Does radiologic response correlate to pathologic response in patients undergoing neoadjuvant therapy for borderline resectable pancreatic malignancy?

BACKGROUND AND OBJECTIVES: In patients with borderline resectable pancreas cancers, clinicians frequently consider radiographic response as the primary driver of whether patients should be offered surgical intervention following neoadjuvant therapy (NT). We sought to determine any correlation between radiographic and pathologic response rates following NT. METHODS: Between 2005 and 2015, 38 patients at a tertiary care referral center underwent NT followed by pancreaticoduodenectomy for borderline resectable pancreas cancer. Radiographic response after the completion of NT and pathologic response after surgery were graded according to RECIST and Evans' criteria, respectively. RESULTS: Preoperatively, 50% of patients underwent chemotherapy alone and 50% underwent chemotherapy and chemoradiation. Radiographically, one patient demonstrated a complete radiologic response, 68.4% (n = 26) of patients had stable disease (SD), 26.3% (n = 10) demonstrated a partial response, and one patient had progressive. Among patients without radiographic response, 77.7% (n = 21) achieved a R0 resection. Of patients with SD on imaging, 26.9% (n = 7) had Evans grade IIB or greater pathologic response. CONCLUSIONS: Our data indicate that approximately one-fourth of patients who did not have a radiologic response had a grade IIB or greater pathologic response. In the absence of metastatic progression, lack of radiographic down-staging following NT should not preclude surgery.

Distant metastasis occurs late during the genetic evolution of pancreatic cancer.

Metastasis, the dissemination and growth of neoplastic cells in an organ distinct from that in which they originated, is the most common cause of death in cancer patients. This is particularly true for pancreatic cancers, where most patients are diagnosed with metastatic disease and few show a sustained response to chemotherapy or radiation therapy. Whether the dismal prognosis of patients with pancreatic cancer compared to patients with other types of cancer is a result of late diagnosis or early dissemination of disease to distant organs is not known. Here we rely on data generated by sequencing the genomes of seven pancreatic cancer metastases to evaluate the clonal relationships among primary and metastatic cancers. We find that clonal populations that give rise to distant metastases are represented within the primary carcinoma, but these clones are genetically evolved from the original parental, non-metastatic clone. Thus, genetic heterogeneity of metastases reflects that within the primary carcinoma. A quantitative analysis of the timing of the genetic evolution of pancreatic cancer was performed, indicating at least a decade between the occurrence of the initiating mutation and the birth of the parental, non-metastatic founder cell. At least five more years are required for the acquisition of metastatic ability and patients die an average of two years thereafter. These data provide novel insights into the genetic features underlying pancreatic cancer progression and define a broad time window of opportunity for early detection to prevent deaths from metastatic disease.

Patterns of EphA2 protein expression in primary and metastatic pancreatic carcinoma and correlation with genetic status.

EphA2 is a transmembrane receptor tyrosine kinase that functions in the regulation of cell growth, survival, angiogenesis, and migration and EphA2 targeting has been proposed as a novel therapeutic strategy for neoplasms that overexpress this protein. EphA2 overexpression has been correlated with increased invasive and metastatic ability in pancreatic cancer cell lines. However, the patterns of EphA2 expression in human pancreatic cancers and associated metastases is unknown, as are the genetics of EphA2 in this tumor type. We collected clinicopathologic data and paraffin-embedded materials from 98 patients with primary and/or metastatic pancreatic cancer and performed immunohistochemical labeling for EphA2 protein. EphA2 protein immunolabeling was found in 207 of 219 samples (95%). The expression was predominantly cytoplasmic, although predominant membranous staining was observed in a minority of cases. When evaluated specifically for labeling intensity, primary and metastatic carcinomas were more strongly positive compared to benign ducts and PanIN lesions (P < 0.00001 and P < 0.01, respectively) and poorly differentiated carcinomas were more strongly positive for EphA2 than well and moderately differentiated tumors (P < 0.005). When primary carcinomas without metastatic disease were specifically compared to carcinomas with associated metastatic disease, the advanced carcinomas showed relatively less strong positive labeling for EphA2 (P < 0.008). Moreover, decreased EphA2 labeling was more commonly found in liver (P < 0.002), lung (P < 0.004) or peritoneal metastases (P < 0.01) as compared to distant lymph node metastases (P < 0.01). Genetic sequencing of the tyrosine kinase domain of EPHA2 in 22 samples of xenograft enriched pancreatic cancer did not reveal any inactivating mutations. However, EPHA2 amplification was found in 1 of 33 pancreatic cancers corresponding to a lymph node metastasis, indicating EPHA2 genomic amplification may underlie EphA2 overexpression in a minority of patients. Our data confirms that EphA2 is overexpressed in pancreatic cancer, but suggests a relative loss of EphA2 in co-existent pancreatic cancer metastases as well as a role for EPHA2 in organ specific metastasis.

Keratinocyte growth conditions modulate telomerase expression, senescence, and immortalization by human papillomavirus type 16 E6 and E7 oncogenes.

Keratinocytes undergo a finite number of divisions in culture before senescing. The high-risk human papillomavirus (HPV) E6 and E7 oncoproteins prevent keratinocyte senescence and extend life span by interacting with p53 and pRb, respectively, and also by transcriptionally activating the human telomerase reverse transcriptase (hTERT) gene, which encodes the catalytic subunit of telomerase. We correlated telomerase activity, which was measured by a highly sensitive and quantitative real-time quantitative-PCR-based telomeric repeat amplification protocol assay, with telomere length and the expression of hTERT, p16(INK4a), and HPV-16 E6 and E7 in keratinocytes grown under two culture conditions. Primary human foreskin keratinocytes (HFKs) cultured in keratinocyte serum-free medium on plastic senesced at approximately 13 population doublings (PDs). Senescence was accompanied by a dramatic increase in p16(INK4A) levels, a marked decrease in telomerase, and only a slight decrease in telomere length. In contrast, HFKs grown in F medium on 3T3 fibroblast feeders maintained elevated telomerase and lower levels of p16(INK4A) for 60 PDs before senescing approximately 81 PDs. E7 was shown to act synergistically with E6 to super induce telomerase expression in a feeder environment-dependent manner. Culture of both HFKs and HFK/16E6E7 cells in the feeder environment significantly increased the number of doublings that these cells could undergo without a significant reduction in telomere length. Finally, transfer of either HFKs or HFK/16E6E7 cells from plastic to the feeder fibroblast culture system significantly induced telomerase activity. This induction in telomerase was fully reversible and largely attributable to the medium. Our results suggest that the influence of keratinocyte culture conditions on the expression of telomerase and p16(INK4A) and on telomere maintenance is responsible, at least partially, for the differences in proliferative capacity, senescence, and HPV-keratinocyte interactions seen in the two culture systems.

Clinicopathologic and genetic characterization of traditional serrated adenomas of the colon.

Traditional serrated adenomas (TSAs) are a type of colorectal polyp with neoplastic potential. Immunohistochemical analysis and sequencing were performed on 24 TSAs from 23 patients to characterize the molecular genetics of TSAs. Abnormal Ki-67 and p53 labeling were observed in 7 (29%) of 24 and 6 (25%) of 24 TSAs, respectively; both types were significantly associated with the presence of conventional epithelial dysplasia (P = .0005 and P = .0001, respectively). Activating KRAS mutation was identified in 11 TSAs (46%) and was mutually exclusive with activating BRAF mutations, which were seen in 7 TSAs (29%). Abnormal p53 nuclear labeling in a TSA was significantly associated with BRAF mutation status (P = .04), whereas no relationship was found for ß-catenin labeling patterns. The overall morphologic features of TSA do not correlate with the genetic status of the KRAS and BRAF genes. However, conventional epithelial dysplasia and abnormal p53 labeling in a TSA are seen more often in the setting of BRAF mutation.

GATA6 activates Wnt signaling in pancreatic cancer by negatively regulating the Wnt antagonist Dickkopf-1.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.

The developmental transcription factor Gata4 is overexpressed in pancreatic ductal adenocarcinoma.

GATA4 is a transcription factor that plays a role in regulating the normal development of many mesoderm and endoderm derived tissues, including the pancreas. Silencing of GATA4 mRNA expression by promoter methylation has been implicated in carcinogenesis of the ovary, lung and colorectum. By contrast, GATA4 mRNA expression is upregulated in pancreatic cancer cell lines and tissues. To further clarify the relationship of GATA4 to pancreatic cancer, we immunolabeled 90 samples of pancreatic ductal adenocarcinoma using a GATA4 specific monoclonal antibody. Both the intensity and percent of labeling was recorded for each carcinoma and correlated to the clinic opathologic features available for each patient. Samples of normal adult (n=26) and fetal pancreatic tissue (n=8) were also immunolabeled for comparison to expression patterns in pancreatic carcinoma tissues. Immunolabeling for GATA4 indicated robust nuclear expression in developing acini in fetal pancreatic tissues, consistent with the role of GATA4 in embryologic development, and in mature pancreatic acinar epithelium. Immunolabeling for GATA4 was also noted within normal duct epithelial cells, although it was always lesser in intensity than for acinar cell nuclei in the same section. Positive GATA4 immunolabeling was seen in 61/90 (68%) infiltrating pancreatic cancers of which 27/90 (30%) showed strong positive labeling. While there was no relationship among GATA4 and patient age, race or pathologic features, we did find a significant association among strong positive labeling and female gender (p=0.01). These findings support previous studies implicating GATA4 in pancreatic cancer and offer new avenues for investigation into this aggressive tumor type.

DPC4 gene status of the primary carcinoma correlates with patterns of failure in patients with pancreatic cancer.

PURPOSE: Contrary to the extensive data accumulated regarding pancreatic carcinogenesis, the clinical and molecular features characteristic of advanced stage (stage III and IV) disease are unknown. A comprehensive study of pancreatic cancers from patients who have succumbed to their disease has the potential to greatly expand our understanding of the most lethal stage of this disease and identify novel areas for intervention. MATERIALS AND METHODS: Rapid autopsies were performed on 76 patients with documented pancreatic cancer. The histologic features of end stage disease were determined and correlated to the stage at initial diagnosis, patterns of failure (locally destructive v metastatic disease) and the status of the KRAS2, TP53, and DPC4 genes. RESULTS: At autopsy, 30% of patients died with locally destructive pancreatic cancer, and 70% died with widespread metastatic disease. These divergent patterns of failure found at autopsy (locally destructive v metastatic) were unrelated to clinical stage at initial presentation, treatment history, or histopathologic features. However, Dpc4 immunolabeling status of carcinoma tissues harvested at autopsy, a sensitive marker of DPC4 genetic status, was highly correlated with the presence of widespread metastasis but not with locally destructive tumors (P = .007). CONCLUSION: Pancreatic cancers are represented by distinct genetic subtypes with significantly different patterns of failure. Determinations of DPC4 status at initial diagnosis may be of value in stratifying patients into treatment regimens related to local control versus systemic therapy.

Frequent genomic copy number gain and overexpression of GATA-6 in pancreatic carcinoma.

Multiple genetic alterations are well recognized as contributing to pancreatic carcinogenesis, although the finding of recurrent copy number changes indicates additional targets remain to be found. The objective of this study was to identify novel targets of genetic alteration that contribute to pancreatic cancer development or progression. We used Representational Oligonucleotide Microarray Analysis (ROMA) to identify copy number changes in pancreatic cancer xenografts, and validated these findings using FISH, quantitative PCR, Western blotting and immunohistochemical labeling. With this approach, we identified a 0.36-Mb amplification at 18q11.2 containing two known genes, GATA-6 and cTAGE1. Using a cutoff value of 3.0 fold compared to haploid controls, copy number gain or amplification was confirmed in 4 of 42 (9.5%) pancreatic carcinomas analyzed. Combined genetic and transcriptional analyses showed consistent overexpression of GATA-6 in all carcinomas with 18q11.2 gain, as well as in the majority of pancreatic cancers examined (17 of 30 cancers, 56.7%) that did not have gain of this region. By contrast, overexpression of cTAGE1 was rare in these same cancers suggesting GATA-6 is the true target of this copy number increase. GATA-6 mRNA overexpression corresponded to robust nuclear protein expression in cancer cell lines and resected tissues consistent with its role as a transcription factor. Intense nuclear labeling was significantly increased in PanIN-3 lesions and infiltrating carcinomas compared to normal duct epithelium (p < 0.000001 and p < 0.003, respectively). Forced overexpression of GATA6 in MiaPaca2 cells resulted in increased proliferation and growth in soft-agar. Gain and overexpression of the development-related transcription factor GATA-6 may play an important and hitherto unrecognized role in pancreatic carcinogenesis.

Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.

There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for approximately 10(6) single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.

Evaluation of GATA-4 and GATA-5 methylation profiles in human pancreatic cancers indicate promoter methylation patterns distinct from other human tumor types.

The GATA-4 and GATA-5 transcription factors are increasingly recognized as playing a role in carcinogenesis of human tumors derived of endodermal and mesodermal origin. The pancreas is derived from endodermal tissues suggesting GATA-4 and GATA-5 gene methylation may play a critical role in the biology of human pancreatic cancer as well. We investigated GATA-4 and -5 by methylation-specific PCR (MSP) in normal and neoplastic pancreatic tissues, including isogenic xenografts or cultured cell lines derived from the coexistent primary cancer and/or metastases in patients with pancreatic carcinoma. The relationship of promoter methylation was correlated with mRNA expression for each gene, and methylation patterns were correlated with known clinicopathologic features of patients. GATA-4 demonstrated a significantly lower methylation frequency than GATA-5 in low passage pancreatic cancer xenografts or cell lines (1/34 versus 21/34, p < 0.001). GATA-4 and -5 were also evaluated in microdissected samples of normal duct epithelium and cancer from pancreas cancer tissues which confirmed infrequent GATA-4 methylation in pancreatic cancers as well as in normal duct epithelium. GATA-4 was frequently overexpressed at the mRNA level with 27 of 30 (90%) pancreatic cancers showing >5.0-fold overexpression compared to normal duct epithelial cells. By contrast, high frequency methylation of GATA-5 was confirmed in pancreatic cancers tissues, but was rarely methylated in normal duct epithelium, indicating hypermethylation of this gene during pancreatic cancer development. GATA-5 mRNA expression did not correlate with its promoter hypermethylation, and treatment with the demethylating agent 5-aza-2'-deoxycytidine only partially restored mRNA expression suggesting additional regulatory mechanisms of GATA-5 expression. The presence of GATA-5 methylation showed a trend towards worse long-term survival (14.0 +/- 9.2 months versus 19.5 +/- 3.9 months, p = 0.06). While hypermethylation of GATA-5 seems to be a universal feature among human tumors, infrequent methylation of GATA-4, and its corresponding overexpression, appears unique to pancreatic cancer from other tumor types reported thus far.

Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation.

Despite recent emerging evidence suggesting that cancer stem cells subsist in a variety of tumors, it is not yet fully elucidated whether postnatal stem cells are directly involved in tumorigenesis. We used murine bone marrow-derived mesenchymal stem cells (BMMSCs) as a model to test a hypothesis that tumorigenesis may originate from spontaneous mutation of stem cells. In this study, we demonstrated that murine BMMSCs, after numerous passages, obtained unlimited population doublings and proceeded to a malignant transformation state, resulting in fibrosarcoma formation in vivo. Transformed BMMSCs colonized to multiple organs when delivered systemically through the tail vein. Fibrosarcoma cells formed by transformed BMMSCs contained cancer progenitors, which were capable of generating colony clusters in vitro and fibrosarcoma in vivo by the second administration. The mechanism by which BMMSCs transformed to malignant cells was associated with accumulated chromosomal abnormalities, gradual elevation in telomerase activity, and increased c-myc expression. Moreover, BMMSCs and their transformed counterpart, fibrosarcoma-forming cells, demonstrated different sensitivity to anti-cancer drugs. BMMSCs/fibrosarcoma transformation system may provide an ideal system to elucidate the mechanism of how stem cells become cancer cells and to screen anti-sarcoma drugs.

The E6AP ubiquitin ligase is required for transactivation of the hTERT promoter by the human papillomavirus E6 oncoprotein.

Most human cancer cells display increased telomerase activity that appears to be critical for continued cell proliferation and tumor formation. The E6 protein of malignancy-associated human papillomaviruses increases cellular telomerase in primary human keratinocytes at least partly via transcriptional activation of the telomerase catalytic subunit, hTERT. In the present study, we investigated whether E6AP, a ubiquitin ligase well known for binding and mediating some of the activities of the E6 oncoprotein, participated in the transactivation of the hTERT promoter. Our results demonstrate that E6 mutants that fail to bind E6AP are also defective for increasing telomerase activity and transactivating the hTERT promoter. More importantly, E6AP knock-out mouse cells and small interfering RNA techniques demonstrated that E6AP was required for hTERT promoter transactivation in both mouse and human cells. Neither E6 nor E6AP bound to the hTERT promoter or activated the promoter in the absence of the partner protein. With all transactivation-competent E6 proteins, induction of the hTERT promoter was dependent upon E box elements in the core promoter. It appears, therefore, that E6-mediated activation of the hTERT promoter requires a complex of E6-E6AP to engage the hTERT promoter and that activation is dependent upon Myc binding sites in the promoter. The recruitment of a cellular ubiquitin ligase to the hTERT promoter during E6-mediated transcriptional activation suggests a role for the local ubiquitination (and potential degradation) of promoter-associated regulatory proteins, including the Myc protein.