Mutations in cancer-relevant genes are ubiquitous in histologically normal endometrial tissue.

OBJECTIVE: Endometrial cancer (EndoCA) is the most common gynecologic cancer and incidence and mortality rate continue to increase. Despite well-characterized knowledge of EndoCA-defining mutations, no effective diagnostic or screening tests exist. To lay the foundation for testing development, our study focused on defining the prevalence of somatic mutations present in non-cancerous uterine tissue. METHODS: We obtained ≥8 uterine samplings, including separate endometrial and myometrial layers, from each of 22 women undergoing hysterectomy for non-cancer conditions. We ultra-deep sequenced (>2000× coverage) samples using a 125 cancer-relevant gene panel. RESULTS: All women harbored complex mutation patterns. In total, 308 somatic mutations were identified with mutant allele frequencies ranging up to 96.0%. These encompassed 56 unique mutations from 24 genes. The majority of samples possessed predicted functional cancer mutations but curiously no growth advantage over non-functional mutations was detected. Functional mutations were enriched with increasing patient age (p = 0.045) and BMI (p = 0.0007) and in endometrial versus myometrial layers (68% vs 39%, p = 0.0002). Finally, while the somatic mutation landscape shared similar mutation prevalence in key TCGA-defined EndoCA genes, notably PIK3CA, significant differences were identified, including NOTCH1 (77% vs 10%), PTEN (9% vs 61%), TP53 (0% vs 37%) and CTNNB1 (0% vs 26%). CONCLUSIONS: An important caveat for future liquid biopsy/DNA-based cancer diagnostics is the repertoire of shared and distinct mutation profiles between histologically unremarkable and EndoCA tissues. The lack of selection pressure between functional and non-functional mutations in histologically unremarkable uterine tissue may offer a glimpse into an unrecognized EndoCA protective mechanism.

Deep molecular tracking over the 12-yr development of endometrial cancer from hyperplasia in a single patient.

Although the progressive histologic steps leading to endometrial cancer (EndoCA), the most common female reproductive tract malignancy, from endometrial hyperplasia are well-established, the molecular changes accompanying this malignant transformation in a single patient have never been described. We had the unique opportunity to investigate the paired histologic and molecular features associated with the 12-yr development of EndoCA in a postmenopausal female who could not undergo hysterectomy and instead underwent progesterone treatment. Using a specially designed 58-gene next-generation sequencing panel, we analyzed a total of 10 sequential biopsy samples collected over this time frame. A total of eight pathogenic/likely pathogenic mutations in seven genes, APC, ARID1A, CTNNB1, CDKN2A, KRAS, PTEN, and TP53, were identified. A PTEN nonsense mutation p.W111* was present in all samples analyzed except histologically normal endometrium. Apart from this PTEN mutation, the only other recurrent mutation was KRAS G12D, which was present in six biopsy samplings, including histologically normal tissue obtained at the patient's first visit but not detectable in the cancer. The PTEN p.W111* mutant allele fractions were lowest in benign, inactive endometrial glands (0.7%), highest in adenocarcinoma (36.9%), and, notably, were always markedly reduced following progesterone treatment. To our knowledge, this report provides the first molecular characterization of EndoCA development in a single patient. A single PTEN mutation was present throughout the 12 years of cancer development. Importantly, and with potential significance toward medical and nonsurgical management of EndoCA, progesterone treatments were consistently noted to markedly decrease PTEN mutant allele fractions to precancerous levels.

A Community-Based Pancreatic Cancer Screening Study in High-Risk Individuals: Preliminary Efficacy and Safety Results.

INTRODUCTION: Pancreatic cancer (PC) screening recommendations have been based on studies performed solely at high-volume academic centers. To make PC screening more widely available, community-based efforts are essential. We implemented a prospective PC screening study in the community of Fairfield County, CT, and report our early safety and efficacy results. METHODS: Eligible individuals were enrolled into an investigator-initiated study and underwent a baseline and 3 annual magnetic resonance imagings/magnetic resonance cholangiopancreatographies (MRIs/MRCPs) with gadolinium, biannual blood donations for biobanking, and assessments for anxiety and depression. All MRIs were presented at a multidisciplinary board to determine whether further investigation was warranted. RESULTS: Seventy-five individuals have been enrolled and 201 MRIs performed over a 2.6-year average length of follow-up. Abnormal pancreatic findings (predominantly small cysts) were detected in 58.7% of the participants. Among these, 6.7% underwent endoscopic ultrasound, with 1 case complicated by postprocedural pancreatitis. One surgical resection was performed on a 4.7-cm intraductal papillary mucinous neoplasm with a focus on low-grade pancreatic intraepithelial neoplasia. One incidental finding of fibrosing mediastinitis was detected. Anxiety and depression scores decreased over the course of this study from 21.4% to 5.4% and 10.7% to 3.6%, respectively. DISCUSSION: This preliminary report supports the feasibility of performing MRI/magnetic resonance cholangiopancreatographies-based PC screening as part of a clinical trial in a community setting. A longer follow-up is needed to better assess safety and efficacy. To the best of our knowledge, this is the first report from a community-based PC screening effort ( clinicaltrials.gov ID: NCT03250078).

Transformed Waldenström Macroglobulinemia Responsive to Tafasitamab Plus Lenalidomide: A Case Report.

The histologic transformation (HT) of Waldenström macroglobulinemia (WM) into diffuse large-cell lymphoma is an uncommon but poor-prognostic event for which there is no standard therapy. Knowledge of this entity is mainly derived from largely retrospective studies, which report abysmal average survival rates even with the utilization of first-line chemoimmunotherapy and especially in patients who meet the high-risk criteria based on prognostic indices used for WM. We present the case of a 75-year-old man with high-risk, transformed WM who was ineligible for standard chemoimmunotherapy (due to pancytopenia and multiple comorbidities) and was consequently treated with tafasitabmab, an anti-CD19 monoclonal antibody plus lenalidomide. Tafasitamab plus lenalidomide (TAF/LEN) is a recently approved therapy for relapsed or refractory de novo diffuse large-cell lymphoma (DLCL) but has not been previously studied in transformed low-grade lymphomas or WM. We show that TAF/LEN resulted in a complete and durable response of the DLCL by PET/CT and a complete bone marrow response of lymphoplasmacytoid cells, including the normalization of complex cytogenetic abnormalities. The extraordinary response of our patient to TAF/LEN suggests that this combination may be an effective and tolerable therapy for transformed WM as well as relapsed or refractory non-transformed WM. Clinical trials of TAF/LN for the treatment of Waldenström macroglobulinemia are recommended.

Correction: Transformed Waldenström Macroglobulinemia Responsive to Tafasitamab Plus Lenalidomide: A Case Report.

[This corrects the article DOI: 10.7759/cureus.32403.].

Glycemic Control as an Early Prognostic Marker in Advanced Pancreatic Cancer.

PURPOSE: Impaired glucose metabolism is present in most patients with pancreatic ductal adenocarcinoma (PDAC). Whereas previous studies have focused on pre-treatment glycemic indices and prognosis in those with concomitant diabetes, the effects of glycemic control during chemotherapy treatment on prognosis, in patients with and without diabetes, have not been well characterized. We examined the relationship between early glycemic control and overall survival (OS) in a cohort of patients with advanced PDAC treated in a community setting. PATIENTS AND METHODS: Seventy-three patients with advanced PDAC (38% with diabetes) receiving chemotherapy while participating in a biobanking clinical trial were included. Clinical characteristics and laboratory results during 1 year were obtained from the electronic medical record. Kaplan-Meier estimate, log-rank test and hazard ratios were computed to assess the effect of glycemic control on OS. The Cox proportional hazards regression model was applied to ascertain the significance of glycemic control with other survival variables. RESULTS: One thousand four hundred eighteen random blood glucose (RBG) values were analyzed. In accord with previous findings, a 50% decline in the serum tumor marker CA 19-9 at any time was predictive of survival (P=0.0002). In univariate analysis, an elevated pre-treatment average RBG, 3-month average RBG (RBG-3) and the FOLFIRINOX regimen were associated with longer survival. Based on ROC analysis (AUC=0.82), an RBG-3 of 120 mg/dl was determined to be the optimal cutoff to predict 12-month survival. In multivariate analysis that included age, stage, BMI, performance status, presence of diabetes, and chemotherapy regimen, only RBG-3 maintained significance: an RBG-3 ≤120 mg/dl predicted for improved OS compared to >120 mg/dl (19 vs. 9 months; HR=0.37, P=0.002). In contrast, an early decline in CA 19-9 could not predict OS. CONCLUSION: Lower glucose levels during the first 3 months of treatment for advanced PDAC predict for improved OS in patients both with and without diabetes. These results suggest that RBG-3 may be a novel prognostic biomarker worthy of confirmation in a larger patient cohort and that studies exploring a possible cause and effect of this novel survival-linked relationship are warranted.

Treatment-emergent neuroendocrine prostate cancer with a germline BRCA2 mutation: identification of a candidate reversion mutation associated with platinum/PARP-inhibitor resistance.

Neuroendocrine prostate cancer (NEPC) is a highly aggressive histologic subtype of prostate cancer associated with a poor prognosis. Its incidence is expected to increase as castration-resistant disease emerges from the widespread use of potent androgen receptor-targeting therapies, such as abiraterone and enzalutamide. Defects in homologous recombination repair genes, such as BRCA1/2, are also being increasingly detected in individuals with advanced prostate cancer. We present the case of a 65-yr-old man with a germline BRCA2 mutation who developed explosive treatment-emergent, small-cell neuroendocrine prostate cancer. He achieved a complete response to platinum-containing chemotherapy, but a limited remission duration with the use of olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, as maintenance therapy. Upon relapse, tumor genomic profiling revealed a novel 228-bp deletion in exon 11 of the BRCA2 gene. The addition of the anti-PD1 drug pembrolizumab to olaparib was ineffective. This case highlights the ongoing challenges in treating neuroendocrine prostate cancer, even in the setting of homologous recombination repair deficiency.

ATM-Mutated Pancreatic Cancer: Clinical and Molecular Response to Gemcitabine/Nab-Paclitaxel After Genome-Based Therapy Resistance.

Metastatic pancreatic cancer (PC) is an aggressive malignancy, with most patients deriving benefit only from first-line chemotherapy. Increasingly, the recommended treatment for those with a germline mutation in a gene involved in homologous recombination repair is with a platinum drug followed by a poly (ADP-ribose) polymerase (poly adenosine phosphate-ribose polymerase [PARP]) inhibitor. Yet, this is based largely on studies of BRCA1/2 or PALB2 mutated PC. We present the case of a 44-year-old woman with ATM-mutated PC who achieved stable disease as the best response to first-line fluorouracil, leucovorin, irinotecan, and oxaliplatin, followed by progression on a PARP inhibitor. In the setting of jaundice, painful hepatomegaly, and a declining performance status, she experienced rapid disease regression with the nonplatinum regimen, gemcitabine plus nab-paclitaxel. Both physical stigmata and abnormal laboratory values resolved, imaging studies showed a reduction in metastases and her performance status returned to normal. Measurement of circulating tumor DNA for KRAS G12R by digital droplet polymerase chain reaction confirmed a deep molecular response. This case highlights that first-line treatment with a platinum-containing regimen followed by PARP inhibition may not be the best choice for individuals with ATM-mutated pancreatic cancer. Additional predictors of treatment response are needed in this setting.

Rapid development and use of patient-specific ctDNA biomarkers to avoid a "rash decision" in an ovarian cancer patient.

Epithelial ovarian cancer (OvCa) is the most lethal female reproductive tract malignancy. A major clinical hurdle in patient management and treatment is that when using current surveillance technologies 80% of patients will be clinically diagnosed as having had a complete clinical response to primary therapy. In fact, the majority of women nonetheless develop disease recurrence within 18 mo. Thus, without more accurate surveillance protocols, the diagnostic question regarding OvCa recurrence remains framed as "when" rather than "if." With this background, we describe the case of a 61-yr-old female who presented with a 3-mo history of unexplained whole-body rash, which unexpectedly led to a diagnosis of and her treatment for OvCa. The rash resolved immediately following debulking surgery. Nearly 1 yr later, however, the rash reappeared, prompting the prospect of tumor recurrence and requirement for additional chemotherapy. To investigate this possibility, we undertook a genomics-based tumor surveillance approach using a targeted 56-gene NGS panel and biobanked tumor samples to develop personalized ctDNA biomarkers. Although tumor-specific TP53 and PTEN mutations were detectable in all originally collected tumor samples, pelvic washes, and blood samples, they were not detectable in any biosample collected beyond the first month of treatment. No additional chemotherapy was given. The rash spontaneously resolved. Now, 2 yr beyond the patient's original surgery, and in the face of continued negative ctDNA findings, the patient remains with no evidence of disease. As this single case report suggests, we believe for the first time that ctDNA can provide an additional layer of information to avoid overtreatment.

Detection of endometrial precancer by a targeted gynecologic cancer liquid biopsy.

Endometrial cancer is the most common gynecologic malignancy in industrialized countries, and both its incidence and its associated mortality are increasing. The "liquid biopsy" is becoming an important transformative precision oncology tool, but barriers intrinsic to blood sampling have limited its use in early cancer detection. We hypothesized that using a more targeted sample for analysis-namely, a uterine lavage-should provide a more sensitive and specific diagnostic test for endometrial cancer. Using a custom 12-gene endometrial cancer panel, molecular analysis of uterine lavage fluid from an asymptomatic 67-yr-old female without histopathologic evidence of premalignant lesions or cancer in her uterine tissue revealed two oncogenic PTEN mutations. Ten months later, the patient returned with postmenopausal bleeding and a single microscopic focus of endometrial cancer. DNA isolated and sequenced from laser-capture microdissected tumor tissue revealed the same two PTEN mutations. These mutations were unlikely to occur by chance alone (P < 3 × 10-7). This illustrative case provides the first demonstration that future, tumor-specific mutations can be identified in an asymptomatic individual without clinical or pathologic evidence of cancer by using already established sequencing technologies but targeted sampling methods. This finding provides the basis for new opportunities in early cancer screening, detection, and prevention.

Epstein-Barr Virus MicroRNA Expression Increases Aggressiveness of Solid Malignancies.

The Cancer Genome Atlas (TCGA) microRNA (miRNA) initiative has revealed a pivotal role for miRNAs in cancer. Utilizing the TCGA raw data, we performed the first mapping of viral miRNA sequences within cancer and adjacent normal tissues. Results were integrated with TCGA RNA-seq to link the expression of viral miRNAs to the phenotype. Using clinical data and viral miRNA mapping results we also performed outcome analysis. Three lines of evidence lend credence to an active role of viral miRNAs in solid malignancies. First, expression of viral miRNA is consistently higher in cancerous compared to adjacent noncancerous tissues. Second, viral miRNA expression is associated with significantly worse clinical outcome among patients with early stage malignancy. These patients are also featured by increased expression of PD1/PD-L1, a pathway implicated in tumors escaping immune destruction. Finally, a particular cluster of EBV-miRNA (miR-BART2, miR-BART4, miR-BART5, miR-BART18, and miR-BART22) is associated with expression of cytokines known to inhibit host response to cancer. Quantification of specific viral miRNAs may help identify patients who are at risk of poor outcome. These patients may be candidates for novel therapeutic strategies incorporating antiviral agents and/or inhibitors of the PD-1/PD-L1 pathway.

Nek6 and Hif-1α cooperate with the cytoskeletal gateway of drug resistance to drive outcome in serous ovarian cancer.

Hypoxia selects the most aggressive and drug-resistant clones in solid malignancies. One of the pivotal transcription factors induced by hypoxia is Hif-1α. However, in serous ovarian cancer (SEOC), Hif-1α expression is not a prognostic biomarker. This study aims to assess the hypothesis that the serine-threonine kinase Nek6 functions as a downstream effector cooperating with Hif-1α in driving ovarian cancer aggressiveness. Nek6 was overexpressed and Hif-1α was silenced in A2780 cells. Nek6 was also stably silenced in Hey cells. The dependence of Nek6 expression on Hif-1α was assayed as a function of hypoxic growth conditions. Nek6 interaction with the cytoskeletal gateway of drug resistance was investigated with far western blot. The co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts was quantified with qPCR in two cohorts of SEOC patients (346 locally treated patients and 344 from the TCGA dataset). Nek6 expression is induced by hypoxia in a Hif-1α dependent fashion. Nek6 directly interacts with GBP-1, thus being a component of the cytoskeletal gateway of drug resistance. Nek6 overexpression increases and silencing decreases the anchorage-independent growth of cultured cells. In SEOC patients, NEK6 expression is significantly correlated with HIF1A. Co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts identifies a subset of SEOC patients characterized by poor outcome and drug resistance. This study demonstrates the functional relevance of Nek6 in the context of the adaptive response to hypoxia in SEOC. This finding may help identify a sub-population of patients at high risk of relapse to standard first-line chemotherapy.

Defining "mutation" and "polymorphism" in the era of personal genomics.

BACKGROUND: The growing advances in DNA sequencing tools have made analyzing the human genome cheaper and faster. While such analyses are intended to identify complex variants, related to disease susceptibility and efficacy of drug responses, they have blurred the definitions of mutation and polymorphism. DISCUSSION: In the era of personal genomics, it is critical to establish clear guidelines regarding the use of a reference genome. Nowadays DNA variants are called as differences in comparison to a reference. In a sequencing project Single Nucleotide Polymorphisms (SNPs) and DNA mutations are defined as DNA variants detectable in >1 % or <1 % of the population, respectively. The alternative use of the two terms mutation or polymorphism for the same event (a difference as compared with a reference) can lead to problems of classification. These problems can impact the accuracy of the interpretation and the functional relationship between a disease state and a genomic sequence. We propose to solve this nomenclature dilemma by defining mutations as DNA variants obtained in a paired sequencing project including the germline DNA of the same individual as a reference. Moreover, the term mutation should be accompanied by a qualifying prefix indicating whether the mutation occurs only in somatic cells (somatic mutation) or also in the germline (germline mutation). We believe this distinction in definition will help avoid confusion among researchers and support the practice of sequencing the germline and somatic tissues in parallel to classify the DNA variants thus defined as mutations.

Class III ß-tubulin in normal and cancer tissues.

Microtubules are polymeric structures composed of tubulin subunits. Each subunit consists of a heterodimer of α- and ß-tubulin. At least seven ß-tubulin isotypes, or classes, have been identified in human cells, and constitutive isotype expression appears to be tissue specific. Class III ß-tubulin (ßIII-tubulin) expression is normally confined to testes and tissues derived from neural cristae. However, its expression can be induced in other tissues, both normal and neoplastic, subjected to a toxic microenvironment characterized by hypoxia and poor nutrient supply. In this review, we will summarize the mechanisms underlying ßIII-tubulin constitutive and induced expression. We will also illustrate its capacity to serve as a biomarker of neural commitment in normal tissues and as a pure prognostic biomarker in cancer patients.

An investigation of common crosslinking agents on the stability of electrospun collagen scaffolds.

Electrospinning is a widely used processing method to form fibrous tissue engineering scaffolds that mimic the structural features of the native extracellular matrix. Electrospun fibers made of collagen have been sought because it is a natural structural protein that supports cell attachment and growth. Yet, conventional solvents used to electrospin collagen can result in the loss of hydrolytic stability and fiber morphology of the scaffold. This study evaluated the effect of commonly used synthetic and natural crosslinking agents, genipin, glutaraldehyde, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and EDC with N-hydroxysulfosuccinimide (EDC-NHS), on electrospun collagen. Crosslinked collagen scaffolds were assessed for structural integrity in an in vitro immersion study for up to 3 months. Their cytocompatibility was evaluated by human mesenchymal stem cell morphology and proliferation. Our results showed that dimensional stability and cytocompatibility of crosslinked electrospun collagen scaffolds are dependent on the type of crosslinking agent used. Collagen scaffolds treated with EDC and EDC-NHS were structurally stable and retained fiber structure for up to 3 months and were cytocompatible. Therefore, EDC and EDC-NHS are favorable crosslinking agents for electrospun collagen that can be utilized in tissue engineering applications.

Herpes virus microRNA expression and significance in serous ovarian cancer.

Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality.

Integrated multidimensional analysis is required for accurate prognostic biomarkers in colorectal cancer.

CRC cancer is one of the deadliest diseases in Western countries. In order to develop prognostic biomarkers for CRC (colorectal cancer) aggressiveness, we analyzed retrospectively 267 CRC patients via a novel, multidimensional biomarker platform. Using nanofluidic technology for qPCR analysis and quantitative fluorescent immunohistochemistry for protein analysis, we assessed 33 microRNAs, 124 mRNAs and 9 protein antigens. Analysis was conducted in each single dimension (microRNA, gene or protein) using both the multivariate Cox model and Kaplan-Meier method. Thereafter, we simplified the censored survival data into binary response data (aggressive vs. non aggressive cancer). Subsequently, we integrated the data into a diagnostic score using sliced inverse regression for sufficient dimension reduction. Accuracy was assessed using area under the receiver operating characteristic curve (AUC). Single dimension analysis led to the discovery of individual factors that were significant predictors of outcome. These included seven specific microRNAs, four genes, and one protein. When these factors were quantified individually as predictors of aggressive disease, the highest demonstrable area under the curve (AUC) was 0.68. By contrast, when all results from single dimensions were combined into integrated biomarkers, AUCs were dramatically increased with values approaching and even exceeding 0.9. Single dimension analysis generates statistically significant predictors, but their predictive strengths are suboptimal for clinical utility. A novel, multidimensional integrated approach overcomes these deficiencies. Newly derived integrated biomarkers have the potential to meaningfully guide the selection of therapeutic strategies for individual patients while elucidating molecular mechanisms driving disease progression.