Marker Of Malignant Transformation Research Articles

CAN KI-67 PREDICT MALIGNANT AND PRE-MALIGNANT ORAL LESION? : AN EXPERIENCE IN A TERTIARY CARE HOSPITAL IN THE CENTRAL INDIA.

Background: Oral cancer ranks the sixth commonest cancer worldwide and the third most common cancer in India. Ki67 index has been suggested to play an important role in early diagnosis and prognosis of potentially malignant disorders. This study aims to understand prognostic role of Ki 67 expression in different categories of oral mucosal neoplastic lesions in Central India, where tobacco chewing is endemic. This could help in early categorization and effective management of patients. Aims: To study the Ki67 expression in terms of Index of Positivity and Intensity score in oral mucosal neoplastic lesions. Setting and Design: Department of Pathology, Tertiary Care Hospital Observational & Prospective study Methods & Material: The expression of Ki-67was studied in parafn- embedded tissue sections of histopathology proven pre- malignant and malignant lesions of oral cavity in ninety four cases. Results: Association of Ki67 Expression Intensity score with the type of lesion was found to be signicant in premalignant and malignant lesions predominantly (p= <0.001).Similarly, Ki67 Index of positivity score was highly statistically signicant (p= <0.001) in association with the type of lesion. Conclusions: the expression of Ki-67 correlates well with the disease progression from dysplasia to carcinoma of the oral cavity. It is therefore a marker of malignant transformation and carcinogenesis in oral premalignant lesions and in future it may serve as a prognostic tool in the early detection of malignancy

Гиперплазия эндометрия и микросателлитная нестабильность: возможности прогнозирования опухолевой трансформации эндометрия

Introduction. Microsatellite instability leads to the appearance of neoantigens, which play a key role in oncogenesis. Given the contribution of microsatellite instability to the development of oncopathology, it seems relevant to define microsatellite instability as a marker of malignant transformation in endometrial hyperplasia, which is recognized as a precancerous process. The aim of the study was to develop a model for predicting the risk of malignant transformation of the endometrium in its hyperplasia based on the tumor microsatellite instability. Materials and methods. We performed a two-stage non-randomized retrospective study using histological, immunohistochemical, and statistical methods. In stage 1, a comparative analysis of the structural features of microsatellite instability in endometrial hyperplasia and the development of a model were performed to determine the risk of tumor transformation. In stage 2, we validated the developed model for the prediction of malignant transformation of endometrial hyperplasia. Results. Samples of endometrial hyperplasia with atypia and endometrial hyperplasia without atypia are characterized by a significantly lower expression of MLH1, PMS2, and MSH6 in relation to normal endometrial samples. The expression of these genes was at a significantly higher level in endometrial samples collected from endometrioid adenocarcinoma. The data on the patients’ age and the average area of immunopositive structures in relation to MMR antigens (i.e., MSH6 and MLH1) formed the basis for the development of a model for calculating the risk of oncogenic transformation. The sensitivity and specificity were 92.9% and 92.3%, respectively. Conclusion.The developed model of oncogenic transformation risk can be used to determine the management options of patients with endometrial hyperplasia. Keywords: microsatellite instability, endometrial hyperplasia, endometrial adenocarcinoma

Telomere Dysfunction Is Associated with Altered DNA Organization in Trichoplein/Tchp/Mitostatin (TpMs) Depleted Cells.

Recently, we highlighted a novel role for the protein Trichoplein/TCHP/Mitostatin (TpMs), both as mitotic checkpoint regulator and guardian of chromosomal stability. TpMs-depleted cells show numerical and structural chromosome alterations that lead to genomic instability. This condition is a major driving force in malignant transformation as it allows for the cells acquiring new functional capabilities to proliferate and disseminate. Here, the effect of TpMs depletion was investigated in different TpMs-depleted cell lines by means of 3D imaging and 3D Structured illumination Microscopy. We show that TpMs depletion causes alterations in the 3D architecture of telomeres in colon cancer HCT116 cells. These findings are consistent with chromosome alterations that lead to genomic instability. Furthermore, TpMs depletion changes the spatial arrangement of chromosomes and other nuclear components. Modified nuclear architecture and organization potentially induce variations that precede the onset of genomic instability and are considered as markers of malignant transformation. Our present observations connect the tumor suppression ability of TpMs with its novel functions in maintaining the proper chromosomal segregation as well as the proper telomere and nuclear architecture. Further investigations will investigate the connection between alterations in telomeres and nuclear architecture with the progression of human tumors with the aim of developing personalized therapeutic interventions.

Podoplanin Expression Independently and Jointly with Oral Epithelial Dysplasia Grade Acts as a Potential Biomarker of Malignant Transformation in Oral Leukoplakia.

Our aim was to evaluate the expression of biomarkers, CD44v6, CD147, EGFR, p53, p63, p73, p16, and podoplanin in oral leukoplakias (OL) and to assess their potential for prediction of malignant transformation (MT). We analyzed the expression of CD44v6, CD147, EGFR, p53, p63, p73, p16, and podoplanin by immunohistochemistry in 52 OL, comprised of 41 low-grade (LG) dysplasia and 11 high-grade (HG) cases. Twelve healthy normal tissues (NT) were also included. Univariate and multivariate analysis were performed to evaluate any association with MT. Variable expression among the studied markers was observed, with a significant increase of high expression from NT to LG and HG cases in CD44v6 (p = 0.002), P53 (p = 0.002), P73 (p = 0.043), and podoplanin (p < 0.001). In multivariate analysis, cases with high podoplanin score showed a significant increased risk of MT (HR of 10.148 (95% CI of 1.503–68.532; p = 0.017). Furthermore, podoplanin combined with binary dysplasia grade obtained a HR of 10.238 (95% CI of 2.06–50.889; p = 0.004). To conclude, CD44v6, p53, p73, and podoplanin showed an increasing expression along the natural history of oral carcinogenesis. Podoplanin expression independently or combined with dysplasia grade could be useful predictive markers of MT in OL.

Altered Expression of Epigenetic Modifiers Identifies Novel Biomarkers and Therapeutic Targets in AL Amyloidosis

Altered Expression of Epigenetic Modifiers Identifies Novel Biomarkers and Therapeutic Targets in AL Amyloidosis

Identification of Novel Targets Based on Splicing Alterations for Undruggable RAS/CDK Signaling Cascade in Multiple Myeloma

Background: RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis. Targeting these pathways represents a novel therapeutic strategy in MM. Our ongoing studies (>420 patients) demonstrate that aberrantly spliced transcript expressions can predict MM patient survival outcomes better than gene expression alone, indicating a significant role of splicing mechanism in MM pathophysiology. These studies also identified intron retentions as the predominant recurrent alterations (~32% of spliced genes were retained introns) in MM. We evaluated splicing alterations associated with pathway-level responses after RAS/CDK inhibition in order to identify and validate novel molecular targets.Methods/results: MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to inhibit RAS and CDK pathways. Our studies demonstrated strong synergistic (IC<0.5) MM cytotoxicity triggered by this combination treatment, which triggered dose-dependent manner G0/G1 phase growth arrest. We assessed early death cascade in MM cells after Ei+Ci treatment, and demonstrated significant priming to selective peptides BIM, BAD, and MS1 or HRK, suggesting dependency on BCL2 and MCL1 or on BCL-XL proteins. Our studies showed that Ei+Ci treatment induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling including c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of Ei and Ci. Patient MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that this combination can overcome the protective effects of the MM BM milieu. Moreover, our in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. We evaluated the effect of Ei+Ci treatment on target gene expression in BM cells isolated from flushed femurs of treated animals with Ei, Ci or Ei+Ci, and observed downregulation of Erk1/2-CDK4/6-dependent gene signature. Therefore, we suggest that these inhibitors selectively target Erk1/2, CDK4/6 and their downstream substrates both in vitro/vivo.We next evaluated aberrantly spliced transcript expression in MM cells, with/without Erk1/2 knockdown (KD) or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. This observation was further supported by principal component analyses: upregulation in response to Erk1/2 KD and downregulation due to treatment with Ei+Ci were considered spliced gene-signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complex and are involved in intron retention processes, a marker of malignant transformation. We compared signature-gene expressions from 558 MM patient samples to the signature-genes in plasma cells from normal donors and observed significant (p<2e-11) upregulation of genes with progression from MGUS to sMM, and, also to overt MM . SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 is linked with poor outcomes. SNRPB proteins are also overexpressed in MM cells. Our studies evaluating SNRPB effects on RNA splicing showed both upregulation of transcripts with full intron retention and transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. We evaluated RAVER1 and SNRPB expression in BM cells from animals treated with Ei and Ci alone or in combination. We observed significant downregulation of RAVER1/SNRPB (p=0.001) in BM samples obtained from animals treated with Ei+Ci. We observed decreased intron retention events in genes in treated samples, consistent with our in vitro analyses in MM cell lines and patient samples. Thus, RAVER1/SNRPB overexpression contributes to the aberrant transcriptome splicing associated with RAS/CDK cascade in MM.Conclusions: Our studies 1) show an association between RNA processing and RAS-CDK pathways in MM, 2) identify a core splicing protein, SNRPB/RAVER1, as a novel target for modulating this cascade, and 3) suggest that targeting spliceosome complexes represents a promising therapy in MM. DisclosuresLetai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Anderson: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

P-020: Altered mRNA splicing identifies novel biomarkers and therapeutic targets in AL (Amyloid light-chain) Amyloidosis

<h3>Background</h3> Amyloid light-chain Amyloidosis (ALA) is a plasma cell (PC) dyscrasia characterized by the accumulation of amyloid fibrils formed by clonal PCs in the bone marrow (BM). While multiple myeloma (MM) is present at the time of diagnosis in 10% of patients with ALA, nearly 30% of MM patients have subclinical amyloid deposits in the BM and/or in vital organs. Differential diagnoses of ALA, monoclonal gammopathy of undetermined significance (MGUS), and MM are challenging because these malignancies share genetic similarities. Unrecognized ALA in an MM setting can be life-threatening due to the side effects of some MM treatment regimens. Thus, a molecular signature selectively associated with ALA is needed to improve patient outcomes. <h3>Methods</h3> To identify a selective gene-signature linked to ALA, we evaluated altered mRNA splicing (AltS) events in 8 ALA, 24 MGUS, 33 smoldering MM, 40 MM patient samples and 10 normal donor plasma cells (NPC). We used the custom pipeline to identify AltS events present only in ALA cells and absent in other PC dyscrasias. Gene and transcript level analyses showed that 1609 genes are aberrantly spliced (894 genes are upregulated and 615 downregulated) in ALA patients compared to NPC (P< 001). <h3>Results</h3> Focusing on upregulated gene-splicing events occurring in the mRNA coding region, we observed significant upregulation of CD200, CDKN2B, and B7H3 splice variants in ALA PCs. Since splicing of these genes was also detected in other PC dyscrasias, custom transcript level analyses were used to identify ALA-specific AltS events. These analyses indicated that ~19% of the mRNAs were aberrantly spliced in each subgroup of patient samples, and ~17% retained introns, a marker of malignant transformation. In ALA patients we identified 1607 unique AltS events on 624 genes, 132 of which retained introns, 429 showed exon skipping alterations, and 250 were subjected to nonsense-mediated decay and degraded. These analyses showed significant upregulation of certain splice variants of CD200 and B7H3 in ALA PCs. These variants are attractive targets for therapy; CD200 and B7H3 are immune checkpoint proteins and targeting them may overcome checkpoint-induced drug resistance. In patient's samples harboring upregulated spliced genes, we evaluated the expression of cis-/trans-splicing molecules. Samples with upregulated AltS variants overexpressed SNRPN70 and RBM8A genes, both of which are part of the U1 spliceosome. Alteration of this complex assembly leads to AltS at the 5'sites of exons, causing intron retention and/or uncontrolled exon skipping. Therefore, targeting these splicing factors has the potential to control AltS in ALA. <h3>Conclusion</h3> Our study (i) identifies splice variants that are selectively expressed in ALA PCs, (ii) provides potential ALA molecular biomarkers to aid the differential diagnoses of ALA and other PC dyscrasias, and (iii) identifies potential therapeutic strategies targeting altered splicing in ALA.

P-089: Identification of novel targets in multiple myeloma for “undruggable” RAS/CDK signaling cascade

<h3>Background</h3> RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis; both pathways are undruggable. We evaluated molecular changes associated with pathway-level responses after RAS/CDK inhibition to identify novel molecular targets. <h3>Method</h3> in our previous studies MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to target RAS/CDK pathways. Our studies indicate strong synergistic (IC<0.5) MM cytotoxicity triggered by Ei+Ci treatment, which in a dose-dependent manner arrested MM cells in G0/G1 phase and activated mitochondrial apoptotic signaling. Ei+Ci treatment decreased Erk1/2, CDK4/6, and p-Erk1/2 levels in MM cells, and also induced inhibition of key targets (c-myc, p-RSK,-RB, E2F1) of RAS/CDK cascade. Our studies in patient samples indicate that MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that Ei+Ci combination can overcome the protective effects of the MM BM milieu. An in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. Our studies therefore suggesting on-target activity of these inhibitors in vitro/vivo. <h3>Results</h3> We evaluated mRNA splicing changes in MM cells, with and without Erk1/2 knockdown or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. Upregulation in response to Erk1/2 knockdown and downregulation due to treatment with Ei+Ci were considered spliced gene signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complexes and are involved in intron retention processes, a marker of malignant transformation. We evaluated expressions of RAVER1 and SNRPB in 558 MM patent samples and 10 normal donor BM PCs and observed significant (p<2e-11) upregulation of both genes in clonal PCs with progression from MGUS to sMM, and to overt MM. SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 has a trend toward poor outcomes. SNRPB proteins are also overexpressed in MM cells. Evaluating SNRPB effects on RNA splicing showed upregulation of transcripts with full intron retention or transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. Thus, SNRPB overexpression contributes to aberrant transcriptome splicing associated with RAS/CDK cascade in MM. <h3>Conclusions</h3> Our studies show an association between RNA processing and RAS-CDK pathways in MM, identify a core splicing protein, SNRPB, as a novel target for modulating this undruggable cascade, and suggest that targeting spliceosome complexes is a promising therapy.

Prediction of Malignant Transformation of WHO II Astrocytoma Using Mathematical Models Incorporating Apparent Diffusion Coefficient and Contrast Enhancement.

Using only increasing contrast enhancement as a marker of malignant transformation (MT) in gliomas has low specificity and may affect interpretation of clinical outcomes. Therefore we developed a mathematical model to predict MT of low-grade gliomas (LGGs) by considering areas of reduced apparent diffusion coefficient (ADC) with increased contrast enhancement. Patients with contrast-enhancing LGGs who had contemporaneous ADC and histopathology were retrospectively analyzed. Multiple clinical factors and imaging factors (contrast-enhancement size, whole-tumor size, and ADC) were assessed for association with MT. Patients were split into training and validation groups for the development of a predictive model using logistic regression which was assessed with receiver operating characteristic analysis. Among 132 patients, (median age 46.5 years), 106 patients (64 MT) were assigned to the training group and 26 (20 MT) to the validation group. The predictive model comprised age (P = 0.110), radiotherapy (P = 0.168), contrast-enhancement size (P = 0.015), and ADC (P < 0.001). The predictive model (area-under-the-curve [AUC] 0.87) outperformed ADC (AUC 0.85) and contrast-enhancement size (AUC 0.67). The model had an accuracy of 84% for the training group and 85% respectively for the validation group. Our model incorporating ADC and contrast-enhancement size predicted MT in contrast-enhancing LGGs.

Characterisation of anal intraepithelial neoplasia and anal cancer in HIV-positive men by immunohistochemical markers p16, Ki-67, HPV-E4 and DNA methylation markers.

Human papillomavirus (HPV)‐induced anal intraepithelial neoplasia (AIN, graded 1‐3) is highly prevalent in HIV‐positive (HIV+) men who have sex with men (MSM), but only a minority of lesions progresses to cancer. Our study aimed to characterise comprehensively anal tissue samples from a cross‐sectional series (n = 104) of HIV+ MSM and longitudinal series (n = 40) of AIN2/3 progressing to cancer using different biomarkers. The cross‐sectional series consisted of 8 normal, 26 AIN1, 45 AIN2, 15 AIN3 and 10 anal squamous cell carcinoma. Tissue sections were immunohistochemically (IHC) stained for p16 (viral transformation marker), Ki‐67 (cellular proliferation marker) and HPV‐E4 (viral production marker). We evaluated the expression of IHC markers and compared it with DNA methylation, a marker for malignant transformation. E4 positivity decreased, whereas p16 and Ki‐67 scores and methylation marker positivity increased (P values < .001) with increasing severity of anal lesions. Within AIN2, a heterogeneous biomarker pattern was observed concerning E4, p16 and methylation status, reflecting the biological heterogeneity of these lesions. In the longitudinal series, all AIN2/3 and carcinomas showed high p16 and Ki‐67 expression, strong methylation positivity and occasional E4 positivity. We earlier showed that high methylation levels are associated with progression to cancer. The observed E4 expression in some AIN2/3 during the course of progression to cancer and absence of E4 in a considerable number of AIN1 lesions make the potential clinical significance of E4 expression difficult to interpret. Our data show that IHC biomarkers can help to characterise AIN; however, their prognostic value for cancer risk stratification, next to objective methylation analysis, appears to be limited.

Expression OF p16INK4a Gene in Premalignant and Malignant Lesions of Oral Cavity

Squamous cell carcinoma is the summits malignant neoplasm of the oral cavity. Tobacco and alcohol is identified as risk factors, but squamous cell carcinoma can occur in patients with no known risk factors. Oral cancer is the sixth most common malignancy and is one of the major causes of cancer morbidity and mortality worldwide. Cancer is caused due to a series of alteration in genetic and epigenetic factors that occur in multiple steps and is influenced by the genetic predisposition of the individual and by exogenous environmental factors. These factors result in a series of molecular alteration, including inactivation of tumor suppressor genes expression of p16 has been proposed as a marker for malignant transformation. The p16 staining was correlated between the control and study groups and p 16 was shown to be increasing expressed in premalignant and less expressed in malignant category and was found to be statistically significant by Fischer’s exact test. This study concluded that p16 was increasingly expressed in premalignant lesions and less expressed in malignant lesion. In the present study 9 of the control cases were p16 negative and one case showed sporadic staining. The study group I showed 1 case of sporadic staining, 6 cases of focal staining and 8 cases of diffuse staining. The study II showed 14 cases of sporadic staining, 6 cases of focal staining and 5 cases of diffuse staining. Hence variations cannot be accurately assessed, but it plays a crucial role in assessing pre-malignant lesions progressing to malignancy. To confirm this, a larger sample study is required. As advances in research have leads to greater understanding of potentially malignant lesions in the oral cavity.

DBD-FISH, an effective marker for detecting genotoxicity in buccal mucosa exfoliated cells of patients with oral cancer

Oral squamous cell carcinoma (OSCC) is characterized by increased genetic instability as an essential variable of event of neoplastic transformation. The aim of this study was to evaluate genomic instability in exfoliated cells from the buccal mucosa of patients with OSCC vs. the control group, using DNA Breakage Detection/Fluorescence In Situ hybridization (DBD-FISH). Exfoliated cells from the buccal mucosa were obtained from 38 patients with oral cancer (case group) and from 10 individuals without oral lesions (control group). DNA damage was evaluated by DBD-FISH using the whole-genome DNA probe and digital imaging analysis. Collaterally, HPV infection was determined utilizing the INNO-LiPA HPV kit. Patients with OSCC showed an increase in the hybridization signal five times more intense than that of the baseline level of DNA damage detected in control individuals. The best cutoff value for predicting oral squamous cell carcinoma was 67.46, and an Odds Ratio (OR) value of 87. HPV detection analysis revealed than one patient with OSCC (2.6%) was positive for HPV. All controls were negative HPV. In conclusion, DBD-FISH permitted the clear visualization of level high of DNA damage in the buccal epithelial cells of patients with OSSC respect to control group. Chromosome instability in oral mucosa may be an individual marker of malignant transformation in OSCC.

Abstract 2076: Phospholipid profiling of clinical prostate tissues reveals targetable alterations in membrane lipid composition accompanying tumorigenesis

Abstract Membranes are unique phospholipid (PL) interfaces that play a central role in cancer cell biology. However, PL composition of clinical tumors, and its dynamic regulation, remains a critical gap in the molecular profiling of this disease. Here, we used mass spectrometry-based spatial imaging and lipidomics to generate PL profiles of clinical prostate tissues upon cancer development and in patient-derived tumor explants (PDEs; n=43) cultured with the current clinical agent enzalutamide. Analysis of more than 100 PL species in tumors and matched normal tissues from 21 patients revealed a complexity of cancer-related changes in the tissue PL landscape. Phosphatidylcholine (PC)-based PLs were the most abundant PL class, and their relative abundance was increased in tumors compared to matched benign tissues. A greater proportion of monounsaturated fatty acid chains was characteristic of PLs in tumors, most evident in the PC and phosphatidylethanolamine (PE) PLs, as was striking elongation of fatty acid chains in the phosphatidylinositol (PI) and phosphatidylserine (PS) classes. The abundance of specific PL species and saturation groups was significantly associated with malignancy in both this and an independent cohort of unmatched patient tissues (n=47). Notably, the PL profile was significantly associated with the ERG molecular subtype and basal proliferative index (Ki67) in tumors. Furthermore, we identified individual PL species that were associated with the antiproliferative response to enzalutamide, in cultured PDEs. In light of these PL features, we proposed acetyl coA carboxylase (ACC) as a novel therapeutic target in prostate cancer. Indeed, blocking lipid biosynthesis and elongation in prostate tumors with an ACC inhibitor (ACCi, PF-05175157) significantly reduced tumor cell proliferation in PDE tissues (n=13), concomitant with increased pACC1 staining and a decreased proportion of longer chain PLs. Our findings suggest that the clinical PCa lipidome is not only a marker of malignant transformation and aggressiveness, but also therapeutic response to enzalutamide. Moreover, defining this unique biology identified further clinically actionable targets that may improve prostate cancer outcomes. Citation Format: Lisa M. Butler, Chui Yan Mah, Jonas Dehairs, Andrew Vincent, Shadrack Mutuku, Xander Spotbeen, Rajdeep Das, Zeyad Nassar, Luke Selth, Paul Trim, Marten Snel, David Lynn, Lisa Horvath, Wayne Tilley, Margaret Centenera, Johannes Swinnen. Phospholipid profiling of clinical prostate tissues reveals targetable alterations in membrane lipid composition accompanying tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2076.

MRS as an Aid to Diagnose Malignant Transformation in Low-Grade Gliomas with Increasing Contrast Enhancement.

Increased contrast enhancement has been used as a marker of malignant transformation in low-grade gliomas. This marker has been found to have limited accuracy because many low-grade gliomas with increased contrast enhancement remain grade II. We aimed to investigate whether MR spectroscopy can contribute to the diagnosis of malignant transformation in low-grade gliomas with increased contrast enhancement. Patients with low-grade gliomas who had contemporaneous MR spectroscopy and histopathology for tumor regions with increased contrast enhancement between 2004 and 2015 were retrospectively reviewed. Clinical data collected were sex and age, Karnofsky Performance Scale, histologic subtypes, isocitrate dehydrogenase 1 mutation status, disease duration, adjuvant therapy, and post-radiation therapy duration. Imaging data collected were contrast-enhancement size, whole-tumor size, MR spectroscopy metabolite ratios, and tumor grades of regions with increased contrast enhancement. Diagnostic values of these factors on malignant transformation of low-grade gliomas were statistically analyzed. A total of 86 patients with 96 MR spectroscopy studies were included. Tumor grades associated with increased contrast enhancement were grade II (n = 42), grade III (n = 27), and grade IV (n = 27). On multivariate analysis, the NAA/Cho ratio was the only significant factor (P < .001; OR, 7.1; 95% CI, 3.2-16.1) diagnostic of malignant transformation. With 0.222 as the cutoff value, the sensitivity, specificity, and accuracy of NAA/Cho for diagnosing malignant transformation were 94.4%, 83.3%, and 89.6%, respectively. MR spectroscopy complements conventional MR imaging in the diagnosis of malignant transformation in a subgroup of low-grade gliomas with increased contrast enhancement.

TRPM7 is overexpressed in human IBD-related and sporadic colorectal cancer and correlates with tumor grade

BackgroundInflammatory bowel disease (IBD) predisposes to colorectal cancer (CRC) with some specific features that distinguish it from sporadic CRC. Magnesium (Mg) homeostasis is severely compromised in IBD patients, which may affect both inflammation and tumor development. Efficient transcellular Mg transport in intestinal cells depends on the transient receptor potential melastatin (TRPM) channels type 6 and 7, but their expression has never been investigated in the context of IBD-related CRC. AimsWe sought to study the expression pattern of TRPM6 and TRPM7 in CRC, and to compare IBD-related cases to sporadic cases. MethodsTRPM6 and TRPM7 protein expression was evaluated by immunohistochemistry in surgical specimens from 16 IBD and 13 NON-IBD CRC patients. ResultsTRPM7 expression was higher in tumor tissue than in the adjacent non-neoplastic tissue in both IBD and NON-IBD patients. Overall, adenocarcinomas showed a higher TRPM7 expression than adenomas. TRPM7 expression also positively correlated with tumor grade. Conversely, TRPM6 expression was higher in tumor tissues in both IBD and NON-IBD CRC, but it did not correlate with tumor stage or grade. ConclusionsWe report a possible participation of TRPM6 and 7 in both IBD-related and sporadic CRC and suggest that TRPM7 might serve as a marker of malignant transformation and lack of differentiation.

The relationship between leptin, leptin receptor, and FGFR1 in primary human breast tumors.

e13578 Background: General/overall obesity and visceral/central obesity are associated with increased breast cancer risk and poorer cancer outcomes; however, the precise etiology of these observations is not fully known. Our previous research suggests that adipose tissue-derived fibroblast growth factor 2 (FGF2) promotes malignant transformation of mammary epithelial cells through the activation of its primary receptor, fibroblast growth factor receptor 1 (FGFR1). Both FGF2 and leptin have been shown to be elevated in the serum in obesity (body mass index (BMI) &gt; 25kg/m2). It is well established that elevated sera leptin promotes breast cancer through multiple mechanisms. Therefore, we hypothesized that tumors that express elevated FGFR1 and leptin receptor (LepR) may have a poorer prognosis in obese patients. Methods: Databases that house gene-sequenced breast cancer tumors and normal breast tissue were analyzed to discern relationships between gene copy number and mRNA levels of leptin, leptin receptor, FGFR1 and janus kinase 2 (Jak2), as Jak2 was shown to be a common downstream mediator of both FGFR1 and leptin receptor activation by Cytoscape network analysis. Furthermore, we tested the efficacy of Jak2 inhibitors for preventing mammary epithelial cell malignant transformation using our well-established models of adipose tissue-stimulated growth in soft agar. The anchorage-independent growth in soft agar is a surrogate marker for malignant transformation. Results: A much stronger significant relationship exists between FGFR1 mRNA and leptin mRNA in primary breast cancer (p = 3.17*10−9) compared with normal breast epithelium (p = 0.0013). In primary breast tumors, gene copy number of FGFR1 positively correlates with leptin copy number (p = 8.956*10−8, r = 0.19). Moreover, two separate Jak2 inhibitors attenuated both leptin+FGF2-stimulated and mouse adipose tissue-stimulated MCF-10A cell transformation. Conclusions: Taken together, these results suggest that elevated sera FGF2 and leptin in obese patients may promote breast tumorigenesis in tumors that express elevated FGFR1 and LepR and that Jak2 inhibitors may be a novel therapeutic option for adiposity-driven breast cancers. Future work will examine the relationship between FGFR1/leptin receptor expression and survival and how these relationships are influenced by BMI status.

Malignant transformation of oral leukoplakia is associated with macrophage polarization

BackgroundMost oral squamous cell carcinomas (OSCC) occur on the basis of oral leukoplakias (OLP). The histologic degree of dysplasia is insufficient for the prediction of OLP malignant transformation. Immunologic parameters are gaining importance for prognostic assessment and therapy of cancer. M2 polarized macrophages were shown to be associated with OSCC progression and inferior prognosis. The current study aims to answer the question if OLP with malignant transformation into OSCC within 5 years differ from OLP without transformation regarding macrophage infiltration and polarization.Methods201 specimens (50 transforming OLP, 53 non-transforming OLP, 49 corresponding OSCC and 49 healthy oral mucosa controls) were processed for immunohistochemistry. Samples were stained for CD68, CD163 and CD11c expression, completely digitalized and computer-assisted cell counting was performed. Epithelial and subepithelial compartments were differentially assessed. Groups were statistically compared using the Mann–Whitney U-test. A cut-off point for the discrimination of transforming and non-transforming OLP was determined and the association between macrophage infiltration and malignant transformation was calculated using the Chi-square test (χ2 test).ResultsMacrophage infiltration and M2 polarization in OLP with malignant transformation within 5 years was significantly increased compared to OLP without malignant transformation (p < 0.05). OSCC samples showed the highest macrophage infiltration and strongest M2 polarization (p < 0.05). Additionally, transforming OLP revealed a significant shift of macrophage infiltration towards the epithelial compartment (p < 0.05). χ2 test revealed a significant association of increased macrophage infiltration with malignant transformation (p < 0.05).ConclusionImmunological changes precede malignant transformation of OLP. Increased macrophage infiltration and M2 polarization was associated with the development of oral cancer in OLP. Macrophage infiltration could serve as predictive marker for malignant transformation.

Abstract 3325: EGFR mutation and protein expression analysis in sinonasal inverted papilloma and squamous cell carcinoma

Abstract Introduction Inverted sinonasal papilloma has been demonstrated to be precursor to a subset of sinonasal squamous cell carcinoma and to carry frequent mutations in EGFR exon 20. The aim of this study was to evaluate EGFR mutation and protein expression as risk marker for malignant transformation of inverted papillomas. Experimental procedures The total number of samples studied was 41 inverted papillomas (ISP) and 32 squamous carcinomas (SCC). We defined patients with one single ISP, with multiple ISP (ISP-ISP) or ISP with malignant transformation (ISP-transformed). In addition, we classified SCC related with ISP (SCC-ISP) and those without relation (SCC de novo). EGFR exon 20 was amplified by PCR and analyzed by Sanger sequencing using the ABI PRISM 3100 and 3730 Genetic Analyzer, (Applied Biosystems, Foster City CA). Immunohistochemistry was performed on an automatic staining workstation (Dako Autostainer Plus; DakoCytomation, Glostrup, Denmark) using the antibody anti-pEGFR clone D7A5 (Cell Signaling Technology, Cambridge, UK). Results were evaluated by two experienced investigators (BV and MM). Results We found EGFR exon 20 mutations in 62% (8/13) ISP, 58% (7/12) ISP-ISP, 63% (10/16) ISP-transformed, 54% (7/13) SCC-ISP and 5% (1/19) SCC de novo. Protein expression of pEGFR was detected in 56% (5/9) ISP, 75% (6/8) ISP-ISP, 47% (7/15) ISP-transformed, 42% (5/12) SCC-ISP and 71% (10/14) SCC de novo. We observed an inverse correlation between EGFR exon 20 mutation and pEGFR expression (p=0.034). Overall survival was significantly better for SCC-ISP compared with SCC de novo (2-year survival 54% and 29% respectively; p=0.030). Conclusions EGFR exon 20 mutations occurred in a high frequency, both for cases with single or multiple ISP and for ISP-transformed, making it a characterizing genetic abnormality for ISP in general. This result also means that the presence of a EGFR exon 20 mutations is not of value as a risk marker for malignant progression. Among the SCC, EGFR exon 20 mutations were notably less and pEGFR expression more frequent in SCC de novo compared to SCC-ISP, suggesting that the EGFR signaling pathway is important in both types of SCC, albeit activated in a different manner. Patients with SCC-ISP had a more favorable clinical course, however, neither EGFR exon 20 mutations nor pEGFR expression demonstrated prognostic value. Nevertheless, EGFR exon 20 mutations can be targeted with specific inhibitors and may be of value for adjuvant therapy for SCC as well as ISP that are difficult to manage. Citation Format: Virginia N. Cabal, Marta Menéndez, Sira Potes-Ares, Blanca Vivanco, Laura Suárez-Fernández, Cristina Riobello, Rocío García-Marín, Fernando López, José Luís Llorente, Mario Hermsen. EGFR mutation and protein expression analysis in sinonasal inverted papilloma and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3325.

Evaluation of nonlinear dynamics of the cell structure damage as a promising method for personalized cancer diagnostics

Background:Modulation interference microscopy is one of the promising technologies for early personalized cancer diagnostics, while it gives the possibility to obtain real-time images of living functioning cells at a nanometer resolution, as well as to assess spatio-temporal changes of their intracellular structures.Aim:To identify the markers of malignant transformation of cells based on a multiple scale spatio-temporal of the data obtained by lase interferometry duringin situregistration of changes in intracellular structures.Materials and methods:The study was performed with the breast adenocarcinoma MCF-7 cell line and human kidney epithelial cells HEK 293 provided by the N.N. Blokhin National Medical Research Centre of Oncology (Moscow, Russia). Changes in fluctuation of the living cell phase thickness were measured with a laser modulation interference microscope MIM-340 (PA UOMZ, Russia). The multifractal detrended fluctuation analysis (MF-DFA) was used to assess the results obtained.Results:By comparison of the scaling exponential spectra and corresponding spectra of the singular fluctuations in the minimal and maximal diameters of the phase pictures of HEK 293 cells and MCF-7 cells, it was shown that the cancer cell dynamics has definite monofractal properties: a linear dependency of the scaling exponentsτfrom the actualqparameter and “dotted” singularity spectrum (the spectral width does not exceed 0.1). The dynamics of healthy cell demonstrated multifractal properties, such as a non-linear dependencyτ(q)and a bell shaped singularity spectrum (the spectral width over 0.1).Conclusion:We have proposed a new approach to assess the intracellular structural damage during oncogenesis. The signs of monofractality are objective criteria of malignization. The results obtained seems promising as early diagnostic markers of cancer transformation (damage) of cells and determination of tumor cell sensitivity / resistance to anti-tumor agents in an individual patient.

Association of polymorphism in P16 and myeloperoxidase genes with susceptibility to oral lesions in North Indian population

BackgroundExpression of P16 and MPO (myeloperoxidase) has been proposed as a marker for malignant transformation. In MPO, a G/A polymorphism at 463 bp upstream of the transcription start site of the enzyme has functional outcome, where G allele results in higher level of MPO expression than A allele. Protein P16, a key regulator in cell cycle, is also commonly associated with cancer. SNP of P16 exon3 (C540G) has been found to be linked with tumor aggressiveness. ObjectiveThe study aimed to investigate SNP of P16C540G and myeloperoxidase (MPO) −463G/A for their potential association in oral lesions with risk to oral squamous cell carcinoma (OSCC). MethodsIn this hospital based case control study, single nucleotide polymorphism in P16 and MPO genes was studied by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) method in patients of oral lesions or OSCC and compared with age matched healthy controls. The test group consisted of 226 patients from both rural and urban areas whereas the control group had 103 individuals who did not show a history of oral lesions or OSCC. Genotype and allele frequencies were analyzed by chi-square test and strength of associations by odds ratio with 95% confidence intervals. ResultsThis case–control study demonstrates that people having P16C540G carrier genotypes 540 (CG/GG) were found to be protected from the development of leukoplakia (P = 0.004, <0.001, OR = 0.2451, 0.0818, 95% CI = 0.097–0.619, 0.0295–0.2272 respectively). Whereas, SNP at G463A of MPO gene was found to be not associated with the risk of oral lesions (P = 0.8351, OR = 0.5667, 95% CI = 0.0968–3.3168). ConclusionOur results suggest that single nucleotide polymorphisms in P16 genes may affect the susceptibility to oral lesions; while genotype of MPO showed no association with the development of oral lesions.