MARIPOSA: phase 3 study of first-line amivantamab + lazertinib versus osimertinib in EGFR-mutant non-small-cell lung cancer.

Third-generation EGFR tyrosine kinase inhibitors (TKIs), such as osimertinib, have demonstrated efficacy in patients with EGFR-mutant non-small-cell lung cancer; however, almost all patients will eventually relapse. Amivantamab is an EGFR-MET bispecific antibody with immune cell-directing activity that targets activating and resistance EGFR mutations and MET mutations and amplifications. In the ongoing CHRYSALIS study (NCT02609776), amivantamab in combination with lazertinib, a potent, brain-penetrant third-generation EGFR TKI, demonstrated antitumor activity in the treatment-naive and osimertinib-relapsed setting. Here the authors present the methodology for the MARIPOSA study (NCT04487080), a phase 3, multicenter, randomized study designed to compare the efficacy and safety of amivantamab and lazertinib combination therapy versus single-agent osimertinib as first-line treatment for EGFR-mutant non-small-cell lung cancer.

Plain language summary Osimertinib is the standard-of-care treatment for patients with non-small-cell lung cancer caused by mutations in the EGFR. However, patients will eventually see their disease return because their tumors will develop new mutations that are resistant to osimertinib treatment. Amivantamab is a new antibody treatment that blocks the EGFR and another receptor called the MET receptor, to stop the growth of lung tumor cells. In an ongoing clinical trial, called the CHRYSALIS study, when amivantamab was given with lazertinib (another drug that blocks the EGFR), lung tumors shrank in patients whose lung cancer had not been previously treated. A new clinical trial called the MARIPOSA study (NCT04487080) aims to compare the antitumor activity and safety of the amivantamab + lazertinib combination versus osimertinib alone in patients with EGFR-mutant non-small-cell lung cancer who have not received treatment for their lung cancer. Trial registration number: NCT04487080 (ClinicalTrials.gov).

Pancreatic cancer stem-like cells display aggressive behavior mediated via activation of FoxQ1.

Subpopulations of cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) have been identified from most tumors, including pancreatic cancer (PC), and the existence of these cells is clinically relevant. Emerging evidence suggests that CSLCs participate in cell growth/proliferation, migration/invasion, metastasis, and chemo-radiotherapy resistance, ultimately contributing to poor clinical outcome. However, the pathogenesis and biological significance of CSLCs in PC has not been well characterized. In the present study, we found that isolated triple-marker-positive (CD44(+)/CD133(+)/EpCAM(+)) cells of human PC MiaPaCa-2 and L3.6pl cells behave as CSLCs. These CSLCs exhibit aggressive behavior, such as increased cell growth, migration, clonogenicity, and self-renewal capacity. The mRNA expression profiling analysis showed that CSLCs (CD44(+)/CD133(+)/EpCAM(+)) exhibit differential expression of more than 1,600 mRNAs, including FoxQ1, compared with the triple-marker-negative (CD44(-)/CD133(-)/EpCAM(-)) cells. The knockdown of FoxQ1 by its siRNA in CSLCs resulted in the inhibition of aggressive behavior, consistent with the inhibition of EpCAM and Snail expression. Mouse xenograft tumor studies showed that CSLCs have a 100-fold higher potential for tumor formation and rapid tumor growth, consistent with overexpression of CSC-associated markers/mediators, including FoxQ1, compared with its parental MiaPaCa-2 cells. The inhibition of FoxQ1 attenuated tumor formation and growth, and expression of CSC markers in the xenograft tumor derived from CSLCs of MiaPaCa-2 cells. These data clearly suggest the role of differentially expressed genes in the regulation of CSLC characteristics, further suggesting that targeting some of these genes could be important for the development of novel therapies for achieving better treatment outcome of PC.

Androgen receptor splice variants contribute to prostate cancer aggressiveness through induction of EMT and expression of stem cell marker genes.

BACKGROUND: The mechanism(s) by which androgen receptor (AR) splice variants contribute to castration-resistant prostate cancer (CRPC) is still lacking. METHODS: Expressions of epithelial-to-mesenchymal transition (EMT) and stem cell markers were molecularly tested using prostate cancer (PCa) cells transfected with AR and AR3 (also known as AR-V7) plasmids or siRNA, and also cultured cells under androgen deprivation therapy (ADT) condition. Cell migration, clonogenicity, sphere-forming capacity was assessed using PCa cells under all experimental conditions and 3,3'-diindolylmethane (DIM; BR-DIM) treatment. Human PCa samples from BR-DIM untreated or treated patients were also used for assessing the expression of AR3 and stem cell markers. RESULTS: Overexpression of AR led to the induction of EMT phenotype, while overexpression of AR3 not only induced EMT but also led to the expression of stem cell signature genes. More importantly, ADT enhanced the expression of AR and AR3 concomitant with up-regulated expression of EMT and stem cell marker genes. Dihydrotestosterone (DHT) treatment decreased the expression of AR and AR3, and reversed the expression of these EMT and stem cell marker genes. BR-DIM administered to PCa patients prior to radical prostatectomy inhibited the expression of cancer stem cell markers consistent with inhibition of self-renewal of PCa cells after BR-DIM treatment. CONCLUSION: AR variants could contribute to PCa progression through induction of EMT and acquisition of stem cell characteristics, which could be attenuated by BR-DIM, suggesting that BR-DIM could become a promising agent for the prevention of CRPC and/or for the treatment of PCa.

Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1.

BACKGROUND: There is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches. METHODS: A primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed. RESULTS: Histopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. Low passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64-66 chromosomes with numerous aberrations. Following subcutaneous and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma. CONCLUSIONS: Although derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human meningioma mouse xenograft models will provide biologically relevant platforms from which to investigate differences in low- vs. high-grade meningioma tumor biology and disease progression as well as to develop novel therapies to improve treatment options for poor prognosis or recurrent meningiomas.

Racial disparities in Human Papillomavirus (HPV) associated head and neck cancer.

PURPOSE: Poorer survival from head and neck squamous cell carcinoma (HNSCC) in African Americans (AA) may be due to disparity in the prevalence of Human Papillomavirus (HPV) but earlier studies often failed to control other etiological factors. We aimed to elucidate whether racial disparities in HPV prevalence and overall survival were due to confounding from smoking or alcohol use. MATERIALS AND METHODS: 385 patients with SCC of the mouth, pharynx, nose, or larynx who had surgical resection at Wayne State University affiliated hospitals were identified through a population-based cancer registry. Formalin fixed paraffin embedded tissue blocks were used to determine the presence of HPV DNA and its genotype using a sensitive broad-spectrum PCR technique. Patients' demographics, tumor characteristics and vital status were obtained through record linkage with the registry data and smoking and alcohol information was abstracted from medical record. Cox's proportional hazard model and unconditional logistic regression models were employed to analyze the overall survival and tumor HPV-positivity, respectively. RESULTS: HPV positivity in oropharyngeal cancer was substantially lower in AA than in other racial groups (odds ratio 0.14, 95% confidence interval (CI) 0.05-0.37) and adjustment for smoking or alcohol did not change this association. However, a significantly increased hazard ratio of death in AA oropharyngeal cancer patients (univariable hazard ratio (HR) 2.55, 95% CI 1.42-4.59) decreased to almost unity (HR 1.49, 95% CI 0.75-2.93) after adjustment for HPV and smoking. CONCLUSIONS: Lower HPV prevalence in AA largely accounts for their poorer survival from oropharyngeal cancer, but not other HNSSC.

Predicting Regression of Barrett's Esophagus-Can All the King's Men Put It Together Again?

The primary pre-neoplastic lesion of the lower esophagus in the vicinity of the gastroesophageal junction (GEJ) is any Barrett's esophageal lesions (BE), and esophageal neoplasia has increased in the US population with predispositions (Caucasian males, truncal obesity, age, and GERD). The responses to BE are endoscopic and screening cytologic programs with endoscopic ablation of various forms. The former have not been proven to be cost-effective and there are mixed results for eradication. A fresh approach is sorely needed. We prospectively followed 2229 mostly male veterans at high risk for colorectal cancer in a 27-year longitudinal long-term study, collecting data on colorectal neoplasia development and other preneoplastic lesions, including BE and spontaneous regression (SR). Another cross-sectional BE study at a similar time period investigated antigenic changes at the GEJ in both BE glandular and squamous mucosa immunohistochemistry and the role of inflammation. Ten of the prospective cohort (21.7%) experienced SR out of a total of forty-six BE patients. Significant differences between SR and stable BE were younger age (p < 0.007); lower platelet levels (p < 0.02); rectal p87 elevation in SR (p < 0.049); a reduced innate immune system (InImS) FEREFF ratio (ferritin: p87 colonic washings) (p < 0.04). Ancillary testing showed a broad range of neoplasia biomarkers. InImS markers may be susceptible to intervention using commonplace and safe medical interventions and encourage SR.

Clinical advances in molecular biomarkers for cancer diagnosis and therapy.

Cancer diagnosis is currently undergoing a paradigm shift with the incorporation of molecular biomarkers as part of routine diagnostic panel. The molecular alteration ranges from those involving the DNA, RNA, microRNAs (miRNAs) and proteins. The miRNAs are recently discovered small non-coding endogenous single-stranded RNAs that critically regulates the development, invasion and metastasis of cancers. They are altered in cancers and have the potential to serve as diagnostic markers for cancer. Moreover, deregulating their activity offers novel cancer therapeutic approaches. The availability of high throughput techniques for the identification of altered cellular molecules allowed their use in cancer diagnosis. Their application to a variety of body specimens from blood to tissues has been helpful for appreciating their use in the clinical context. The development of innovative antibodies for immunohistochemical detection of proteins also assists in diagnosis and risk stratification. Overall, the novel cancer diagnostic tools have extended their application as prognostic risk factors and can be used as targets for personalized medicine.

Characteristics and survival of head and neck cancer by HPV status: a cancer registry-based study.

Elucidation of the role of human papillomavirus (HPV) in the etiology and prognosis of squamous carcinomas of the head and neck (HNSCC) is essential to optimize prevention and treatment strategies for this disease. We analyzed 385 HNSCC tissue blocks identified through a population-based cancer registry in Metropolitan Detroit for HPV DNA using a broad-spectrum PCR technique (SPF10-LiPA25) to correlate with patient and tumor characteristics and overall survival. Overall, HPV DNA (any type) was detected in 29.4% of all HNSCC, but it was significantly more prevalent (50.6%) in oropharyngeal sites (N=81), where 90% of HPV were type 16, than in other sites. HPV prevalence (any type) in oropharyngeal sites was highest in patients with a negative smoking indicator, Caucasians and in regional tumor stage. Likewise, only in oropharyngeal sites did patients overall positive to HPV show significantly better survival compared with HPV-negative patients, notably among those who had been irradiated. The best and the worst survival from cancer in oropharyngeal sites were found, respectively, among HPV-positive patients with negative smoking indicator and among HPV-negative patients with positive smoking indicator. The results of this study revealed that the presence of HPV DNA was associated with patients' specific characteristics and better overall survival exclusively in oropharyngeal sites. To define the fraction of HNSCC preventable by HPV vaccination or amenable to less aggressive treatment, however, tobacco exposure and HPV markers other than DNA presence need to be taken into account.

ERLIN2 promotes breast cancer cell survival by modulating endoplasmic reticulum stress pathways.

BACKGROUND: Amplification of the 8p11-12 region has been found in approximately 15% of human breast cancer and is associated with poor prognosis. Previous genomic analysis has led us to identify the endoplasmic reticulum (ER) lipid raft-associated 2 (ERLIN2) gene as one of the candidate oncogenes within the 8p11-12 amplicon in human breast cancer, particularly in the luminal subtype. ERLIN2, an ER membrane protein, has recently been identified as a novel mediator of ER-associated degradation. Yet, the biological roles of ERLIN2 and molecular mechanisms by which ERLIN2 coordinates ER pathways in breast carcinogenesis remain unclear. METHODS: We established the MCF10A-ERLIN2 cell line, which stably over expresses ERLIN2 in human nontransformed mammary epithelial cells (MCF10A) using the pLenti6/V5-ERLIN2 construct. ERLIN2 over expressing cells and their respective parental cell lines were assayed for in vitro transforming phenotypes. Next, we knocked down the ERLIN2 as well as the ER stress sensor IRE1α activity in the breast cancer cell lines to characterize the biological roles and molecular basis of the ERLIN2 in carcinogenesis. Finally, immunohistochemical staining was performed to detect ERLIN2 expression in normal and cancerous human breast tissues RESULTS: We found that amplification of the ERLIN2 gene and over expression of the ERLIN2 protein occurs in both luminal and Her2 subtypes of breast cancer. Gain- and loss-of-function approaches demonstrated that ERLIN2 is a novel oncogenic factor associated with the ER stress response pathway. The IRE1α/XBP1 axis in the ER stress pathway modulated expression of ERLIN2 protein levels in breast cancer cells. We also showed that over expression of ERLIN2 facilitated the adaptation of breast epithelial cells to ER stress by supporting cell growth and protecting the cells from ER stress-induced cell death. CONCLUSIONS: ERLIN2 may confer a selective growth advantage for breast cancer cells by facilitating a cytoprotective response to various cellular stresses associated with oncogenesis. The information provided here sheds new light on the mechanism of breast cancer malignancy.

Dual color multiplex TTF-1 + Napsin A and p63 + CK5 immunostaining for subcategorizing of poorly differentiated pulmonary non-small carcinomas into adenocarcinoma and squamous cell carcinoma in fine needle aspiration specimens.

BACKGROUND: The distinction of lung adenocarcinoma (ADC) from squamous cell carcinoma (SCC) has important therapeutic implications. Napsin A is a recently developed marker, which has shown high specificity for lung tissue in the surgical pathology specimens. In this study, we have evaluated whether the use of a panel of novel multiplex cocktails of TTF-1 + Napsin A and p63 + CK5 for dual color immunostaining will improve the diagnostic accuracy of lung adenocarcinoma and squamous cell carcinoma in fine needle aspiration (FNA) specimens, usually with relatively scant microfragments of diagnostic material. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded, adequately cellular FNA cell blocks with a confirmed diagnosis of either ADC (n = 22), SCC (n = 20) or poorly differentiated carcinoma (PDC; n = 7), from a total of 49 consecutive cases, were studied. All these cases had subsequently confirmed diagnosis in biopsies or resection specimens. The sections were immunostained with two color methods of TTF-1 + Napsin A and p63 + CK5 multiplex cocktails. The presence of one or more unequivocal individual tumor cells with convincing brown nuclear TTF-1 and red cytoplasmic Napsin A staining, and cells with brown nuclear p63 and membranous / cytoplasmic CK5 staining were interpreted as 'positive'. RESULTS: All 20 FNA cell blocks from SCC cases were positive for dual stain p63 + CK5 and negative for dual stain TTF-1 + Napsin A. The sensitivity and specificity of the dual immunoexpressions of p63 + CK5 for SCC of lung FNAs were both 100%. All 22 ADC cases were positive with dual stain of TTF-1 + Napsin A and negative for dual stain of p63 + CK5. On follow-up of the surgical pathology specimens, 22 cases were confirmed as ADC. The sensitivity of the dual immunoexpression of TTF-1 + Napsin A for ADC of lung FNAs was 100% and the specificity was also 100%. Of the seven PDC cases, five cases that were positive for dual stain p63 + CK5 and negative for dual stain TTF-1 + Napsin A could be categorized as SCC. Two of the seven (2 / 7) PDC cases were positive for dual stain TTF-1 + Napsin A and negative for dual stain p63 + CK5, consistent with ADC. CONCLUSIONS: Simultaneous coordinate or individual immunostaining for Napsin A / TTF-1 in ADC and p63 / CK5 in SCC demonstrated high sensitivity and specificity. The panel with multiplex Napsin A / TTF-1 and p63 / CK5 dual color immunostains could specifically subcategorize PDC into ADC and SCC in lung FNA specimens. Multiplex dual color Napsin A / TTF-1 and p63 / CK5 immunostaining is especially recommended for evaluation of FNA specimens with relatively scant cellularity.

3,3'-Diindolylmethane enhances taxotere-induced growth inhibition of breast cancer cells through downregulation of FoxM1.

Emerging evidence suggests that the transcription factor Forkhead Box M1 (FoxM1) is associated with aggressive human carcinomas, including breast cancer. Because elevated expression of FoxM1 has been observed in human breast cancers, FoxM1 has attracted much attention in recent years as a potential target for the prevention and/or therapeutic intervention in breast cancer. However, no information is currently available regarding how downregulation of FoxM1 could be achieved for breast cancer prevention and therapy. Here, we report for the first time that 3,3'-diindolylmethane (DIM), a nontoxic dietary chemopreventive agent could effectively downregulate FoxM1 in various breast cancer cell lines. Using gene transfection, real-time reverse transcription-PCR, Western blotting, invasion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we found that DIM could enhance Taxotere-induced growth inhibition of breast cancer cells, and decreased invasive capacity of breast cancer cells was observed after either treatment alone or the combination. These effects were associated with downregulation of FoxM1. We also found that knock down of FoxM1 expression by small interfering RNA (siRNA) transfection increased DIM-induced cell growth inhibition, whereas over-expression of FoxM1 by cDNA transfection attenuated DIM-induced cell growth inhibition, suggesting the mechanistic role of FoxM1. Most importantly, the combination treatment significantly inhibited tumor growth in severe combined immunodeficiency (SCID) mice, and the results were correlated with the downregulation of FoxM1 in tumor remnants. We conclude that inactivation of FoxM1 and its target genes by DIM could enhance the therapeutic efficacy of Taxotere in breast cancer, which could be a useful strategy for the prevention and/or treatment of breast cancer.

Restoring sensitivity to oxaliplatin by a novel approach in gemcitabine-resistant pancreatic cancer cells in vitro and in vivo.

Oxaliplatin (OxP) has been used in combination therapy with gemcitabine for the treatment of pancreatic cancer (PC), but the beneficial effect was marginal, which is believed to be due to de novo and acquired drug resistance of PC. Here, we report our in vitro and in vivo preclinical evidence in support of chemosensitization of drug-resistant cells by a nontoxic chemopreventive agent (genistein). Genistein pretreatment together with low concentration of OxP showed significant reduction in cell viability and colony formation concomitant with increased apoptosis (p < 0.01), which was highly synergistic. Drug resistance of PC is allegedly linked with both constitutive and OxP-induced activation of NF-κB, and we found that inactivation of (nuclear factor kappa B) NF-κB by genistein before treatment of cells with OxP was required for cell killing, which was consistent with the downregulation of NF-κB and its downstream antiapoptotic genes (Bcl-2, XIAPs and survivin). Most importantly, our in vivo experiments using orthotopic mouse model showed significant reduction in tumor size (p < 0.01) and reduction of locoregional lymph node metastasis by combination treatment. These results were also consistent with inactivation of NF-κB and the downregulation of NF-κB downstream genes, decreased proliferation marker (Ki-67) and increased apoptosis (TUNEL) in tumor remnants, all of which was consistent with in vitro findings. From these results, we conclude that genistein sensitizes drug-resistant PC to OxP, which is mechanistically linked with inactivation of NF-κB signaling, resulting in greater antitumor effects, and thus our data suggest that this approach could be useful in improving the treatment outcome for patients diagnosed with PC.

Protein kinase Cδ and caspase-3 modulate TRAIL-induced apoptosis in breast tumor cells.

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL-induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL-induced apoptosis was significantly inhibited in PKCδ overexpressing MCF-7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase-8 activation, a first step in TRAIL-induced apoptosis, thus preventing TRAIL-induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase-8 activation leading to TRAIL-induced apoptosis. To determine if caspase-3-induced PKCδ cleavage reverses its inhibition on caspase-8, we developed stable cell lines that either expresses wild-type PKCδ (MCF-7/cas-3/PKCδ) or caspase-3 cleavage-resistant PKCδ mutant (MCF-7/cas-3/PKCδ mut) utilizing MCF-7 cells expressing caspase-3. Cells that overexpress caspase-3 cleavage-resistant PKCδ mutant (MCF-7/cas-3/PKCδmut) significantly inhibited TRAIL-induced apoptosis when compared to wild-type PKCδ (MCF-7/cas-3/PKCδ) expressing cells. In MCF-7/cas-3/PKCδmut cells, TRAIL-induced caspase-8 activation was blocked leading to inhibition of apoptosis when compared to wild-type PKCδ (MCF-7/cas-3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase-8 activation leading to inhibition of TRAIL-induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase-3 sensitizes cells to TRAIL-induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy.

Patient and tumor factors at diagnosis in a multi-ethnic primary head and neck squamous cell carcinoma cohort.

BACKGROUND: A long-term objective is to refine patient diagnosis and prognosis to address heterogeneity in head and neck squamous cell carcinoma (HNSCC) through incorporation of patient and tumor factors. This study examined histopathology and demographic variables at primary diagnosis (early vs. late stage) in a HNSCC patient population with a higher than usual percentage of African American (AA) subjects. METHODS: The primary HNSCC cohort was drawn from a diverse patient population and constructed through re-review of the primary biopsy. Nine specific histopathology and patient factors (race, gender, age) at primary HNSCC diagnosis were evaluated. Logistic regression analyses incorporated univariate and multivariable modeling. RESULTS: Race, gender, pattern of invasion, tumor necrosis, perineural invasion, site, and tumor grade were included in the first multivariable model. The final multivariable model retained gender, race, grade, site, and perineural invasion as independent risk factors for late stage with goodness-of-fit, the area under the curve (AUC), as 0.691. CONCLUSIONS: This report emphasizes patient and tumor characteristics of race, gender, site, perineural invasion, grade, and pattern of invasion as independent factors of advanced stage HNSCC. Pattern of invasion and necrosis are also important tumor characteristics of late stage disease. These factors may offer clinical perspectives when evaluating patients with indeterminate stage.

A novel cross-talk between CXCR4 and PI4KIIIα in prostate cancer cells.

Chemokine signaling regulates cell migration and tumor metastasis. CXCL12, a member of the chemokine family, and its receptor, CXCR4, a G protein coupled receptor (GPCR), are key mediators of prostate-cancer (PC) bone metastasis. In PC cells androgens activate CXCR4 gene expression and receptor signaling on lipid rafts, which induces protease expression and cancer cell invasion. To identify novel lipid-raft-associated CXCR4 regulators supporting invasion/metastasis, we performed a SILAC-based quantitative proteomic analysis of lipid-rafts derived from PC3 stable cell lines with overexpression or knockdown of CXCR4. This analysis identified the evolutionarily conserved phosphatidylinositol 4-kinase IIIα (PI4KIIIα), and SAC1 phosphatase that dephosphorylates phosphatidylinositol-4-phosphate as potential candidate CXCR4 regulators. CXCR4 interacted with PI4KIIIα membrane targeting machinery recruiting them to the plasma membrane for PI4P production. Consistent with this interaction, PI4KIIIα was found tightly linked to the CXCR4 induced PC cell invasion. Thus, ablation of PI4KIIIα in CXCR4-expressing PC3 cells reduced cellular invasion in response to a variety of chemokines. Immunofluorescence microscopy in CXCR4-expressing cells revealed localized production of PI4P on the invasive projections. Human tumor studies documented increased PI4KIIIα expression in metastatic tumors vs. the primary tumor counterparts, further supporting the PI4KIIIα role in tumor metastasis. Furthermore, we also identified an unexpected function of PI4KIIIα in GPCR signaling where CXCR4 regulates PI4KIIIα activity and mediate tumor metastasis. Altogether, our study identifies a novel cross-talk between PI4KIIIα and CXCR4 in promoting tumor metastasis and suggests that PI4KIIIα pharmacological targeting may have therapeutic benefit for advanced prostate cancer patients.

Clinical Implication of MicroRNAs in Molecular Pathology: An Update for 2018.

MicroRNAs (miRNAs) are poised to provide diagnostic, prognostic, and therapeutic targets for several diseases including malignancies for precision medicine applications. The miRNAs have immense potential in the clinical arena because they can be detected in the blood, serum, tissues (fresh and formalin-fixed paraffin-embedded), and fine-needle aspirate specimens. The most attractive feature of miRNA-based therapy is that a single miRNA could be useful for targeting multiple genes that are deregulated in cancers, which can be further investigated through systems biology and network analysis that may provide cancer-specific personalized therapy.

Myelolipoma of the Pelvis: A Case Report and Review of Literature.

Myelolipomas are uncommon, benign tumors which typically occur in the adrenal glands and consist of mature adipose tissue and benign hematopoietic components. Myelolipomas can occur outside of the adrenal glands, but the presacral region, retroperitoneum, pelvis, and mediastinum are unusual locations for these tumors. It is important to recognize this entity in these locations since they can attain massive sizes leading to pressure symptoms and need to be differentiated from the malignant tumors like liposarcomas. We present a myelolipoma case in the presacral region. Our case illustrates the clinical approach of these tumors in such unusual locations.

Methylation of multiple genes as diagnostic and therapeutic markers in primary head and neck squamous cell carcinoma.

OBJECTIVE: To examine epigenetic events of aberrant promoter methylation as diagnostic markers in primary head and neck squamous cell carcinoma using a novel multigene approach. Promoter methylation-mediated silencing is a hallmark of several established tumor suppressor genes. Changes in DNA methylation have been reported to occur early in carcinogenesis and therefore are potentially important early indicators of existing disease. DESIGN: A multicandidate gene probe panel interrogated DNA for aberrant methylation status in 22 cancer genes using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Aberrant promoter hypermethylation was confirmed using methylation-specific polymerase chain reaction after bisulfite treatment. SETTING: Primary care medical center. SUBJECTS: We examined fresh-frozen primary head and neck tumor specimens from 28 patients, including 21 late-stage (19 stage IV and 2 stage III) and 7 early-stage (6 stage II and 1 stage I) tumors. RESULTS: Promoter hypermethylation was observed in 14 of the 28 patients (50%). Genes for RARB, APC, and CHFR were most frequently hypermethylated, occurring in 11 (39%) for RARB, 7 (25%) for CHFR, and 6 (21%) for APC. Aberrant methylation of CHFR was solely a stage IV event. Methylation-specific polymerase chain reaction after bisulfite treatment with conventional and real-time polymerase chain reaction confirmed aberrant methylation for RARB and CHFR. CONCLUSIONS: Promoter methylation profiling of primary head and neck squamous cell carcinoma using multiple target genes identified RARB, APC, and CHFR as frequent epigenetic events. The clinical implications of these genes as diagnostic and treatment biomarkers are highly relevant as attractive targets for cancer therapy, given the reversible nature of epigenetic gene silencing.

HPV-associated differential regulation of tumor metabolism in oropharyngeal head and neck cancer.

HPV-positive oropharyngeal cancer patients experience significantly lower locoregional recurrence and higher overall survival in comparison with HPV-negative patients, especially among those who received radiation therapy. The goal of the present study is to investigate the molecular mechanisms underlying the differential radiation sensitivity between HPV-negative and HPV-positive head and neck squamous cell carcinoma (HNSCC). Here, we show that HPV-negative HNSCC cells exhibit increased glucose metabolism as evidenced by increased production of lactate, while HPV-positive HNSCC cells effectively utilize mitochondrial respiration as evidenced by increased oxygen consumption. HPV-negative cells express HIF1α and its downstream mediators of glucose metabolism such as hexokinase II (HKII) and carbonic anhydrase IX (CAIX) at higher levels, while the expression level of cytochrome c oxidase (COX) was noticeably higher in HPV-positive HNSCC. In addition, the expression levels of pyruvate dehydrogenase kinases (PDKs), which inhibit pyruvate dehydrogenase activity, thereby preventing entry of pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle, were much higher in HPV-negative HNSCC compared to those in HPV-positive cells. Importantly, a PDK inhibitor, dichloroacetate, effectively sensitized HPV-negative cells to irradiation. Lastly, we found positive interactions between tonsil location and HPV positivity for COX intensity and COX/HKII index ratio as determined by immunohistochemical analysis. Overall survival of patients with HNSCC at the tonsil was significantly improved with an increased COX expression. Taken together, the present study provides molecular insights into the mechanistic basis for the differential responses to radiotherapy between HPV-driven vs. spontaneous or chemically induced oropharyngeal cancer.

Fine-mapping loss of gene architecture at the CDKN2B (p15INK4b), CDKN2A (p14ARF, p16INK4a), and MTAP genes in head and neck squamous cell carcinoma.

OBJECTIVE: To identify the extent and the smallest region of loss for CDKN2B(INK4b), CDKN2A(ARF,INK4a), and MTAP. Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines and are common in squamous cell carcinoma of the head and neck (HNSCC). This complex region encodes the tumor suppressor genes cyclin-dependent kinase 2B (CDKN2B) (p15(INK4b)) and CDKN2A (p14(ARF), p16(INK4a)) and the housekeeping gene methylthioadenosine phosphorylase (MTAP). DESIGN: A targeted probe panel designed to finely map the region of 9p21 loss comprised 3 probes for CDKN2B(INK4b), 7 for CDKN2A(ARF, INK4a), and 3 for MTAP and was interrogated using the multiplex ligation-dependent probe amplification assay (MLPA). The MLPA genomic copy number alterations for CDKN2A were validated using real-time polymerase chain reaction. SUBJECTS: Six HNSCC primary (A) and recurrent or metastatic (B) cell lines were examined: UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B. RESULTS: Cell line UMSCC-11B retained all 9p loci tested in the region. Cell lines UMSCC-17A/B indicated homozygous deletion of CDKN2A(ARF, INK4a) starting at p16(INK4) exon 1alpha to include exons 2 and 3. Homozygous loss was indicated for CDKN2B(INK4b) and CDKN2A(ARF,INK4a) in UMSCC-11A, and UMSCC-81A. Cell line UMSCC-81B indicated retention of all 9p loci except for exon 1alpha (p16(INK4a)). Selective loss of the 3' end of MTAP was observed in UMSCC-11A. Genomic alterations by fine-mapping MLPA were validated at the DNA level for CDKN2A. CONCLUSIONS: We identified exon 1alpha (p16(INK4a)) as the smallest region of loss in the CDKN2A(ARF, INK4a) gene. The frequency and precise loss of CDKN2B(INK4b), CDKN2A(ARF, INK4a), and MTAP in the prognosis of 9p21-deleted HNSCC may provide impetus for use of these targets as therapeutic biomarkers in head and neck cancer.