Jessica Hicks

Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia.

A multistep model of carcinogenesis has recently been proposed for pancreatic ductal adenocarcinomas. In this model, noninvasive precursor lesions in the pancreatic ductules accumulate genetic alterations in cancer-associated genes eventually leading to the development of an invasive cancer. The nomenclature for these precursor lesions has been standardized as pancreatic intraepithelial neoplasia or PanIN. Despite the substantial advances made in understanding the biology of invasive pancreatic adenocarcinomas, little is known about the initiating genetic events in the pancreatic ductal epithelium that facilitates its progression to cancer. Telomeres are distinctive structures at the ends of chromosomes that protect against chromosomal breakage-fusion-bridge cycles in dividing cells. Critically shortened telomeres can cause chromosomal instability, a sine qua non of most human epithelial cancers. Although evidence for telomeric dysfunction has been demonstrated in invasive pancreatic cancer, the onset of this phenomenon has not been elucidated in the context of noninvasive precursor lesions. We used a recently described in situ hybridization technique in archival samples (Meeker AK, Gage WR, Hicks JL, Simon I, Coffman JR, Platz EA, March GE, De Marzo AM: Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining. American Journal of Pathology 2002, 160:1259-1268) for assessment of telomere length in tissue microarrays containing a variety of noninvasive pancreatic ductal lesions. These included 82 PanIN lesions of all histological grades (24 PanIN-1A, 23 PanIN-1B, 24 PanIN-2, and 11 PanIN-3) that were selected from pancreatectomy specimens for either adenocarcinoma or chronic pancreatitis. Telomere fluorescence intensities in PanIN lesions were compared with adjacent normal pancreatic ductal epithelium and acini (62 of 82 lesions, 76%), or with stromal fibroblasts and islets of Langerhans (20 of 82 lesions, 24%). Telomere signals were strikingly reduced in 79 (96%) of 82 PanINs compared to adjacent normal structures. Notably, even PanIN-1A, the earliest putative precursor lesion, demonstrated a dramatic reduction of telomere fluorescence intensity in 21 (91%) of 23 foci examined. In chronic pancreatitis, reduction of telomere signal was observed in all PanIN lesions, whereas atrophic and inflammatory ductal lesions retained normal telomere length. Telomere fluorescence intensity in PanIN lesions did not correlate with proliferation measured by quantitative Ki-67-labeling index or topoisomerase IIalpha expression. Thus, telomere shortening is by far the most common early genetic abnormality recognized to date in the progression model of pancreatic adenocarcinomas. Telomeres may be an essential gatekeeper for maintaining chromosomal integrity, and thus, normal cellular physiology in pancreatic ductal epithelium. A critical shortening of telomere length in PanINs may predispose these noninvasive ductal lesions to accumulate progressive chromosomal abnormalities and to develop toward the stage of invasive carcinoma.

Xp11 Translocation renal cell carcinoma in adults: Expanded clinical, pathologic, and genetic spectrum

The recently recognized Xp11 translocation renal cell carcinomas (RCCs), all of which bear gene fusions involving the TFE3 transcription factor gene, comprise at least one-third of pediatric RCC. Only rare adult cases have been reported, without detailed pathologic analysis. We identified and analyzed 28 Xp11 translocation RCC in patients over the age of 20 years. All cases were confirmed by TFE3 immunohistochemistry, a sensitive and specific marker of neoplasms with TFE3 gene fusions, which can be applied to archival material. Three cases were also confirmed genetically. Patients ranged from ages 22 to 78 years, with a strong female predominance (F:M=22:6). These cancers tended to present at advanced stage; 14 of 28 presented at stage 4, whereas lymph nodes were involved by metastatic carcinoma in 11 of 13 cases in which they were resected. Previously not described and distinctive clinical presentations included dense tumor calcifications such that the tumor mimicked renal lithiasis, and obstruction of the renal pelvis promoting extensive obscuring xanthogranulomatous pyelonephritis. Previously unreported morphologic variants included tumor giant cells, fascicles of spindle cells, and a biphasic appearance that simulated the RCC characterized by a t(6;11)(p21;q12) chromosome translocation. One case harbored a novel variant translocation, t(X;3)(p11;q23). Five of 6 patients with 1 or more years of follow-up developed hematogenous metastases, with 2 dying within 1 year of diagnosis. Xp11 translocation RCC can occur in adults, and may be aggressive cancers that require morphologic distinction from clear cell and papillary RCC. Although they may be uncommon on a percentage basis, given the vast predominance of RCC in adults compared with children, adult Xp11 translocation RCC may well outnumber their pediatric counterparts.

Telomere Length Abnormalities Occur Early in the Initiation of Epithelial Carcinogenesis

Purpose: Telomeres help maintain chromosomal integrity. Dysfunctional telomeres can cause genetic instability in vitro and an increased cancer incidence in telomerase knock out mouse models. We recently reported that telomere shortening was a prevalent alteration in human prostate, pancreas, and breast cancer precursor lesions. In the present study, we address whether the previous findings are broadly applicable to human epithelial cancer precursors in general. Experimental Design: Surgical specimens of epithelial cancer precursor lesions from the urinary bladder, esophagus, large intestine, oral cavity, and uterine cervix were examined using a recently developed technique for direct in situ telomere length assessment in formalin-fixed human tissue specimens. Results: Widespread telomere length abnormalities were nearly universal (97.1% of cases) in the preinvasive stages of human epithelial carcinogenesis in all sites examined in this series, with telomere shortening the predominant abnormality (88.6% of cases). Conclusions: Telomere length abnormalities appear to be one of the earliest and most prevalent genetic alterations acquired in the multistep process of malignant transformation. These findings support a model whereby telomere dysfunction induces chromosomal instability as an initiating event in many, perhaps most, human epithelial cancers. Together with previous findings from the prostate and pancreas, the percentage of intraepithelial neoplasia lesions showing telomere length abnormalities is 95.6%. The implications of these findings include the potential that telomere length assessment in situ may be a widely useful biomarker for monitoring disease prevention strategies and for improved early diagnosis.

Tracking the clonal origin of lethal prostate cancer

Recent controversies surrounding prostate cancer overtreatment emphasize the critical need to delineate the molecular features associated with progression to lethal metastatic disease. Here, we have used whole-genome sequencing and molecular pathological analyses to characterize the lethal cell clone in a patient who died of prostate cancer. We tracked the evolution of the lethal cell clone from the primary cancer to metastases through samples collected during disease progression and at the time of death. Surprisingly, these analyses revealed that the lethal clone arose from a small, relatively low-grade cancer focus in the primary tumor, and not from the bulk, higher-grade primary cancer or from a lymph node metastasis resected at prostatectomy. Despite being limited to one case, these findings highlight the potential importance of developing and implementing molecular prognostic and predictive markers, such as alterations of tumor suppressor proteins PTEN or p53, to augment current pathological evaluation and delineate clonal heterogeneity. Furthermore, this case illustrates the potential need in precision medicine to longitudinally sample metastatic lesions to capture the evolving constellation of alterations during progression. Similar comprehensive studies of additional prostate cancer cases are warranted to understand the extent to which these issues may challenge prostate cancer clinical management.

Hypermethylation of the Human Glutathione S-Transferase-π Gene (GSTP1) CpG Island Is Present in a Subset of Proliferative Inflammatory Atrophy Lesions but Not in Normal or Hyperplastic Epithelium of the Prostate : A Detailed Study Using Laser-Capture Microdissection

Somatic inactivation of the glutathione S-transferase-pi gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in approximately 70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-alpha (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA lesions. These data support the hypothesis that atrophic epithelium in a subset of PIA lesions may lead to high-grade PIN and/or adenocarcinoma. Because these atrophic lesions are so prevalent and extensive, even though only a small subset contains this somatic DNA alteration, the clinical impact may be substantial.

Nuclear MYC Protein Overexpression is an Early Alteration in Human Prostate Carcinogenesis

The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.

PTEN Protein Loss by Immunostaining: Analytic Validation and Prognostic Indicator for a High Risk Surgical Cohort of Prostate Cancer Patients

Purpose: Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome FISH spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemistry (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease. Experimental Design: PTEN IHC was validated by employing formalin fixed and paraffin-embedded isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and 97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then assessed on 376 prostate tumor samples, and PTEN FISH or high resolution single nucleotide polymorphism microarray analysis was done on a subset of these cases. Results: PTEN protein loss, as assessed as a dichotomous IHC variable, was highly reproducible, correlated strongly with adverse pathologic features (e.g., Gleason score and pathologic stage), detected between 75% and 86% of cases with PTEN genomic loss, and was found at times in the absence of apparent genomic loss. In a cohort of 217 high risk surgically treated patients, PTEN protein loss was associated with decreased time to metastasis. Conclusion: These studies validate a simple method to interrogate PTEN status in clinical specimens and support the utility of this test in future multicenter studies, clinical trials, and ultimately perhaps for routine clinical care. Clin Cancer Res; 17(20); 6563–73. ©2011 AACR.

Small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well-differentiated pancreatic neuroendocrine tumors

Poorly differentiated neuroendocrine carcinomas (NECs) of the pancreas are rare malignant neoplasms with a poor prognosis. The aim of this study was to determine the clinicopathologic and genetic features of poorly differentiated NECs and compare them with other types of pancreatic neoplasms. We investigated alterations of KRAS, CDKN2A/p16, TP53, SMAD4/DPC4, DAXX, ATRX, PTEN, Bcl2, and RB1 by immunohistochemistry and/or targeted exomic sequencing in surgically resected specimens of 9 small cell NECs, 10 large cell NECs, and 11 well-differentiated neuroendocrine tumors (PanNETs) of the pancreas. Abnormal immunolabeling patterns of p53 and Rb were frequent (p53, 18 of 19, 95%; Rb, 14 of 19, 74%) in both small cell and large cell NECs, whereas Smad4/Dpc4, DAXX, and ATRX labeling was intact in virtually all of these same carcinomas. Abnormal immunolabeling of p53 and Rb proteins correlated with intragenic mutations in the TP53 and RB1 genes. In contrast, DAXX and ATRX labeling was lost in 45% of PanNETs, whereas p53 and Rb immunolabeling was intact in these same cases. Overexpression of Bcl-2 protein was observed in all 9 small cell NECs (100%) and in 5 of 10 (50%) large cell NECs compared with only 2 of 11 (18%) PanNETs. Bcl-2 overexpression was significantly correlated with higher mitotic rate and Ki67 labeling index in neoplasms in which it was present. Small cell NECs are genetically similar to large cell NECs, and these genetic changes are distinct from those reported in PanNETs. The finding of Bcl-2 overexpression in poorly differentiated NECs, particularly small cell NEC, suggests that Bcl-2 antagonists/inhibitors may be a viable treatment option for these patients.

DNA Hypomethylation Arises Later in Prostate Cancer Progression than CpG Island Hypermethylation and Contributes to Metastatic Tumor Heterogeneity

Hypomethylation of CpG dinucleotides in genomic DNA was one of the first somatic epigenetic alterations discovered in human cancers. DNA hypomethylation is postulated to occur very early in almost all human cancers, perhaps facilitating genetic instability and cancer initiation and progression. We therefore examined the nature, extent, and timing of DNA hypomethylation changes in human prostate cancer. Contrary to the prevailing view that global DNA hypomethylation changes occur extremely early in all human cancers, we show that reductions in (5me)C content in the genome occur very late in prostate cancer progression, appearing at a significant extent only at the stage of metastatic disease. Furthermore, we found that, whereas some LINE1 promoter hypomethylation does occur in primary prostate cancers compared with normal tissues, this LINE1 hypomethylation is significantly more pronounced in metastatic prostate cancer. Next, we carried out a tiered gene expression microarray and bisulfite genomic sequencing-based approach to identify genes that are silenced by CpG island methylation in normal prostate cells but become overexpressed in prostate cancer cells as a result of CpG island hypomethylation. Through this analysis, we show that a class of cancer testis antigen genes undergoes CpG island hypomethylation and overexpression in primary prostate cancers, but more so in metastatic prostate cancers. Finally, we show that DNA hypomethylation patterns are quite heterogeneous across different metastatic sites within the same patients. These findings provide evidence that DNA hypomethylation changes occur later in prostate carcinogenesis than the CpG island hypermethylation changes and occur heterogeneously during prostate cancer progression and metastatic dissemination.

Telomere Length Assessment in Human Archival Tissues : Combined Telomere Fluorescence in Situ Hybridization and Immunostaining

A method was developed to assess human telomere lengths at the individual cell level in tissue sections from standard formalin-fixed paraffin-embedded tissues. We coupled this method with immunofluorescence to allow the simultaneous identification of specific cell types. Validation of this in situ quantification method showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. The assay requires very few cells ( approximately 10 to 15). Thus, small tissue samples, including clinical biopsies, can be easily accommodated. In addition, the cells under study need not be actively cycling and there is no requirement for tissue disaggregation or cell culture. This method provides a more accurate assessment of telomere lengths than Southern blotting because confounding contributions from undesired cell types within tissue samples are avoided. Using this technique, we were able to perform the first comparison of relative telomere lengths in matched tumor versus normal epithelial cells within archival human prostate tissues.