DNA Index Research Articles

Aneuploidy in children with relapsed B-cell precursor acute lymphoblastic leukaemia: clinical importance of detecting a hypodiploid origin of relapse.

Aneuploidy is common in paediatric B-cell precursor acute lymphoblastic leukaemia (ALL). Specific subgroups, such as high hyperdiploidy (>50 chromosomes or DNA Index ≥1·16) and hypodiploidy (<45 chromosomes), predict outcome of patients after primary treatment. Whether aneuploidy has a prognostic value for relapsed disease is yet to be determined. Using DNA index and centromere screening by multiplex ligation-dependent probe amplification, we investigated aneuploidy in 413 children treated for first relapse of B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. Ten-year event-free survival of patients with high hyperdiploid relapses approached 70%, whereas it was only 40% in low hyperdiploid relapses. Three patients with apparent hyperdiploid relapse had TP53 mutations. In these cases, array-based allelotyping revealed a hypodiploid origin with absence of the hypodiploid founder clone (masked hypodiploidy). Collectively, patients with evident or masked hypodiploid relapses showed an extremely low event-free survival rate of 9%. Importantly, the current relapse risk stratification did not identify cases with masked hypodiploidy as high-risk patients, due to their favourable clinical presentation. In multivariate analysis, hypodiploidy proved to be an independent prognostic factor. This finding supports stratification of relapses with hypodiploid origin into high-risk arms in future trials or allocation of patients to alternative treatment approaches.

Sobrevida global de pacientes con leucemia aguda en el Hospital de Especialidades Pediátricas de Chiapas, México

At the 10th anniversary of the Hospital de Especialidades Pediátricas in Chiapas, Mexico, it was important to assess the 5-year acute leukemia overall survival under the Seguro Popular program (Popular Insurance). A descriptive and survival study of 210 acute leukemia patients diagnosed and treated during 2008-2012 was performed. Kaplan-Meier survival curves were developed for all patients, each leukemia type (B, T and myeloid) and for B type related to risk group, age, sex, leukocytes, cell markers, DNA index, karyotype, and translocations. Age, gender and proportion of leukemia types (B = 85%; M = 10%; T = 5%), were similar to other parts of the country. At the end of the 5-year treatment, 20% of the patients were alive, 53% had died and 27% had abandoned the treatment. Global survival was 42% (B = 45%; T = 20%; M = 10%) (median: 38.8 months; confidence interval of 95% = 28.9-48.7). Very high-risk median survival was 7.7 versus 47 months. There was no difference between standard and high-risk groups. The initial leukocyte count < 50,000/mL and CD10 positive were related to better B survival; no other variables were related. At the time of death, 29% of patients were in remission. Global survival of acute leukemia at Hospital de Especialidades Pediátricas under the Seguro Popular during its first 5 years was surprisingly poor given the medical resources available through the insurance. Early mortality, death during remission and high desertion rates contributed to these results. A detailed revision of treatment protocols and reasons for abandoning treatment is mandatory.

DNA ploidy status, S-phase fraction, and p53 are not independent prognostic factors for survival in endometrioid endometrial carcinoma FIGO stage I-III.

To assess the effects on relative survival of established and new prognostic factors in stage I-III grade 1-3 endometrioid endometrial carcinoma and in the subgroup of stage I grade 1-2. This was a population-based, retrospective study including all women (n=1113) in the western Swedish healthcare region diagnosed with International Federation of Gynecology and Obstetrics (FIGO) stage I-III grade 1-3 endometrioid endometrial carcinoma in 2006-2011. Histology, grade, stage, and age were prospectively reported to the regional clinical and national cancer registers. DNA ploidy and S-phase fraction were analyzed by flow cytometer. S-phase fraction cut-off was set at ≥8%. Tumor biopsies were classified as diploid if there was one G0/G1 peak or the DNA index was 1.0±0.04. Overexpression of p53 as determined by immunohistochemistry was positive if strong nuclear staining was found in >30% of the neoplastic cells. Based on univariable statistical analyses we found that 5-year relative survival was significantly associated with S-phase fraction, DNA ploidy, p53, stage, grade, and age. Excess mortality for S-phase fraction ≥8%, aneuploidy, and p53 overexpression was 8, 14, and 8 and times higher, respectively. However, in a multivariable regression model, adjusted for stage, grade, and age, S-phase fraction, DNA ploidy, and p53 were not statistically independent prognostic factors (p=0.413, p=0.107, p=0.208, respectively) for 5-year relative survival in stage I-III grade 1-3 endometrioid endometrial carcinoma. In a subgroup analysis of stage I grade 1-2, aneuploidy identified a subgroup with impaired 5-year relative survival. We can conclude that S-phase fraction, DNA ploidy, and p53 overexpression did not improve identification of high-risk patients by stage, grade, and age in stage I-III endometrioid endometrial carcinoma. In stage I, aneuploidy and grade 2 predicted lower relative survival rates than other variables.

Rapid assessment of hyperdiploidy in plasma cell disorders using a novel multi‐parametric flow cytometry method

Trisomies of odd numbered chromosomes are seen in nearly half of patients with multiple myeloma (MM) and typically correlate with a hyperdiploid state and better overall survival (OS). We compared DNA ploidy of monoclonal plasma cells (as a surrogate for the presence of trisomies) assessed simultaneously by PCPRO (plasma cell proliferative index), a novel method that estimates DNA index by multi-parametric flow cytometry to fluorescence in situ hybridization (FISH) in 1703 patients with plasma cell disorders. The distribution of ploidy was hyperdiploid: 759 (45%), diploid 765 (45%), hypodiploid: 71 (4%), tetraploid/near-tetraploid: 108 (6%). FISH identified trisomies in 82% (621/756) of patients with hyperdiploidy by PCPRO and no trisomy by FISH was observed in 88% (730/834) of patients without hyperdiploidy. 95% (795/834) of patients without hyperdiploidy on PCPRO had one or less trisomy by FISH. Sensitivity and specificity of PCPRO for detecting hyperdiploidy was 86% (621/725) and 84% (730/865), respectively. Sensitivity increased to 94% (579/618) for patients with more than one trisomy. Newly diagnosed MM patients with hyperdiploidy on PCPRO (147/275) had better OS compared to nonhyperdiploid patients (median not reached vs 59 months, P = 0.008) and better progression free survival (median: 33 vs 23 months, P = 0.03). Within the hyperdiploidy group, patients with high-hyperdiploidy (DNA index: 1.19-1.50) versus those with low-hyperdiploidy (DNA index: 1.05-1.18) had superior OS (3 year OS of 88% vs 68% P = 0.03). Ploidy assessment by flow cytometry can provide rapid, valuable prognostic information and also reduces the number of copy number FISH probes required and hence the cost of FISH.

Cytotoxic and genotoxic effects in mechanics occupationally exposed to diesel engine exhaust

Diesel engine exhaust (DEE), which is the product of diesel combustion, is considered carcinogenic in humans. It comprises toxic gases, polycyclic aromatic hydrocarbons (PAHs) and particulate matter which can reach the pulmonary parenchyma and trigger various diseases, including cancer. The aim of the present study was to evaluate the potential cytotoxic and genotoxic effects of DEE exposure on peripheral blood and buccal epithelial cells in mechanics occupationally exposed to DEE. We recruited 120 exposed mechanics and 100 non-exposed control individuals. Significant differences were observed between the two groups in terms of percentage of tail DNA and damage index (DI) in the alkaline comet assay; levels of biomarkers by cytokinesis-block micronucleus cytome (CBMN-Cyt) assay; frequency of micronucleus (MN), nucleoplasmic bridge (NPB), nuclear bud (NBUD) and apoptotic cells (APOP) and levels of biomarkers for micronucleus, karyorrhexis (KRX), karyolysis (KRL) and condensed chromatin (CC) by the buccal micronucleus cytome (BM-Cyt) assay. A significant and positive correlation was found between the frequency of MN in lymphocytes and buccal cells in the exposed group. Also, there was a significant correlation between age and percentage of tail DNA and DI in the comet assay, APOP and MN in the CBMN-Cyt assay and NBUD and MN in the BM-Cyt assay. Additionally, we found a positive and significant correlation of MN frequency in lymphocytes and buccal cells and age and MN frequency in lymphocytes with the time of service (years). Regarding lifestyle-related factors, a significant correlation was observed between meat and vitamin consumption and NBUD formation on CBMN-Cyt and between meat consumption and MN formation on CBMN-Cyt. Of the BM-Cyt biomarkers, there was a correlation between alcohol consumption and NBUD formation and between binucleated cell (BN), pyknosis (PYC), CC and KRL occurrence and family cancer history. These results are the first data in Colombia on the cytotoxic and genotoxic effects induced by continuous exposure to DEE and thus showed the usefulness of biomarkers of the comet, CBMN-Cyt and BM-Cyt assays for human biomonitoring and evaluation of cancer risk in the exposed populations.

Combined DNA Index System (CODIS)-Based analysis of untested sexual assault evidence in Palm Beach County Florida

In August 2015, the Palm Beach County Sheriff’s Office (PBSO) initiated a CODIS-Based Sexual Assault Evidence Testing Initiative, hereto referred to as the Initiative, as a direct result of the ongoing national inquiry about the number of untested sexual assault cases having DNA evidence stored in law enforcement agency vaults. The Initiative was designed to research sexual assault related evidence including the estimated 1800 untested sexual assault kits stored in the custody of Palm Beach County Law Enforcement Agencies to determine if probative evidence was available, conduct DNA testing on the evidence, and enter all eligible DNA profiles into the CODIS database. Between December 2015 and July 2018 more than 5500 cases were researched and evaluated resulting in evidence from 1,558 cases spanning a 43-year period being tested at a cost of $1,032,496. Nearly 44% of all cases tested provided CODIS-eligible profiles and 36% of those eligible profiles generated a CODIS hit. The positive impact of this Initiative, while already realized by Initiative-related arrests, will continue to grow as additional CODIS hits are generated and investigated by PBSO law enforcement partners. Ultimately, the Initiative will provide information to criminal investigators and the courts including exonerating the innocent, bringing closure to victims, and helping convict the true perpetrators.

Value of the concentration and integrity of serum cell-free DNA for the clinical diagnosis of esophageal carcinoma

Objective: To explore the diagnostic value of serum cell-free DNA (cfDNA) concentration and integrity for esophageal carcinoma. Methods: Venous blood samples from 68 patients with esophageal cancer, 36 patients with benign esophageal lesions and 45 healthy subjects were collected. Circulating cfDNA was verified through quantitative real-time PCR (Alu-qPCR) using Alu-115 and Alu-247 primers. DNA integrity index was calculated as the ratio of Alu-qPCR results (Alu247/115). Concentrations of carcino-embryonic antigen (CEA) and squamous cell carcinoma associated antigen (SCC) were detected by chemiluminescence analyzer assay. Statistical analysis was performed using Mann-Whitney U test, Kruskal-Wallis H test and Spearman correlation test. The Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficiency of each index to esophageal carcinoma. Results: The median absolute serum Alu115 and the Alu247/115 index (1 162.0 ng/ml, 0.57) in esophageal cancer group were significantly higher than those in benign esophageal disease group (496.7 ng/ml, 0.43) and in healthy control group (432.3 ng/ml, 0.42) (all P<0.01, respectively). The Alu115 and Alu247/115 index of serum DNA in benign esophageal disease group were no statistically different from those in the healthy control group (all P>0.05, respectively). The levels of cfDNA and its integrity were not significantly correlated with age, gender, tumor differentiation, or disease stage according to American Joint Committee on Cancer (AJCC) staging system in the esophageal cancer group (all P>0.05). The serum Alu247/115 index of Stage Ⅲ patients was higher than that of Stage Ⅰ~Ⅱ patients(P<0.05). The serum Alu247/115 index of Stage Ⅳ was higher than that of Stage Ⅲ(P<0.05). In the esophageal cancer group, both of serum Alu115 and Alu247/115 index had no correlation with CEA or SCC (all P>0.05). The area under the ROC curve (AUC) of Alu115 and Alu247/115 index were 0.867 and 0.854, respectively, which were both higher than that of CEA (0.622) and SCC (0.753). The addition of Alu115 or Alu247/115 index to CEA and SCC detection increased the sensitivity of the diagnosis of esophageal cancer by 95.6% and 94.1%, respectively. Conclusions: The detection of serum cfDNA concentration and integrity is helpful to the early diagnosis and monitoring of esophageal cancer. Their diagnostic value of esophageal cancer is better than that of the traditional tumor markers CEA and SCC.

Prognostic Value of DNA Index By Flow Cytometry for Minimal Residual Disease in Childhood B-Cell Acute Lymphoblastic Leukemia

Background: Prognostic factors in B-ALL represent a critical role for treatment stratification. High hyperdiploids (>50 chromosomes) have been related to better outcomes, and hypodiploids to worse prognosis. DNA Index (DI) quantifies the DNA from leukemic blast by flow cytometry and has been correlated to the number of chromosomes. Our aim was to demonstrate the prognostic value of DI for minimal residual disease in childhood B-ALL.Methods: 26 blood samples from newly diagnosed childhood B-ALL cases were analyzed between November 2017 and February 2018. DI was evaluated by flow cytometry in samples with Propidium Iodide and leukemic blasts were identified by CD19/CD22/CD10/CD20 antibodies. DI was calculated as the ratio of mean fluorescence of pathologic B blasts and normal cells (T, NK lymphocytes, neutrophils and monocytes) in G0/G1 phase. We stablished three categories: diploid (0.95 - 1.05, 46 chromosomes), hypodiploid (<0.95) and hyperdiploid (>1.05). 8-colors FacsCanto II BD flow cytometer was also used to evaluate minimal residual disease with 0.0025% threshold of detection.Results: 26 cases were evaluated and 2 died during induction treatment. Median age was 8 years (4mo - 16years) and 54% were males. At diagnosis, 62% showed DI diploid and 38% DI hyperdiploid, no hypodiploids cases were detected. Clinical characteristics were similar between both groups. Median DI in the hyperdiploids cases was 1.25 (R: 1.12 - 1.64). We only detected two hyperdiploids cases by conventional karyotype. All patients received the same BFM-based protocol. After induction, all cases achieved complete remission and 46% had MRD negative at the 28th day. DI diploid and hyperdiploid cases achieved 47% and 44% MRD negative, respectively. At the end of consolidation (R: 6-8 Mo), 77% cases achieved MRD negative, and between categories, 62% of DI diploid cases had MRD negative and 100% of DI hyperdiploid cases were negative for MRD detection by flow (p=0.02).Conclusions: The hyperdiploid DNA index by flow cytometry is associated with minimal residual disease negative at the end of consolidation.Flow cytometry offers an alternative over the conventional karyotype to detect good prognosis groups among B-ALL cases. DisclosuresNo relevant conflicts of interest to declare.

Fast P(RMNE): Fast forensic DNA probability of random man not excluded calculation

High throughput sequencing (HTS) of DNA forensic samples is expanding from the sizing of short tandem repeats (STRs) to massively parallel sequencing (MPS). HTS panels are expanding from the FBI 20 core Combined DNA Index System (CODIS) loci to include SNPs. The calculation of random man not excluded, P(RMNE), is used in DNA mixture analysis to estimate the probability that a person is present in a DNA mixture. This calculation encounters calculation artifacts with expansion to larger panel sizes. Increasing the floating-point precision of the calculations allows for increased panel sizes but with a corresponding increase in computation time. The Taylor series higher precision libraries used fail on some input data sets leading to algorithm unreliability. Herein, a new formula is introduced for calculating P(RMNE) that scales to larger SNP panel sizes while being computationally efficient (patent pending).

Identifying migrant remains in South Texas: policy and practice

In 2012, Texas surpassed Arizona in migrant deaths. The majority of deaths occurred in the Rio Grande Valley, specifically in Brooks County, Texas. Brooks County is one of the poorest in the state and was overwhelmed with deaths, without appropriate resources to follow the state laws pertaining to the investigation of unidentified human remains. Until 2013, most remains that were not immediately identified were buried without collecting DNA samples and the location of burials was not recorded. Our paper outlines the difficulties searching for these burials, the struggles of the families of the missing, and the collaborative approaches to facilitating identifications in South Texas. Community outreach combined with geophysical surveys guide which cemeteries are in need of exhumations. Once cemeteries are surveyed, archaeological methods are employed to exhume remains and document burials. Remains are taken to the Forensic Anthropology Center at Texas State for processing, analysis, and identification efforts. Undergraduate and graduate students clean remains and wash clothing and personal effects.After skeletal analysis, all information regarding the remains, including photographs of personal effects, are uploaded to the National Missing and Unidentified Persons System (NamUs) and a DNA sample is submitted to the University of North Texas for inclusion in the Combined DNA Index System (CODIS) DNA database. However, CODIS lacks DNA family reference samples from many families of the missing due to families living outside the US or because they do not feel comfortable providing a DNA sample in the presence of law enforcement. Therefore, it is necessary to work with non-governmental organizations who specialize in collecting missing persons reports and DNA samples from the families of the missing. Working collaboratively with multiple agencies, identification of migrant remains is possible.

Renalase and lupus nephritis: disease activity and histopathological classification

AimTo measure the level of serum renalase and to clarify its relation to lupus nephritis (LN) activity and histopathological classification.Patients and methodsThis study was carried out on 40 patients with systemic lupus erythematosus (SLE), diagnosed according to systemic lupus international collaborating clinics classification criteria (SLICC) criteria, and 20 healthy controls. They were 20 patients without nephritis and 20 patients with LN (17 active and three inactive LN). Venous blood samples were taken from all participants for complete blood count, erythrocyte sedimentation rate, kidney function, anti-double-stranded DNA, C3, C4, and renalase level. The serum renalase levels were determined by enzyme-linked immunosorbent assay. Assessments of protein in 24-h urine collection and protein/creatinine (P/C) ratio were done. Renal biopsies were obtained from patients with LN, with staging and activity and chronicity indices assessment. SLE disease activity was measured by Systemic Lupus Erythematosus Disease Activity Index, and LN activity was estimated by renal Systemic Lupus Erythematosus Disease Activity Index.ResultsRenalase levels were higher in patients with LN than both patients with SLE without LN and control group. The serum renalase levels of patients with LN were positively correlated with P/C ratio, 24-h proteinuria and C3, but negatively correlated with Systemic Lupus Erythematosus Disease Activity Index. For patients with active LN, there was no significant correlation between their serum renalase levels and the indicators of renal activity, including erythrocyte sedimentation rate, proteinuria, P/C ratio, anti-double-stranded DNA, C3, C4, and activity index of renal biopsy. The median of renalase as a marker for diagnosis of LN was 134.65, with a cutoff value of 100 μg/ml.ConclusionSerum renalase may be involved in LN pathogenesis but was not a good predictor for either LN activity or various stages of LN histopathology.

Genetic and physiological effects of the natural radioactive gas radon on the epiphytic plant Tillandsia brachycaulos

Radon (222Rn) is the most abundant natural radioactive gas in nature and triggers carcinogenesis. Few reports exist on whether radon can damage plants as it does animals. Therefore, we chose Tillandsia brachycaulos, a common indicator plant, as the material to detect the physiological and genetic changes caused by radon. With an increase in radon concentration, DNA indices (tail length, tail DNA, tail moment and Olive tail moment) from the comet assay and malondialdehyde (MDA) content increased significantly, suggesting that T. brachycaulos inevitably suffered from radiation damage. However, neither the leaf relative conductivity nor the soluble protein content changed significantly with radon fumigation, and no dose-dependent effect existed between the chlorophyll content and radon concentration, indicating that T. brachycaulos had resistance to radon stress. Foliar trichomes most likely excluded the pollutant from plants because DNA damage in T. brachycaulos with trichomes manually removed was considerably greater than that with trichomes. Moreover, the antioxidant enzyme system further reduced the damage of radon to plants because the activity of superoxide dismutase (SOD) increased significantly with the radon concentration.

Abstract 655: Hyperdiploidy in plasma cell disorders using multi-parametric flow cytometry (MFC) vs. FISH

Abstract Introduction: Hyperdiploidy on FISH portends a good prognosis in myeloma. We compared hyperdiploidy determined by MFC to that by conventional FISH. Methods: We studied 1711 patients with plasma cell disorders who had simultaneous FISH and DNA index testing. Monotypic plasma cells in bone marrow are identified by MFC with antibodies to CD19, CD38, CD45, CD138, kappa and lambda; followed by staining by DAPI. DNA index is determined by dividing the measured DNA content of the G0/G1 abnormal plasma cells by the DNA content of the normal G0/G1 plasma cells present. Plasma cells with DNA content index of &amp;lt;0.95 are hypodiploid and G0/G1 DNA content index of &amp;gt;1.05 are considered hyperdiploid. Results: Distribution DNA index and FISH results are described in Table 1. Sensitivity and specificity of DNA index by hyperdiploidy was 86% and 83%. Cohen's Kappa coefficient for concordance was 0.69. Sensitivity increased to 94% for those with 2 or more trisomies and 97% for 3 or more trisomies. Of the 104 patients with trisomy and non-hyperdiploid DNA index, 62% patients had monosomy (mono) or deletion (del) 13q. Overall, DNA index was lower in patients with mono 13/del 13q (1.01; 0.97-1.11) compared to those without (1.07; 1.0-1.19), p&amp;lt;0.001. After excluding patients with mono 13/del 13q, sensitivity and specificity of hyperdiploidy by DNA index were 92% and 82%, kappa = 0.73. Hyperdiploidy was seen in 55% of 272 patients with newly diagnosed myeloma. Sensitivity and specificity were 88% and 93%, kappa = 0.78. After median follow-up of 2.3 yrs, those with hyperdiploid DNA index had better progression free survival at 2,3 and 5 years, 62%, 45% and 21% compared to 50%, 33% and 13% in non-hyperdiploid group, p=0.05. Median overall survival (OS) was not reached; estimated 2, 3 and 5 year OS was 83%, 76% and 63% vs. 76%, 64% and 44%, respectively; p=0.08. Conclusions: DNA index by MFC is a rapid method to determine hyperdiploidy, with potential for replacing trisomy testing by FISH, especially when coupled with monosomy FISH probes. Hyperdiploid N=768 n/N (%)Diploid N=762 n/N (%)Hypodiploid N=78 n/N (%)P1P2Any trisomy 619/765 (81)96/758(13)8/76 (11)&amp;lt;0.001&amp;lt;0.001One chromosome42587733191429-111362Others12204Two chromosomes8525-Three or more chromosomes492131Any translocation124/744 (17)566/748 (76)53/74 (72)&amp;lt;0.001&amp;lt;0.001t(4;14)106313t(11;14)2240127t(14;16)5325t(14;20)38-t(6;14)8104Unknown partner76524Trisomy + Translocation95/743 (13)60/744 (8)6/74 (8)0.0090.002Monosomy 13/17 or deletion 13q/17p252/759 (33)295/744 (40)54/74 (73)&amp;lt;0.0010.0001Monosomy 13/Deletion 13q208/717 (29)282/718 (39)20/70 (29)Monosomy 17/Deletion 17p70/749 (9)50/735 (7)20/72 (28)P1= three groups; P2= hyperdiploid vs. non-hyperdiploid Citation Format: Surbhi Sidana, Nidhi Tandon, Dragan Jevremovic, Rhett P. Ketterling, Angela Dispenzieri, Morie A. Gertz, Francis K. Buadi, Martha Q. Lacy, William Morice, Curtis A. Hanson, Michael Timm, Patricia Greipp, Linda B. Baughn, David Dingli, Suzanne R. Hayman, Wilson I. Gonsalves, Prashant Kapoor, Robert A. Kyle, Nelson Leung, Ronald S. Go, John A. Lust, S.Vincent Rajkumar, Shaji K. Kumar. Hyperdiploidy in plasma cell disorders using multi-parametric flow cytometry (MFC) vs. FISH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 655.

Abstract 2112: Cell by cell immuno- and cancer marker profiling for non-small cell lung cancer (NSCLC) tissue sample using non-enzymatic tissue dissociation and high-parameter flow cytometry

Abstract Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 80-85% cases of lung cancer. It has poor prognosis as most NSCLC patients are at advanced stage when diagnosed. The development of immunotherapy blocking the PD-1/PD-L1 ligand pathway, in recent years, has shed light on new treatment for lung cancer. An in-depth understanding of the interaction between immune system and tumor will help to identify novel markers for lung cancer treatment. In this study, we performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples for circulating tumor cells (CTCs). The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization (IncellDx IncellPREPTM). The single cell suspensions were stained simultaneously with multiple (&amp;gt;20) immune check point markers (including PD-1*, PD-L1*, TIM-3*, LAG-3*, and CTLA-4*), cancer markers (such as EGFR* and ALK* fusion protein), and a cell cycle dye. The samples were interrogated on a novel, innovative, high parameter, spectral flow cytometer Cytek AuroraTM. Markers on subsets of immune cell and cancer cell populations were investigated. Our results showed the association of high levels of immune check point marker and cancer marker expressions with the high level of aneuploidy (indicated by DNA index &amp;gt;1.05), and the aggressiveness of the cancer (indicated by the number of CTCs). High numbers of CTCs were associated with aneuploidy, increased Post G0-G1%, and high expression of LAG-3, TIM-3, and PD-1. Multi-parametric flow cytometry allows simultaneous profiling of multiple immune and cancer markers on cancer samples at the single cell level. The knowledge acquired from these studies will enhance our understanding of cancer immune system, cancer cells, and the interaction between immune system and the cancer. It will potentially transform patient diagnosis, disease monitoring, and drug discovery. Our study demonstrated a powerful method to study solid tumors that may provide important information for successful precision cancer immunotherapy. *PD-1: programmed death-1; *PD-L1: programmed death-ligand 1; *TIM-3: mucin domain-3-containing molecule-3; *LAG-3: lymphocyte-activation gene-3; *CTLA-4: cytotoxic T-lymphocyte antigen-4; *EGFR: epidermal growth factor receptor; *ALK: anaplastic lymphoma kinase Citation Format: Xiaoyang Wang, Pin-I Chen, Maria Jaimes, Huimin Gu, Keith Shults, Fariba Fazeli, Janine Fernandez, Vishal Sharma, Kalyan Handique, Bruce K. Patterson. Cell by cell immuno- and cancer marker profiling for non-small cell lung cancer (NSCLC) tissue sample using non-enzymatic tissue dissociation and high-parameter flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2112.

PO-227 The effect of chronic mild hypoxia on genomic instability in HER2-overexpression breast cancer cell line SK-BR-3

IntroductionGenomic instability (GIS) is a major tumorigenesis driving factor. Both - amplification of ERBB2 in breast cancer, and acute or cycling tumour hypoxia have been shown to result in increased GIS. Data concerning effects of chronic and mild hypoxia on GIS in cancer cells are conflicting and scarce. Therefore the aim of this study was to explore effects of chronic mild hypoxia (cmH) on GIS in HER2-positive breast cancer cell line SK-BR-3.Material and methodsState of GIS was characterised by proportion of micronuclei containing cells (immunostaining with anti-tubulin and DAPI), chromosomal breakpoints, copy number alterations (Illumina CytoSNP12 v2-1 Bead Chip), expression of genome stability related genes (qPCR); relative telomere length (RTL) (qPCR) after prolonged cultivation of SK-BR-3 (three passages) in hypoxia (2% O2) or normoxia (control).Results and discussionsProlonged exposure to cmH resulted in significant 3.3-fold increase of micronuclei containing cells (hypoxia vs normoxia: 25.38 and 5.86 micronuclei per 1000 cells). Initial adaptation to cmH manifested as contraction of genome and decrease of chromosomal breakpoints (normoxia vs hypoxia: DNA index 2.06 and 1.90; breakpoint count: 4573 and 2678). Further cultivation in cmH resulted in additional reduction of DNA index (1.89) and increase in number of breakpoints (3037). CmH increased expression of ATM dependent DDR genes, significantly decreased expression of dsDNA-break reparation genes (H2AFX, BRCA1, FANCD2) and had no significant effect on aneuploidy–related gene expression. Initial exposure to cmH resulted in major increase of RTL (from 1.1 to 7.8), but further culture in hypoxia showed gradual decrease of RTL (down to 1.3). Low expression levels of telomerase (TERT) through all passages did not change significantly.ConclusionInitial adaptation to hypoxia was characterised by increased RTL, contraction of genome and decrease of genomic heterogeneity. Initial selection of hypoxia-fit SK-BR-3 subpopulations was followed by increased formation of chromosomal rearrangements. CmH in SK-BR-3 activated ATM-CHEK2 branch of DDR and decreased expression of genes involved in reparation of dsDNA breaks. Low expression levels of TERT through all passages did not change significantly and did not correlate with the RTL.

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Genetic risk analysis for an individual according to the theory of programmed onset, illustrated by lung and liver cancers

ObjectiveTo investigate cancer association in the genome and the genetic risk of death from major cancers according to the theory of programmed onset for an individual. MethodsAlleles of 15 randomly selected short tandem repeat (STR) loci, including D6S1043, D12S391, CSF1PO, D7S820, D2S1338, D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D16S539 and D19S433, were determined in 50 patients with lung cancer and 50 patients with liver cancer. The onset age of patients with and without the alleles was compared with Cox regression. Frequencies of significant alleles from Cox regression between the cancer group and control population were analysed through logistic regression for cross-validation. The death probability in an individual carrying two or one of two cancer-related alleles or not carrying two cancer-related alleles was calculated with outcomes of case-control studies translated into the results of the cohort studies. ResultsIt was confirmed that D18S51-20 was a lung cancer-related allele and that D21S11-30.2 and D6S1043-18 were liver cancer-related alleles. Probabilities of death from lung or liver cancers ranged from 0.115 to 0.395, respectively, for those who carry and/or do not carry D18S51-20, D21S11-30.2, D6S1043-18. ConclusionsA more efficient method could be devised for genetic risk analysis according to the theory of programmed onset. The analysis of the CODIS-STR loci (STR loci listed in the US combined DNA indexing system) as genetic markers may provide an efficient and reliable approach to estimate an individual's genetic predisposition.

PO-228 Comprehensive molecular characterisation of TNBCs expressing HORMAD1, a driver of homologous recombination deficiency

IntroductionGenomic instability (GIS) is a major tumorigenesis driving factor. Both - amplification of ERBB2 in breast cancer, and acute or cycling tumour hypoxia have been shown to result in increased GIS. Data concerning effects of chronic and mild hypoxia on GIS in cancer cells are conflicting and scarce. Therefore the aim of this study was to explore effects of chronic mild hypoxia (cmH) on GIS in HER2-positive breast cancer cell line SK-BR-3.Material and methodsState of GIS was characterised by proportion of micronuclei containing cells (immunostaining with anti-tubulin and DAPI), chromosomal breakpoints, copy number alterations (Illumina CytoSNP12 v2-1 Bead Chip), expression of genome stability related genes (qPCR); relative telomere length (RTL) (qPCR) after prolonged cultivation of SK-BR-3 (three passages) in hypoxia (2% O2) or normoxia (control).Results and discussionsProlonged exposure to cmH resulted in significant 3.3-fold increase of micronuclei containing cells (hypoxia vs normoxia: 25.38 and 5.86 micronuclei per 1000 cells). Initial adaptation to cmH manifested as contraction of genome and decrease of chromosomal breakpoints (normoxia vs hypoxia: DNA index 2.06 and 1.90; breakpoint count: 4573 and 2678). Further cultivation in cmH resulted in additional reduction of DNA index (1.89) and increase in number of breakpoints (3037). CmH increased expression of ATM dependent DDR genes, significantly decreased expression of dsDNA-break reparation genes (H2AFX, BRCA1, FANCD2) and had no significant effect on aneuploidy–related gene expression. Initial exposure to cmH resulted in major increase of RTL (from 1.1 to 7.8), but further culture in hypoxia showed gradual decrease of RTL (down to 1.3). Low expression levels of telomerase (TERT) through all passages did not change significantly.ConclusionInitial adaptation to hypoxia was characterised by increased RTL, contraction of genome and decrease of genomic heterogeneity. Initial selection of hypoxia-fit SK-BR-3 subpopulations was followed by increased formation of chromosomal rearrangements. CmH in SK-BR-3 activated ATM-CHEK2 branch of DDR and decreased expression of genes involved in reparation of dsDNA breaks. Low expression levels of TERT through all passages did not change significantly and did not correlate with the RTL.

Forensic efficiency estimate and phylogenetic analysis for Chinese Kyrgyz ethnic group revealed by a panel of 21 short tandem repeats.

Short tandem repeats (STRs) with a high level of polymorphisms and convenient detection method play an indispensable role in human population and forensic genetics. Recently, we detected the 21 autosomal non-combined DNA index system (non-CODIS) STR loci in a Kyrgyz ethnic group, calculated their forensic parameters and analysed its genetic relationships with reference populations from China. In total, 168 alleles were observed at 21 non-CODIS STRs with corresponding allelic frequencies from 0.0016 to 0.4788. No significant deviations at these STRs were observed from the Hardy–Weinberg equilibrium. The values of cumulative power of discrimination and probability of exclusion for all the 21 non-CODIS STRs were 0.99999999999999999998835 and 0.9999994002, respectively. Furthermore, the analyses of phylogenetic trees, genetic distances and interpopulation differentiations demonstrated that the Kyrgyz group had relatively close genetic relationships with the Uygur and Kazak groups. These 21 non-CODIS STRs were characterized by high genetic diversities in the Kyrgyz group and could be applied as a robust tool for individual identification and kinship testing in forensic sciences.

A 21-plex system of STRs integrated with Y-STR DYS391 and ABO typing for forensic DNA analysis

The ABO blood group is considered to be one of the most important markers in forensic testing. Additionally, autosomal short tandem repeat (STR) genotyping continues to be recognized as the dominant technique for determination of human identity, and Y chromosome STR (Y-STR) analysis is becoming increasingly important in solving criminal cases. In this paper, we describe an integrated amplification system of ABO, autosomal STR, and Y-STR genotyping in a single reaction. This system allows for the simultaneous detection of 18 autosomal STR loci (13 Combined DNA Index System [CODIS] loci, as well as D2S1338, D6S1043, D12S391, Penta D, and Penta E), the ABO blood group locus, the Y-STR locus DYS391, and the sex-determining amelogenin locus. Primers were designed and optimized for amplicon sizes from 80–420 bp using a five-dye fluorescent design with the fifth dye reserved for the internal size standard. Sensitivity assays resulted in successful amplification of genomic DNA in the range of 0.250–2.000 ng. A total of 90 individuals from the Chinese Han population were studied using the system and forensic genetic data are presented. We conclude that this integrated system could be an updated and improved solution for forensic DNA analysis.