Gary A. Ulaner

Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium

Telomerase extends telomeric repeats at the ends of linear chromosomes, thereby prolonging the replicative capacity of cells. To investigate possible regulatory mechanisms of telomerase, we measured telomerase enzyme activity, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in normal and neoplastic ovary, endometrium and myometrium. Telomerase activity was detected in most malignancies and in normal endometrium but not in myometrial leiomyoma, normal myometrium or normal ovary. hTR was expressed in all tissue samples. hTERT mRNA was expressed in many tissue samples, and no tissue sample exhibited telomerase activity without expressing hTERT mRNA. However, the presence of hTR and hTERT mRNA was not sufficient for telomerase activity. Alternate splicing of hTERT produced mRNAs lacking critical reverse transcriptase (RT) motifs in both normal and neoplastic tissues. Only tissues expressing hTERT containing complete A and B RT motifs demonstrated telomerase activity. Finally, several normal ovarian tissues and myometrial leiomyomas lacked telomerase activity despite expressing hTR and hTERT containing complete A and B RT motifs. This was not seen in ovarian and myometrial malignancies, where the expression of hTR and hTERT containing complete A and B RT motifs was sufficient for telomerase activity. We conclude that in ovarian and uterine tissues, the presence of a functional telomerase complex is regulated at multiple levels, including hTERT transcription and alternative splicing of hTERT transcripts. The lack of telomerase activity in several normal but not malignant tissues expressing hTR and hTERT containing complete A and B RT motifs suggests that there are further mechanisms for suppressing telomerase activity downstream of hTERT transcription and mRNA splicing, and these mechanisms have been lost during neoplastic transformation. Int. J. Cancer 85:330–335, 2000.©2000 Wiley‐Liss, Inc.

Tissue-specific alternate splicing of human telomerase reverse transcriptase (hTERT) influences telomere lengths during human development

Direct genetic manipulations have shown that telomerase‐mediated telomere elongation plays a key role in determining cellular replicative capacity and senescence. The mechanisms regulating the production of an active telomerase enzyme are still predominantly unknown, although roles for transcriptional control of hTERT, alternative‐splicing of hTERT transcripts, and post‐translational phosphorylation of hTERT protein have been advocated. Here we show that hTERT is alternatively spliced in specific patterns by different tissue types during human development. Alternative splicing often prohibits the expression of hTERT protein containing functional reverse transcriptase domains. In these instances, telomerase activity is absent, leading to shortening of telomeres. The specific pattern of hTERT mRNA variants in human development provides evidence that alternative splicing is non‐random and participates in the regulation of telomerase activity.

Divergent patterns of telomere maintenance mechanisms among human sarcomas: Sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas

Two types of telomere maintenance mechanisms (TMMs) have been described in human tumors: telomerase activation and alternative lengthening of telomeres (ALT). Although the vast majority of epithelial tumors rely on telomerase activation, many mesenchymal tumors rely on ALT for telomere maintenance, but within this tumor group, the TMMs used by translocation‐associated sarcomas have not been systematically studied. We studied telomere lengths and telomerase expression and activity in 30 uncultured tumor samples and in 10 cell lines of Ewings sarcoma, a prototypical translocation‐associated sarcoma, and compared the data to an identical analysis of 60 osteosarcomas, the most common type of sarcoma lacking a specific translocation. Telomerase activity was demonstrated in 21 Ewings sarcoma tumor samples (70%) and in 9 of 10 Ewings sarcoma cell lines. Evidence of ALT, indicated by the presence of long and heterogeneous telomeres, was observed only in the cell line without telomerase activity and in none of the 30 Ewings sarcoma tumor samples. The 9 Ewings sarcoma patients whose tumors lacked detectable telomerase activity did not differ significantly from the remaining patients in age, stage, EWSR1–FLI1 fusion type, prevalence of TP53 point mutations, or overall survival. The low prevalence of ALT in Ewings sarcoma contrasted sharply with our data on TMMs in 60 osteosarcomas, which showed ALT in 38 of 60 cases (P < 0.0001). The present study, together with emerging published data on other sarcoma types, suggests that a predominance of telomerase activation in the absence of ALT may characterize sarcomas with specific chromosomal translocations (such as Ewings sarcoma), whereas a high prevalence of ALT appears typical of sarcomas with nonspecific complex karyotypes (such as osteosarcoma).

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites.

The mouse insulin‐like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11‐13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent‐specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males × C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF‐associated factors that may be needed to coordinate Igf2 imprinting.

Is Methylene Diphosphonate Bone Scan Necessary for Initial Staging of Ewing Sarcoma if 18F-FDG PET/CT Is Performed?

OBJECTIVE The purpose of this study was to determine whether methylene diphosphonate (MDP) bone scans are necessary during initial staging in patients with Ewing sarcoma (ES) in whom (18)F-FDG PET/CT is performed. MATERIALS AND METHODS A retrospective review was performed of patients who underwent FDG PET/CT and MDP bone scan before treatment of newly diagnosed ES from January 2004 to November 2012. Studies were reviewed to document suspected primary and metastatic malignancy. Pathology and imaging follow-up were used to determine the presence or absence of disease at suspected sites. RESULTS Sixty patients were identified in whom FDG PET/CT and MDP bone scans were performed before treatment of newly diagnosed ES. Forty-four primary malignancies had a lytic CT appearance, three were sclerotic, and 13 involved only soft tissue. In 11 of 12 patients with osseous metastases, these were detected on PET/CT, with the one false-negative occurring in a sclerotic primary tumor; in nine of 12 patients with osseous metastases, these were detected on MDP bone scan, with the three false-negatives occurring in patients with lytic primary tumors. Only one of 13 patients with a soft-tissue primary malignancy had bone metastases on both bone scan and PET/CT. PET/CT also showed that eight patients had lung metastases and three patients had lymph node metastases, which were not evident on MDP bone scan. CONCLUSION When ES is lytic, MDP bone scan does not add to staging performed by FDG PET/CT; thus, MDP bone scanning may be omitted. However, when ES is sclerotic, MDP bone scan may detect osseous metastases not detected by FDG PET/CT.

Detection of Internal Mammary Adenopathy in Patients With Breast Cancer by PET/CT and MRI

OBJECTIVE The purpose of this study was to assess the prevalence of internal mammary node (IMN) adenopathy in patients with breast cancer and compare breast MRI and PET/CT for detection of IMN adenopathy. MATERIALS AND METHODS This retrospective study included 90 women who underwent MRI and PET/CT before neoadjuvant chemotherapy for clinical stage IIA through IIIA disease. MRI and PET/CT examinations were read independently by two readers trained in breast imaging and nuclear medicine. All patients underwent follow-up MRI at the end of chemotherapy, and 10 with hypermetabolic IMNs underwent follow-up PET/CT. Histology was not obtained. Women were considered to have IMN adenopathy when nodes seen on MRI or having standardized uptake value (SUV) greater than mediastinal blood pool decreased in either size or SUV (or both) after treatment. Features including lymphovascular invasion, tumor quadrant(s), and axillary adenopathy were compared between presence and absence of IMN adenopathy using Fishers exact test. Prevalence was determined on the basis of the percentage of patients with IMN adenopathy by either modality. The McNemar test compared the prevalence of IMN adenopathy on MRI to its prevalence on PET/CT. RESULTS Prevalence of IMN adenopathy was 16% (14/90) by MRI and 14% (13/90) by PET/CT (p = 0.317). After chemotherapy, IMN adenopathy resolved in 12 of 14 patients (86%). In two patients with poor responses in primary tumors, IMN adenopathy persisted, and both patients developed metastatic disease within 6 months. At 3 years, survival was significantly worse in patients with IMN adenopathy than in those without (85.7% vs 53.3%, respectively; p = 0.009). CONCLUSION In women with advanced breast cancer receiving neoadjuvant chemo-therapy, prevalence of IMN adenopathy was 16%, equally detected by breast MRI and PET/CT. Identification of IMN adenopathy may affect treatment and provides prognostic information.

Value of second-opinion review of outside institution PET-CT examinations

Objective This study aimed to determine whether second-opinion reviews of PET/CT examinations by subspecialists alter reporting of malignant findings. Materials and methods This IRB-approved study compared 240 fluorine-18 fluorodeoxyglucose PET/CT consecutively dictated reports by two nuclear medicine subspecialists against the original outside institution reports. Subspecialist reviews documented whether malignant findings on the outside report were malignant and noted additional malignant findings not described on the outside report. The final diagnosis of malignancy or benignity was determined by pathology when available, otherwise by imaging follow-up. Results A total of 22 findings (in 20 reports) called suspicious/malignant on the outside reports were deemed benign by subspecialist review. A final diagnosis was available for 20 of 22 findings by pathology (n=3) or follow-up imaging (n=17). The subspecialist review was accurate in 20 (100%) of 20 cases where a final diagnosis was available. The subspecialist review called 11 findings (in 11 reports) suspicious/malignant that were not described or deemed benign on the outside reports. Definitive diagnosis was available for 10 of 11 findings by pathology (n=7) or follow-up imaging (n=3). The second-opinion report was accurate in seven (70%) of 10 cases where a final diagnosis was available. Conclusion In 31 (13%) of 240 fluorine-18 fluorodeoxyglucose PET/CT examinations performed at an outside institution, subspecialist review resulted in at least one discordant opinion of malignancy. For 28 discrepant cases where a final diagnosis was available, the subspecialist review defined malignancy or benignity correctly in 25 (89%) of 28 cases. This provides evidence for the cost and effort invested in performing second-opinion reviews of PET/CT studies.

89Zr-Trastuzumab PET/CT for Detection of Human Epidermal Growth Factor Receptor 2–Positive Metastases in Patients With Human Epidermal Growth Factor Receptor 2–Negative Primary Breast Cancer

Purpose The aim of this study was to determine if imaging with 89Zr-trastuzumab, a human epidermal growth factor receptor 2 (HER2)–targeting PET tracer, can detect HER2-positive metastases in patients with HER2-negative primary breast cancer. Methods As part of an institutional review board–approved, prospective clinical trial of 89Zr-trastuzumab PET/CT (ClinicalTrials.gov identifier NCT02286843), a second group of 11 patients with HER2-negative primary breast cancer and known metastatic disease were recruited. Patients with confirmed HER2-negative primary breast cancer underwent 89Zr-trastuzumab PET/CT to screen for 89Zr-trastuzumab–avid lesions suggestive of unsuspected HER2-positive metastases. 89Zr-trastuzumab–avid lesions on PET/CT were biopsied and pathologically examined to determine HER2 status. Results All 11 patients had confirmed HER2-negative primary breast cancer. Four patients demonstrated suspicious foci on 89Zr-trastuzumab PET/CT. Of these 4 patients, 1 patient had biopsy-proven HER2-positive metastases. The other 3 patients with suspicious 89Zr-trastuzumab–avid foci had biopsy demonstrating a metastasis that was HER2-negative and were considered false-positive 89Zr-trastuzumab PET foci. Combined with a published report of the first 9 patients, there have been a total of 20 HER2-negative primary breast cancer patients, with 3 patients (15%) having pathologically confirmed HER2-positive distant metastases and 6 (30%) with suspicious 89Zr-trastuzumab–avid foci that were HER2-negative on pathology, which were thus considered false-positive 89Zr-trastuzumab findings. Conclusions This second group of patients confirms the proof of concept that 89Zr-trastuzumab PET/CT detects unsuspected HER2-positive metastases in a subset of patients with HER2-negtive primary breast cancer. False-positive 89Zr-trastuzumab–avid foci present a challenge to using this tracer.

Unilateral Suppression of Brown Fat on FDG PET/CT in Horner Syndrome.

A 29-year-old woman underwent resection of a left anterior mediastinal thymoma and pleurectomy. Postsurgical FDG PET/CT scan demonstrated FDG avidity in the right neck and upper thoracic fat but relatively absent FDG-avid fat in the left neck and upper thorax. Bilateral FDG-avid fat was also apparent in the lower chest and upper abdomen. After surgery, the patient demonstrated Horner syndrome, with left-sided ptosis, miosis, and facial anhidrosis. It is hypothesized that left-sided sympathetic nerves were compromised during surgery, leading to Horner syndrome and denervation of ipsilateral brown fat. The unilateral FDG avidity should not be mistaken for malignancy.

Fdg Pet/ct Assesses the Risk of Femoral Pathological Fractures in Patients With Metastatic Breast Cancer

Purpose Assessment of pathological fracture risk is critical to optimize the use of prophylactic orthopedic fixation to prevent pathological fractures. Better prediction of pathological fracture risk is needed. We evaluated if quantitative measures of FDG avidity can assess femoral pathological fracture risk in patients with metastatic breast cancer (MBC). Patients and Methods A Health Insurance Portability and Accountability Act–compliant retrospective case-control study was performed under institutional review board waiver. Patients with MBC who received an FDG PET/CT from January 2008 to December 2014 and had pathological fracture of the proximal femur within 3 months of PET/CT were selected as cases. Patients with MBC who had an FDG PET/CT in 2013 were sequentially screened in reverse chronological order to identify patients with proximal femoral metastases on PET/CT but no subsequent pathological fracture to serve as a control group. The prespecified goal was to have twice the number of controls as cases. Target lesions in the proximal femur, from femoral head to 5 cm below the lesser trochanter, were analyzed on FDG PET/CT for SUVmax, SUVmean, metabolic tumor volume, and total lesion glycolysis. Wilcoxon rank sum test was used to compare continuous variables in cases and controls. A nonparametric receiver operating characteristic analysis was performed to assess the ability of quantitative FDG measurements to differentiate between cases and controls. Results In 27 cases with pathological fracture and 55 controls without pathological fracture, all 4 quantitative measures of FDG avidity were statistically different between cases and controls (P < 0.001). A total lesion glycolysis of 81 could differentiate between fracture and nonfracture patients with accuracy, sensitivity, and specificity of 0.83, 0.85, and 0.80, respectively. Conclusions Quantitative measures of FDG avidity may help identify breast cancer patients at high risk of subsequent pathological fracture of the proximal femur.