Real-World Analysis of Post-Progression Treatment Patterns and Outcomes for EGFR Mutation-Positive Patients Treated with First-Line Osimertinib.

Introduction: The use of osimertinib in the first-line (1L) setting is an effective treatment option for sensitizing EGFR-mutations (EGFRm+) and has significantly altered the standard of care practice for EGFRm+ disease in Canada. Unfortunately, acquired resistance to osimertinib is almost universal, and outcomes are disparate. Post-progression treatment patterns and the outcome of real-world Canadian EGFRm+ patients receiving 1L osimertinib were the focus of this retrospective review. Methods: The Glans-Look Lung Cancer Research database was used to identify and collect demographic, clinical, treatment, and outcome data on EGFRm+ patients who received 1L osimertinib in the Canadian province of Alberta between 2018 and 2022. Results: A total of 150 patients receiving 1L osimertinib were identified. In total, 86 developed progressive disease, with 56 (65%) continuing systemic therapy, 73% continuing osimertinib, and 27% switching to second-line (2L) systemic therapy. Patients were similar both in clinical characteristics at 1L osimertinib initiation and patterns of treatment failure at progression; those continuing 1L osimertinib post-progression had a longer time to progression (13.5 vs. 8.8 months, p = 0.05) and subsequent post-osimertinib initiation survival (34.7 vs. 22.8 months, p = 0.11). Conclusions: The continuation of osimertinib post-progression is an effective disease management strategy for select real-world EGFRm+ patients, providing continued clinical benefit, potentially due to different underlying disease pathogenesis.

Primary cutaneous NUT carcinoma with BRD4::NUTM1 fusion.

Nuclear protein in testis (NUT) carcinoma, molecularly defined by the NUTM1 gene rearrangement, is most commonly reported in young adults in the sinonasal tract, nasopharynx, or thorax. At these sites, NUT carcinoma is an extremely aggressive malignancy with dismal prognosis. Recently, five cases of primary cutaneous NUT adnexal carcinoma have been reported with BRD3 and NSD3 fusion partners. Although NUT adnexal carcinomas are shown to have metastatic potential, they may behave less aggressively than extracutaneous NUT carcinomas. We report a case of a 59-year-old man who underwent a biopsy of a 3-cm plantar mass, which showed BRD4::NUTM1 fusion. The tumor was a poorly differentiated dermal neoplasm showing cytologic atypia, large vesicular nuclei with prominent nucleoli, conspicuous mitotic activity, and foci of necrosis. Immunohistochemically, the tumor showed positivity for keratins, EMA, SOX10, and NUT, with patchy smooth muscle actin. Molecular testing revealed BRD4::NUTM1 rearrangement. With no alternative primary identified by imaging, a diagnosis of primary cutaneous NUT carcinoma was favored. We hope to contribute to the limited body of knowledge on this entity, with emphasis on recognition as well as studying and defining its prognostic differences from extracutaneous NUT carcinomas.

Prospective Validation of ThyroSPEC Molecular Testing of Indeterminate Thyroid Nodule Cytology Following Diagnostic Pathway Optimization.

Background: Molecular testing for cytologically indeterminate thyroid nodules (ITNs) is often reported with incomplete data on clinical assessment and ultrasound malignancy risk (USMR) stratification. This study aimed to clinically validate the diagnostic accuracy of a novel molecular test, assess the incremental preoperative malignancy risk of other clinical factors, and measure the impacts of introducing molecular testing at the population level. Methods: Comprehensive clinical data were collected prospectively for the first 615 consecutive patients with ITNs in a centralized health care system following implementation of a reflexive molecular test. Clinical data include patient history, method of nodule discovery, clinical assessment, USMR, cytology, molecular testing, and surgery or follow-up along with surgeon notes on surgical decision-making. Accuracy of molecular testing and the impact of the introduction of molecular testing were calculated. A multivariable regression model was developed to identify which clinical factors have the most diagnostic significance for ITNs. Results: A locally developed, low-cost molecular test achieved a negative predictive value (NPV) of 76-91% [confidence interval, CI 66-95%] and a positive predictive value (PPV) of 46-65% [CI 37-75%] in ITNs using only residual material from standard liquid cytology fine-needle aspiration (FNA). Sensitivity was highest (80%; [CI 63-92%]) in the American Thyroid Association (ATA) intermediate-suspicion ultrasound category, and lowest (46%; [CI 19-75%]) in the ATA high-suspicion ultrasound category. Following implementation of molecular testing, diagnostic yield increased by 14% (p = 0.2442) and repeat FNAs decreased by 24% (p = 0.05). Mutation was the primary reason for surgery in 76% of resected, mutation-positive patients. High-risk mutations were associated with a 58% (p = 0.0001) shorter wait for surgery. Twenty-six percent of patients with a negative molecular test result underwent surgery. Multivariable regression highlighted molecular testing and USMR as significantly associated with malignancy. Conclusions: Molecular testing improves preoperative risk stratification but requires further stratification for intermediate-risk mutations. Incorporation of clinical factors (especially USMR) with molecular testing may increase the sensitivity for detection of malignancy. Introduction of molecular testing offers some clinical benefits even in a low resection rate setting, and directly influences surgical decision-making. This study illustrates the importance of the local diagnostic pathway in ensuring appropriate integrated use of molecular testing for best outcomes.

Consensus Recommendations to Optimize the Detection and Reporting of NTRK Gene Fusions by RNA-Based Next-Generation Sequencing.

The detection of gene fusions by RNA-based next-generation sequencing (NGS) is an emerging method in clinical genetic laboratories for oncology biomarker testing to direct targeted therapy selections. A recent Canadian study (CANTRK study) comparing the detection of NTRK gene fusions on different NGS assays to determine subjects' eligibility for tyrosine kinase TRK inhibitor therapy identified the need for recommendations for best practices for laboratory testing to optimize RNA-based NGS gene fusion detection. To develop consensus recommendations, representatives from 17 Canadian genetic laboratories participated in working group discussions and the completion of survey questions about RNA-based NGS. Consensus recommendations are presented for pre-analytic, analytic and reporting aspects of gene fusion detection by RNA-based NGS.

The impact of population-based EGFR testing in non-squamous metastatic non-small cell lung cancer in Alberta, Canada.

OBJECTIVES: While Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors have been shown to be effective in phase III randomized trials, the value of targeted therapies has been challenging to evaluate at the population-level. We examined the impact of population-level EGFR testing and treatment on survival outcomes among non-squamous metastatic Non-Small Cell Lung Cancer (NSCLC) patients. MATERIALS AND METHODS: Real-world, population-level data were collected from all de novo non-squamous metastatic NSCLC patients in Alberta, Canada from 2004 to 2020. EGFR testing data were collected through Alberta Precision Laboratories. Differences in survival rates and overall survival (OS) pre (2004-2012) and post initiation (post) (2013-2019) testing periods were evaluated using interrupted time series analyses. The impact of testing and subsequent treatment was evaluated using multivariable Cox Proportional Hazards models. RESULTS: In total, 4,578 non-squamous metastatic NSCLC patients were diagnosed pre-EGFR testing and 4,457 patients were diagnosed post-EGFR testing (2013-2019). Among patients diagnosed in the pre-EGFR testing period, the 6-month, 1-year, and 2-year survival probabilities were 0.39 (95 % CI: 0.38-0.41), 0.22 (95 % CI: 0.21-0.23), and 0.09 (95 % CI: 0.08-0.10), while the survival probabilities for patients diagnosed in the post-EGFR testing period were 0.45 (95 % CI: 0.43-0.46), 0.29 (95 % CI: 0.27-0.30), and 0.16 (95 % CI: 0.15-0.17), respectively. After adjusting for baseline patient and clinical characteristics, OS in the post-EGFR period was significantly improved compared to the pre-EGFR period (HR: 0.81; 95 % CI: 0.78-0.85). Among patients who were treated with systemic therapy, those tested for an EGFR mutation had significantly greater survival than patients who were not tested HR of 0.81 (95 % CI: 0.70-0.95). CONCLUSION: These results show the considerable impact of population-based molecular testing and subsequent targeted therapies on survival among metastatic NSCLC patients. The estimates here can be used in future studies to evaluate the population-level cost-effectiveness of testing and treatment.

CANTRK: A Canadian Ring Study to Optimize Detection of NTRK Gene Fusions by Next-Generation RNA Sequencing.

The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

Real-World Management and Outcomes of Crizotinib-Treated ROS1-Rearranged NSCLC: A Retrospective Canadian Cohort.

The use, safety and effectiveness of crizotinib as part of the management of ROS1-rearranged NSCLC patients in a real-world Canadian clinical cohort was the focus of this retrospective review. Twenty-one ROS1-rearranged patients with advanced/metastatic disease receiving crizotinib between 2014-2020 were identified; crizotinib demonstrated tolerability and effectiveness in this population where outcomes were similar to those described in other crizotinib-treated real-world cohorts, but lower than those of the PROFILE 1001 clinical trial population. Systemic anti-cancer therapy prior to crizotinib initiation occurred in half of the study cohort, with platin-pemetrexed and immune checkpoint inhibitors being most common. Platin-pemetrexed showed good effectiveness in this cohort, but despite high prevalence of upregulated PD-L1 expression, immune checkpoint inhibitors showed poor effectiveness in his cohort. Among all systemic therapies received, crizotinib showed the most effective disease control, although longer intervals between diagnosis and crizotinib initiation were more common among those showing a lack of clinical response to crizotinib, and patients with brain metastases at the time of crizotinib initiation also showed increased diagnosis to crizotinib initiation intervals and decreased clinical response to crizotinib. This study reveals crizotinib has clinical benefit, but timely identification of ROS1-rearrangements and initiation targeted therapies appears important to maximize outcome in this population.

Systematic population-based identification of NTRK and RET fusion-positive thyroid cancers.

Objective: The aim of the study was to identify patients with NTRK fusion-positive or RET fusion/mutation-positive thyroid cancers, who could benefit from neurotrophic tyrosine kinase receptor (NTRK) or receptor tyrosine kinase (RET) inhibitors. Methods: Patients were identified in the Calgary prospective thyroid cancer database (N= 482). Patients were 'pre-screened' with clinically available MassARRAY® BRAF test, Colon Panel, Melanoma Panel, or ThyroSPEC™. Mutation-negative tumors were 'screened' for NTRK fusions and RET fusions/mutations with the Oncomine™ Comprehensive Assay v3 (OCAv3). Results: A total of 86 patients were included in 1 of 2 separate analyses. Analysis A included 42 patients with radioactive iodine (RAI)-resistant distant metastases. After pre-screening, 20 BRAF and RAS mutation-negative patients underwent OCAv3 screening, resulting in the detection of 4 patients with NTRKfusions and 4 patients with RET fusions (8/20, 40% of analyzed patients). Analysis B included 44 patients, 42 with American Thyroid Association (ATA) high and intermediate risk of recurrence and 2 with medullary thyroid carcinoma. During pre-screening, 1 patient with an NTRK fusion, 1 patient with a RET fusion, and 30 patients with BRAF mutations were identified. The remaining 9 patients received OCAv3 screening, resulting in detection of 1 patient with an NTRKfusion and 1 with a RET fusion (4/11, 36% of analyzed patients). Conclusions: Our findings indicate a higher rate of NTRK fusions and RETfusions in patients with thyroid cancer with RAI-resistant distant metastases and ATA high or intermediate risk of recurrence. This highlights the importance of early screening to enable intervention with a NTRK or RET inhibitor.

A Subset of Pancreatoblastomas May Arise From an Adenomatous Precursor: An Ampullary Pancreatoblastoma and Adjacent Adenoma With a Shared Molecular Phenotype in an Adult Patient.

ABSTRACT: Pancreatoblastomas are rare pediatric tumors. In adults, they are exceedingly rare and seem to have a worse prognosis. Most are sporadic, though rare, cases occur in patients with familial adenomatous polyposis. Unlike pancreatic ductal adenocarcinomas, pancreatoblastomas are not believed to arise from dysplastic precursor lesions. Clinical history, along with endoscopic, pathological, and molecular findings, was reviewed for a 57-year-old male patient with an ampullary mass who presented with obstructive jaundice. Microscopic examination showed a pancreatoblastoma subjacent to an adenomatous polyp with intestinal differentiation and low-grade dysplasia. Both tumors had abnormal p53 (complete loss) and nuclear ß-catenin immunostaining. Mutational panel analysis showed an identical CTNNB1 (p.S45P) mutation in both. This case adds to our understanding of the pathogenesis of these rare tumors and suggests that a subset may arise from an adenomatous precursor. In addition, this case is just the second pancreatoblastoma to originate in the duodenal ampulla, and the preceding case suggests that an ampullary location leads to earlier diagnosis. Moreover, this case highlights the difficulty in diagnosing pancreatoblastoma on limited tissue specimens and illustrates the need to include pancreatoblastoma in the differential diagnosis in all tumors in and around the pancreas, including those in adult patients.

Retrospective Real-World Outcomes for Patients With ALK-Rearranged Lung Cancer Receiving ALK Receptor Tyrosine Kinase Inhibitors.

INTRODUCTION: This study explored the use, safety, and efficacy of initial use of an ALK-inhibiting targeted therapy (ALK tyrosine kinase inhibitor [TKI]) in patients with ALK-rearranged NSCLC in a population-based, real-world clinical population within the province of Alberta, Canada. METHODS: Demographic, clinical, treatment, and outcome data of the patients with advanced or metastatic ALK-rearranged NSCLC receiving their first ALK TKI between 2014 and 2019 were included in the analysis. RESULTS: A total of 92 patients with ALK-rearranged NSCLC treated with ALK TKI (78% crizotinib, 22% alectinib) were identified. In the ALK-rearranged cohort, 1-year survival rate was 73% and median overall survival (OS) and progression-free survival (PFS) were 48.5 months and 17.0 months, respectively. An objective response rate of 49% was observed, and adverse events were reported in 70% of the patients, primarily of low grade (84%). Case-matched comparison to patients with ALK-wildtype disease treated with cytotoxic chemotherapy revealed the benefit of ALK TKI in the context of an ALK rearrangement (ALK-rearranged versus ALK-wildtype) (median post-treatment initiation OS: 46.8 versus 14.2 mo, p < 0.001). Outcomes, measured from the time of ALK TKI initiation, differed by Eastern Cooperative Oncology Group (ECOG) (ECOG < 2 versus ECOG ≥ 2) (median OS: not reached versus 6.8 mo, p < 0.001; median PFS 17.6 versus 7.4 mo, p = 0.02), disease presentation (relapsed versus de novo) (median PFS: 30.8 versus 15.0 mo, p = 0.04), and brain metastasis onset (brain metastases development during ALK TKI versus baseline brain metastases) (not reached versus 12.8 mo, p = 0.04). CONCLUSIONS: Clinical trials have firmly established that ALK TKIs are safe, well tolerated, and effective; these findings reveal that their impact in a real-world setting is just as profound. The availability and use of ALK TKI therapies contribute to the impressive gains in survival experienced by contemporary patients with ALK-rearranged disease, rendering patients with this oncodriven form of NSCLC among the longest surviving patients with lung cancer.

Identification of high-impact gene-drug pairs for pharmacogenetic testing in Alberta, Canada.

OBJECTIVES: To facilitate decision-making and priority-setting related to Alberta's Pharmacogenomics (PGx) testing implementation strategy by identifying gene-drug pairs with the highest potential impact on prescribing practices in Alberta. PATIENTS AND METHODS: Annual drug dispensing data for Alberta from 2012 to 2016 for 57 medications with PGx-based prescribing guidelines were obtained, along with population estimates and demographics (age and ethnicity). Frequencies of actionable PGx genotypes by ethnicity were obtained from the Pharmacogenomics Knowledgebase (PharmGKB). Annual dispensing activity for each of the 57 medications was calculated for the full population (all ages) and children/youth (0-19 years). Alberta ethnicity data were cross-referenced with genetic frequency data for each of the main ethnic groups from PharmGKB to estimate the proportion of individuals with actionable genotypes. Actionable genotype proportions and drug dispensing frequencies were collectively used to identify high impact gene-drug pairs. RESULTS: We found (a) half of the drugs with PGx-based prescribing guidelines, namely, analgesics, proton pump inhibitors, psychotropics, and cardiovascular drugs, were dispensed at high frequencies (>1% of the entire population), (b) the dispensing rate for about one-third of these drugs increased over the 5-year study period, (c) between 1.1 and 45% of recipients of these drugs carried actionable genotypes, and (d) the gene-drug pairs with greatest impact in Alberta predominatly included CYP2C19 or CYP2D6. CONCLUSIONS: We uncovered specific patterns in drug dispensing and identified important gene-drug pairs that will inform the planning and development of an evidenced-based PGx testing service in Alberta, Canada. Adaptation of our approach may facilitate the process of evidence-based PGx testing implementation in other jurisdictions.

Fulminant Bacterial Myocarditis Presenting as Myocardial Infarction.

A previously healthy man presented with inferior myocardial infarction and recent upper respiratory tract infection. Bacteremia was detected and treated; however, the patient developed refractory polymorphic ventricular tachycardia storm and shock. Clinical autopsy revealed the diagnosis of isolated bacterial myocarditis. (Level of Difficulty: Beginner.).

Combined loss of TFF3 and PTEN is associated with lethal outcome and overall survival in men with prostate cancer.

BACKGROUND: Trefoil Factor 3 (TFF3) has been implicated in Prostate Cancer (PCa) progression. However, its prognostic value and association with other biomarkers have not been fully explored. We assessed the combined value of TFF3 and PTEN in two cohorts: one is managed surgically for localized PCa and the second is managed non-surgically by androgen deprivation therapy for advanced disease. DESIGN: 228 radical prostatectomies (RP) and 318 transurethral resection of prostate (TURP) samples were assessed by immunohistochemistry (IHC) for TFF3 and by IHC and fluorescent in situ hybridization (FISH) for PTEN. Results of biomarkers expression were correlated with various pathological and clinical outcome parameters including biochemical recurrence (BCR) in the RP cohort and cancer-specific mortality (PCSM) and overall survival (OS) in the TURP cohort. RESULTS: TFF3 expression was detected in 131/226 (57.9%) RP samples and 148/318 (46.5%) of TURP cases. In general, TFF3 positivity was less frequently observed with advanced Gleason Groups. TFF3 expression was also assessed in relation to PTEN expression. Only 15-16% of TFF3-expressed cases were present in association with complete loss of PTEN expression in the TURP and localized cohorts, respectively. Loss of TFF3 expression was not related to BCR after RP, but was prognostic in the non-surgical cohort and associated with decrease OS and PCSM (HR 2.31, CI: 1.67-3.18, p < 0.0001) and (HR 3.99, CI: 2.43-6.56; p < 0.0001), respectively. Adjusting for Gleason score, combined loss of TFF3/PTEN was most associated with OS (HR 2.33, CI: 1.49-3.62; p < 0.0001) and PCSM (HR = 3.44, CI: 1.75-6.78, p < 0.0001). CONCLUSION: The study documents for the first time significant association for combined status of TFF3 expression and PTEN loss in OS and PCSM in patients not managed by surgical intervention. Prospective assessment of PTEN and TFF3 may provide further insight into molecularly subtyping PCa and aid in stratifying patients at risk for lethal disease.

Human pegivirus-1 associated leukoencephalitis: Clinical and molecular features.

Etiologic diagnosis is uncertain in 35% to 50% of patients with encephalitis, despite its substantial global prevalence and disease burden. We report on 2 adult female patients with fatal leukoencephalitis associated with human pegivirus-1 (HPgV-1) brain infection. Neuroimaging showed inflammatory changes in cerebral white matter. Brain-derived HPgV-1 RNA sequences clustered phylogenetically with other pegiviruses despite an 87-nucleotide deletion in the viral nonstructural (NS)2 gene. Neuropathology disclosed lymphocyte infiltration and gliosis predominantly in brain white matter. HPgV-1 NS5A antigen was detected in lymphocytes as well as in astrocytes and oligodendrocytes. HPgV-1 neuroadaptation should be considered in the differential diagnosis of progressive leukoencephalitis in humans. Ann Neurol 2018;84:789-795.

High alpha-methylacyl-CoA racemase (AMACR) is associated with ERG expression and with adverse clinical outcome in patients with localized prostate cancer.

Alpha-methylacyl-CoA racemase (AMACR) is a well-characterized marker extensively utilized in prostate cancer (PCA) diagnosis. However, the prognostic value of AMACR expression and its relation to TMPRSS2-ERG gene rearrangement as one of the most common molecular alterations in PCA is not fully explored. AMACR expression was investigated in a cohort of 218 men with localized PCA treated by radical prostatectomy and correlated with ERG and various clinical and pathological parameters. In vitro studies assessed AMACR changes to ERG knockdown and other related genes. In addition, bioinformatics validated the significance of AMACR/ERG expression and assessed relevant genetic signatures in relation to AMACR/ERG expression. AMACR expression was significantly associated with disease progression and with ERG (p ∼0). Seventeen percent of cancer foci showed negative/weak AMACR expression while being ERG positive. High AMACR expression was significantly associated with positive surgical margins (p = 0.01), specifically in tumors with lower Gleason score <7, with ∼95 % exhibiting positive surgical margin (p = 0.008). High AMACR showed marginal association with PSA biochemical recurrence (BCR) (p = 0.06) which was slightly more pronounced in ERG-positive tumors (p = 0.04). This was validated in other public cohorts. However, in this cohort, the association with BCR was not statistically significant in multivariate analysis (p = 0.09). Using in vitro cellular models, AMACR messenger RNA (mRNA) expression, but not protein levels, showed an association with ERG expression. We report for the first time a significant association between AMACR and ERG with prognostic implication. Patients with high AMACR/ERG-positive PCA may be at higher risk for disease progression, and additional studies in larger cohorts are needed to confirm the above findings. Functional studies investigating the molecular pathways connecting AMACR and ERG may provide an additional insight into PCA progression pathways.

AKT loss in human epithelial cells treated with severe hypoxia.

Cancer cells which can survive and or proliferate in hypoxia may be resistant to anti-cancer treatment. In our previous work, we showed that we could group cell lines treated with severe hypoxia into either hypoxia-induced cell cycle arrest-sensitive or resistant phenotypes, and hypoxia-induced cell death (HCD)-sensitive or resistant phenotypes. We showed that the resistant phenotypes were associated with high levels of active-AKT in late hypoxia and sensitive cells were associated with decreased or undetectable levels of AKT in late hypoxia. We have now extended our findings to numerous other cell lines. We show that HCD and loss of AKT is cell density dependent, and both AKT1 and AKT2 isoforms are lost in late hypoxia. Loss of AKT is most likely due to regulated degradation, as transcription of AKT isoforms is unchanged in hypoxia, and AKT is not significantly translocated to the nucleus to account for its disappearance from cytoplasmic lysates. Interestingly, inhibitors of proteosome, calpain or caspase-mediated proteolysis did not significantly block AKT loss. Inhibition of autophagy using diverse lysosome-targeted autophagy inhibitors also did not block AKT loss, however autophagy inhibitors which block general PI3K activity, such as 3-methyladenine or LY294002, were effective inhibitors of AKT loss in late hypoxia. Interestingly, those inhibitors also blocked HCD in an HCD-sensitive cancer cell line. Inhibitors of proteolytic pathways which did not block AKT loss also did not block HCD in HeLa. Our investigations support a model by which AKT is a major switch involved in regulating hypoxia-induced cell death.

Identification and characterization of demethylase JMJD1A as a gene upregulated in the human cellular response to hypoxia.

Hypoxia is commonly found in human solid cancers and serves as a selective environment for the survival of aggressive cancer cells and as protection from anti-cancer therapies. In addition to a shift to anaerobic metabolism, the cellular response to hypoxia includes cessation of cell division and/or cell death. These mechanisms have still not been defined. Identification of the members of hypoxia-induced growth arrest pathways remain incomplete. We have undertaken an expression microarray analysis of the cellular response to hypoxia in diverse cell lines. An identified cohort of genes is reliably upregulated in various cells in response to hypoxia, as validated by reverse-transcriptase polymerase chain reaction (RT-PCR). One of the upregulated targets corresponds to an expressed sequence tag encoded by JMJD1A (a gene also known as JHDM2A), which has been identified as a histone demethylase that regulates the transcription of androgen receptor targets. We confirm, by RT-PCR, the upregulation of JMJD1A after hypoxia and desferroxamine treatment in multiple cell lines. We also show that JMJD1A is predominantly, but not exclusively, a nuclear protein. Immunofluorescent staining of HeLa cells shows a shift of cytoplasmic JMJD1A into the nucleus on hypoxia treatment. Immunohistochemical staining has revealed that JMJD1A is widely expressed in tissues, even in cells that are not known to express the androgen receptor, and is significantly increased in smooth muscle cells upon hypoxia treatment.

Signaling and apoptosis differences between severe hypoxia and desferoxamine treatment of human epithelial cells.

The mechanisms by which cells undergo proliferation arrest or cell death in response to hypoxia are still not completely understood. Originally, we showed that HeLa and Hep3B carcinoma cells undergo different proliferation responses in hypoxia. We now show that these 2 cell lines also have different cell death responses to severe hypoxia, with HeLa showing both cell cycle arrest and apoptosis (as early as 12 h after hypoxia treatment), and Hep3B showing resistance to both. Hypoxia-induced apoptosis in Hela was associated with decreases of both phospho-S473- and -T308-AKT and loss of AKT function, whereas Hep3B cells were resistant to hypoxia-induced apoptosis and did not lose phospho-AKT or AKT function. We then decided to test if our observations were confirmed using a hypoxia mimic, desferoxamine. Desferoxamine treatment yielded cell cycle arrest in HeLa and moderate arrest in Hep3B but, surprisingly, did not induce notable apoptosis of either cell line with up to 24 h of treatment. Hypoxia-treated normal human mammary epithelial cells also showed hypoxia-induced apoptosis. Interestingly, in these cell lines, there was a complete correlation between loss of phospho-AKT and (or) total AKT, and susceptibility to hypoxia-induced apoptosis. Our data suggests a model in which regulated loss of active AKT at a precise time point in hypoxia may be associated with apoptosis in susceptible cells.

Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines.

Flow cytometric analysis of fibroblasts, normal breast epithelial cells and breast or other cancer cell lines identified variation in the abilities of cell lines to undergo cell cycle arrest as a response to hypoxia. Human mammary epithelial cells (HMEC), normal fibroblasts (Hs68 and WI38), HeLa cervical carcinoma and HTB-30 breast carcinoma cells arrest in G(1)/S in response to severe hypoxia. Hep3B hepatocellular carcinoma cells did not exhibit orderly G(1)/S arrest in response to severe hypoxia. We found a general decrease in p16(INK4a) (p16) mRNA levels, with an associated decrease in p16 protein levels in both normal cells and in cancer cells, regardless of their cell cycle response to hypoxia. p27 protein levels did not correlate with the cell line's ability to enter a hypoxic G(1)/S arrest. Furthermore, cell lines that underwent G(1)/S arrest showed decreased expression of hypoxia inducible factor 1 (HIF-1alpha) and at least one member of INK4 or Sdi cell cycle kinase inhibitors families after 12-24 h of hypoxia. Conversely, Hep3B, which did not exhibit orderly hypoxia-associated G(1)/S arrest, also did not show decreased HIF-1alpha, INK4 or Sdi protein levels in hypoxia. Furthermore, Hep3B showed constitutive activating phosphorylation of Akt and inhibitory phosphorylation of GSK3beta, which was the opposite pattern to that exhibited by the cell lines showing the G(1)/S arrest phenotype. Inhibition of GSK3beta by lithium chloride treatment of HeLa cells converted the HIF-1alpha, p16 and p27 loss to levels unchanged by hypoxic exposure. Our results suggest that regulation of the cell cycle during hypoxia in either normal or cancer cells is not simply due to up-regulation of cell cycle kinase inhibitors. Furthermore, decreased protein expression of HIF-1alpha, p16 and p27 was associated with both a hypoxia-induced G(1)/S arrest phenotype and increased GSK3beta activity.