCR. The methylation status was evaluated for an association with cancer, and between SEs and NSEs. RESULTS: We found differential methylation pattern in SEs and NSEs. MGMT, VGF, ER-ß and FKBP4 were predominately methylated in NSEs compared with SEs. APC and hMLH1 are shown to be significantly more methylated in both subtypes in comparison with NT. When combining APC, hMLH1, ER-ß and FKBP4, it is possible to identify 86% of the NSEs, whereas only 7% of the SEs. CONCLUSIONS: Our results indicate that the methylation profile of cancer-associated genes in testicular cancer correlates with histological types and show cancer-specific pattern for certain genes. Further methylation analysis, in a larger cohort is needed to elucidate their role in testicular cancer development and potential for therapy, early detection and disease monitoring.

Quantitative detection of promoter hypermethylation as a biomarker of acute kidney injury during transplantation.

Aberrant promoter hypermethylation, also known as epigenetics, is thought to be a promising biomarker approach to diagnose malignancies. Kidney repair after injury is a recapitulation of normal morphogenesis, with similarities to malignant transformation. We hypothesized that changes in urine epigenetics could be a biomarker approach during early kidney transplant injury and repair. We examined urine DNA for aberrant methylation of two gene promoters (DAPK and CALCA) by quantitative methylation-specific polymerase chain reaction from 13 deceased and 10 living donor kidney transplant recipients on postoperative day 2 and 65 healthy controls. Results were compared with clinical outcomes and to results of the kidney biopsy. Transplant recipients were significantly more likely to have aberrant hypermethylation of the CALCA gene promoter in urine than healthy controls (100% vs 31%; P < .0001). There was increased CALCA hypermethylation in the urine of deceased versus living donor transplants (21.60 +/- 12.5 vs 12.19 +/- 4.7; P = .04). Furthermore, there was a trend toward increased aberrant hypermethylation of urine CALCA in patients with biopsy-proven acute tubular necrosis versus acute rejection and slow or prompt graft function (mean: 20.40 +/- 6.9, 13.87 +/- 6.49, 17.17 +/- 13.4; P = .67). However, there was no difference of CALCA hypermethylation in urine of patients with delayed graft function versus those with slow or prompt graft function (16.9 +/- 6.2 vs 18.5 +/- 13.7, respectively; P = .5). There was no aberrant hypermethylation of DAPK in the urine of transplant patients. Urine epigenetics is a promising biomarker approach for acute ischemic injury in transplantation that merits future study.

Quantitative assessment of promoter methylation profiles in thyroid neoplasms.

CONTEXT: Cancer-specific molecular markers are needed to supplement the cytopathological assessment of thyroid tumors, because a majority of patients with cytologically indeterminate nodules currently undergo thyroidectomy without a definitive diagnosis. OBJECTIVE: The aim of this study was the quantitative assessment of promoter hypermethylation and its relation to the BRAF mutation in thyroid tumors. DESIGN: Quantitative hypermethylation of Rassf1A, TSHR, RAR-beta2, DAPK, S100, p16, CDH1, CALCA, TIMP3, TGF-beta, and GSTpi was tested on a cohort of 82 benign and malignant thyroid tumors and five thyroid cancer cell lines. SETTING: The study was conducted at a tertiary research hospital. PATIENTS: Patients underwent surgical resection for a thyroid tumor from 2000 to 2003 at our institution. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURE: Final surgical pathology diagnosis was the main outcome measure. RESULTS: Thyroid tumors showed hypermethylation for the following markers: Rassf1A, TSHR, RAR-beta2, DAPK, CDH1, TIMP3, and TGF-beta. A trend toward multiple hypermethylation was evident in cancer tissues, with hypermethylation of two or more markers detectable in 25% of hyperplasias, 38% of adenomas, 48% of thyroid cancers, and 100% of cell lines. A rank correlation analysis of marker hypermethylation suggests that a subset of these markers is epigenetically modified in concert, which may reflect an organ-specific regulation process. Furthermore, a positive correlation was found between the BRAF mutation and RAR-beta2, and a negative correlation was found between the BRAF mutation and Rassf1A. CONCLUSIONS: Methylation-induced gene silencing appears to affect multiple genes in thyroid tissue and increases with cancer progression. Additional markers with better discriminatory power between benign and malignant samples are needed for the diagnostic assessment of cytologically indeterminate thyroid nodules.

Dihydropyrimidine dehydrogenase mRNA level correlates with the response to 5-fluorouracil-based chemo-immuno-radiation therapy in human oral squamous cell cancer.

The measurement of the intra-tumoral level of thymidylate synthetase (TS), and dihydropyrimidine dehydrogenase (DPD), may be useful in predicting tumor sensitivity to 5-fluorouracil (5-FU). In this study, we examined the mRNA levels of DPD and TS in 28 oral squamous cell carcinomas (SCC), and 22 salivary gland tumors by semi-quantitative reverse transcription polymerase chain reaction. Then we examined the correlation of the responsiveness of the patients with oral SCC to 5-FU with the intra-tumoral levels of DPD and TS mRNA. All specimens were obtained at the biopsy before treatment, and then the patients were treated by oral administration of a 5-FU compound (UFT), the irradiation of cobalt-60 (upto 60 Gy) and injection of an immuno-potentiator (OK-432). Intra-tumoral levels of DPD mRNA in the patients who showed CR (complete response) and PR (partial response) were significantly lower than those in the patients who showed NC (no change). However, intra-tumoral levels of DPD mRNA did not correlate with the local recurrence of the tumor during the observation period after initial treatment with or without surgical resection of the residual tumors. On the other hand, TS mRNA levels in the tumors did not correlate with any clinico-pathological parameters. These observations suggest that intra-tumoral levels of DPD mRNA may predict the tumor response to 5-FU-based chemo-immuno-radiation therapy in the patients with oral SCC.

Role of HGF/c-met system in invasion and metastasis of oral squamous cell carcinoma cells in vitro and its clinical significance.

We examined the role of the hepatocyte growth factor (HGF)/c-met system on invasion and metastasis of oral squamous cell carcinoma (SCC) cells. In monolayer culture, exogenous HGF marginally affected the growth of oral SCC cells (BHY, HN, IH) and human gingival epithelial cells (GE). In type I collagen matrix, however, HGF significantly enhanced the invasive growth of the cancer cells (p < 0.05). We detected the expression of c-met (HGF receptor) mRNA in all of the cancer cells, but not in human gingival fibroblasts (GF). Oral SCC cells did not secret HGF protein into the medium, but GF secreted a large amount of HGF protein (15 ng/ml). Furthermore, HGF markedly enhanced the migration of cancer cells in a Transwell invasion chamber. Then, we examined the serum levels of HGF in oral SCC patients, or HGF concentrations in oral cancer tissues. Serum levels of HGF in the patients were significantly higher than those in healthy volunteers (p < 0.05). After initial treatment, all of the tumor-free survivors showed a marked decline in the serum HGF levels. Furthermore, HGF concentrations in metastatic cancer tissues were significantly higher than those of non-metastatic cancer tissues and normal gingiva (p < 0.01). These results suggest that HGF plays an important role in invasion and metastasis of oral SCC cells as a paracrine factor, and an elevated HGF level in the cancer tissue can be a predictive marker for metastasis formation in patients with oral SCC.

Low-dose retinoic acid enhances in vitro invasiveness of human oral squamous-cell-carcinoma cell lines.

Retinoids inhibit the proliferation of several types of tumour cells, and are used for patients with several malignant tumours. In this study, we examined the effect of retinoic acids (RAs) on the invasive potentials of the oral squamous cell carcinoma (SCC) cells, BHY and HNt. BHY cells expressed all of retinoid nuclear receptors (RARalpha, beta, gamma, and RXRalpha) and cytoplasmic retinoic acid binding proteins (CRABP1 and CRABP2). HNt cells lacked the expression of RARbeta, but expressed other nuclear receptors and CRABPs. All-trans retinoic acid (ATRA) and 13-cis retinoic acid (13-cisRA) (10(-6)and 10(-7)M) inhibited the growth of the cells, but low-dose ATRA and 13-cisRA (10(-8)M) marginally affected the growth of the cells. Surprisingly, low-dose RAs enhanced the activity of tissue-type plasminogen activator (tPA), and activated pro-matrix metalloproteinases (proMMP2 and proMMP9). Activation of proMMP2 and proMMP9 was inhibited by aprotinin, a serine-proteinase, tPA inhibitor. Furthermore, low-dose RAs enhanced the in vitro invasiveness of BHY cells. These results indicate that low-dose RAs enhances the in vitro invasiveness of oral SCC cells via an activation of proMMP2 and proMMP9 probably mediated by the induction of tPA.

Diabetes and tumor formation in transgenic mice expressing Reg I.

To examine the effect of overexpressed regenerating gene (Reg) I on pancreatic beta-cells, we generated transgenic mice expressing Reg I in islets (Reg-Tg mice). Three lines of Reg-Tg mice were established. In line-1 Reg-Tg mice, the expression level of Reg I mRNA in islets was 7 times higher than those in lines 2 and 3 of Reg-Tg mice, and line 1 mice developed diabetes by apoptosis of beta-cells, as well as various malignant tumors. In addition to the decrease in beta-cells, compensatory islet regeneration and proliferation of ductal epithelial cells were observed in line-1 Reg-Tg mice. Because Reg I protein was secreted primarily into pancreatic ducts from acinar cells, it may primarily stimulate the proliferation of ductal epithelial cells, and not beta-cells, and their differentiation into islets. Moreover, the tumor-promoting activity of Reg I protein should be considered for its possible clinical applications.

Significant correlation between matrix metalloproteinase activity and tumor necrosis factor-alpha in salivary extravasation mucoceles.

We examined the matrix metalloproteinase (MMP) activity and tumor necrosis factor (TNF)-alpha in luminal fluid of 18 extravasation mucoceles and in saliva from Wharton's duct of five patients by means of gelatin zymography and enzyme immunoassay, respectively. The luminal fluid showed a high level of MMP activity compared with the saliva. Quantitative determination by enzyme immunoassay revealed that the luminal fluid contained higher levels of TNF-alpha than the saliva. In addition, the amount of TNF-alpha in luminal fluid exhibited a direct correlation with MMP activity estimated by densitometric analysis of gelatin zymograms. Since TNF-alpha stimulates the production of MMPs from cells such as fibroblasts, these results suggest that TNF-alpha is one of the causal molecules that enhance the accumulation of proteolytic enzymes in luminal fluid of mucoceles.

Expression of integrin subunits in normal and malignant human salivary gland cell clones and its regulation by transforming growth factor-beta 1.

In this study, we have examined the expression of integrin subunits in normal and malignant human salivary gland cell clones as well as its regulation by transforming growth factor-beta 1 (TGF-beta 1). By the analysis using immunofluorescence staining, an SV40 immortalized normal human salivary gland duct cell clone (NS-SVDC) with no tumorigenic ability by s.c. implantation into nude mice was identified to express the integrin beta 1, alpha 2, alpha 3 and alpha 6 subunits on the cell surface, while the expression of these subunits, except for beta 1 subunit, was reduced or completely diminished in a neoplastic human salivary gland duct cell clone (HSGc) with tumorigenic but not metastatic potential in nude mice and metastatic cell clones derived after in vitro exposure of HSGc to N-methyl-N-nitrosourea. In addition, immunoblot analysis also exhibited the same results as those obtained with immunofluorescence staining. The alpha 1 subunit was not demonstrable in any of the cell clones by both techniques. TGF-beta 1 augmented the expression of the beta 1 subunit in NS-SV-DC, while HSGc and metastatic cell clones demonstrated no changes in the expression of the beta 1 subunit in response to TGF-beta 1. These findings, therefore, suggest that there is an inverse relationship between the malignancy and the expression mode of integrin subunits, especially alpha 2 subunit, in human salivary gland cell clones with varying degrees of malignant potential, and that TGF-beta 1 is a positive regulatory factor in the expression of the beta 1 subunit in normal but not malignant cell clones.

Increased matrix metalloproteinase-2 activity induced by TGF-beta 1 in duct cells of human salivary gland is associated with the development of cyst formation in vivo.

Proteolytic enzyme activity has been shown to be important for cyst formation. In this study, we constructed a cyst-like structure in vivo and analyzed molecular mechanisms involved in the development of the lesion. When SV40-immortalized duct cells of normal human salivary gland (NS-SV-DC) were treated with TGF-beta 1 at a concentration of 1 ng/ml or 5 ng/ml followed by co-inoculation with Matrigel into the backs of nude mice, they formed large cysts containing fluid when 5 ng/ml of TGF-beta 1 was used. Analysis of the fluid demonstrated high MMP activity. Immunohistochemical staining exhibited strong reactivity with anti-MMP-2 antibody in TGF-beta 1 (5 ng/ml)-treated NS-SV-DC. Northern blot analysis indicated that the expression of TGF-beta 1 and MMP-2 mRNAs in cells was greatly enhanced by treatment with 5 ng/ml TGF-beta 1. These findings suggest that the in vivo cyst formation by TGF-beta 1-treated cells is associated with continuous induction of MMP-2 activity.

Proteolytic enzymes in salivary extravasation mucoceles.

We examined the content of type IV collagenases and plasminogen activators (PAs) in luminal fluid of four extravasation mucoceles by means of gelatin or casein zymography. Immunohistochemical staining for type IV collagenases and PAs was performed to identify the possible source of these enzymes. The luminal fluid examined by gelatin zymography showed a high level of type IV collagenases compared with saliva from Wharton's duct. PAs were detected in only one of the cases by casein zymography, and none was detected in the saliva. By immunohistochemical staining, type IV collagenases and PAs were detected in duct and myoepithelial cells of the adjacent salivary gland and in macrophages and fibroblasts of the cyst wall. The results suggest that proteolytic enzymes are involved in the pathogenesis of mucoceles.

Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential.

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85-93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.