Loss Of Heterozygosity Analysis Research Articles

Clinical and Molecular-Based Approach in the Evaluation of Hepatocellular Carcinoma Recurrence after Radical Liver Resection.

Simple SummaryHepatocellular carcinoma (HCC) recurrence is still a major issue after liver resection. While several clinical factors were found to be associated with tumor recurrence, HCC pathogenesis is a complex process of accumulation of somatic genomic alterations, which has not been completely understood, especially with respect to tumor recurrence. The aim of this study is to complement potentially predictive clinical factors with next-generation sequencing genomic profiling and loss of heterozygosity analysis. We confirmed that serum bilirubin level, number of HCC nodules and size of the larger nodule are linked to a higher risk of tumor recurrence. Loss of heterozygosity in the PTEN loci was found to be associated with a lower risk of HCC recurrence.Background: Hepatic resection remains the treatment of choice for patients with early-stage HCC with preserved liver function. Unfortunately, however, the majority of patients develop tumor recurrence. While several clinical factors were found to be associated with tumor recurrence, HCC pathogenesis is a complex process of accumulation of somatic genomic alterations, which leads to a huge molecular heterogeneity that has not been completely understood. The aim of this study is to complement potentially predictive clinical and pathological factors with next-generation sequencing genomic profiling and loss of heterozygosity analysis. Methods: 124 HCC patients, who underwent a primary hepatic resection from January 2016 to December 2019, were recruited for this study. Next-generation sequencing (NGS) analysis and allelic imbalance assessment in a case-control subgroup analysis were performed. A time-to-recurrence analysis was performed as well by means of Kaplan–Meier estimators. Results: Cumulative number of HCC recurrences were 26 (21%) and 32 (26%), respectively, one and two years after surgery. Kaplan–Meier estimates for the probability of recurrence amounted to 37% (95% C.I.: 24–47) and to 51% (95% C.I.: 35–62), after one and two years, respectively. Multivariable analysis identified as independent predictors of HCC recurrence: hepatitis C virus (HCV) infection (HR: 1.96, 95%C.I.: 0.91–4.24, p = 0.085), serum bilirubin levels (HR: 5.32, 95%C.I.: 2.07–13.69, p = 0.001), number of nodules (HR: 1.63, 95%C.I.: 1.12–2.38, p = 0.011) and size of the larger nodule (HR: 1.11, 95%C.I.: 1.03–1.18, p = 0.004). Time-to-recurrence analysis showed that loss of heterozygosity in the PTEN loci (involved in the PI3K/AKT/mTOR signaling pathway) was significantly associated with a lower risk of HCC recurrence (HR: 0.35, 95%C.I.: 0.13–0.93, p = 0.036). Conclusions: multiple alterations of cancer genes are associated with HCC progression. In particular, the evidence of a specific AI mutation presented in 20 patients seemed to have a protective effect on the risk of HCC recurrence.

ABCL-349: Genetic Instability of Microsatellite Loci in Primary Mediastinal B-cell Lymphoma

Context: Loss of heterozygosity (LOH) in STR loci, elevated microsatellite alteration at selected tetranucleotides (EMAST) and instability of mononucleotide tandem repeats (MSI - microsatellite instability) are intrinsic features of diverse tumor cells. For primary mediastinal B-cell lymphoma (PMBCL) these molecular factors to determine the prognosis of the disease have not been described yet. Objective: To analyze the frequency of LOH, EMAST, and MSI in tumor cells of PMBCL patients at diagnosis and to check possible association with disease features. Design: STR profiles of the DNA of tumor cells were analyzed on a cohort of 36 PMBCL patients undergoing treatment at the National Research Center for Hematology (Moscow, Russia). Setting: DNA was isolated from tumor biopsy samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission or from the buccal epithelium. STR-profiles for LOH and EMAST analysis were assessed by PCR with COrDIS Plus multiplex kit for amplification of 19 STR-markers and amelogenin loci (Gordiz Ltd, Russia). COrDIS MSI (BAT-25, BAT-26, NR-21, NR-24 and NR-27) kit was used for MSI testing. The fragment analysis was performed on ABI3130 Genetic Analyzer. Patients: Inclusion criteria: de novo diagnosed PMBCL patients. There were 10 males and 26 females, the median age at presentation was 30 years (range 21–61). Main outcome measures: Progression-free survival (PFS) and overall survival (OS) at 24 months from diagnosis were used to assess the independent impact of the genetic instability as a risk factor. Results: LOH was revealed in 21 out of 36 patients (58%). EMAST was found in 18 patients (50%); in 12 of them EMAST was accompanied by LOH, and in one by MSI. MSI was found in three patients only. OS and PFS were highest in the group with stable STR profile of a tumor, but not formally significant because of the small sample size. Conclusions: The high incidence of LOH and EMAST in PMBCL can cause standard therapy failure in a significant number of patients. EMAST as a variant of microsatellite instability might be useful for further consideration as a predictor of response to checkpoint inhibitor therapy.

Genome-Wide Analysis of Mitotic Recombination in Budding Yeast.

DNA break lesions pose a serious threat to the integrity of the genome. Eukaryotic cells can repair these lesions using the homologous recombination pathway that guides the repair reaction by using a homologous DNA template. The budding yeast Saccharomyces cerevisiae is an excellent model system with which to study this repair mechanism and the resulting patterns of genomic change resulting from it. In this chapter, we describe an approach that utilizes whole-genome sequencing data to support the analysis of tracts of loss-of-heterozygosity (LOH) that can arise from mitotic recombination in the context of the entire diploid yeast genome. The workflow and the discussion in this chapter are intended to enable classically trained molecular biologists and geneticists with limited experience in computational methods to conceptually understand and execute the steps of genome-wide LOH analysis as well as to adapt and apply them to their own specific studies and experimental models.

A comprehensive DNA panel next generation sequencing approach supporting diagnostics and therapy prediction in neurooncology

Recent updates in the classification of central nervous system (CNS) tumors have increased the need for molecular testing. Assessment of multiple alterations in parallel, complex combinations of gene sequence and chromosomal changes, as well as therapy prediction by identification of actionable mutations are the major challenges. We here report on a customized next generation sequencing (NGS)-based DNA panel assay that combines diagnostic and predictive testing and -as a comprehensive approach- allows for simultaneous single nucleotide variant (SNP) / small insertion/deletion (InDel), copy number variation (CNV) and loss of heterozygosity (LOH) detection. We analyzed formalin-fixed and paraffin-embedded (FFPE) DNA from a total of 104 patients with CNS tumors. After amplicon capture-based library preparation, sequencing was performed on the relatively cost-efficient Illiumina MiniSeq platform and evaluated with freely available bioinformatical tools. 57 genes for exonic SNP/InDel calling (19 of those in intronic regions for CNV analysis), 3 chromosomal arms and 4 entire chromosomes for CNV and LOH analysis were covered. Results were extensively validated. Our approach yielded high accuracy, sensitivity and specificity. It led to refined diagnoses in a relevant number of analyzed cases, reliably enabled complex subclassifications (e.g. for medulloblastomas) and identified actionable targets for clinical use. Thus, our single-platform approach is an efficient and powerful tool to comprehensively support molecular testing in neurooncology. Future functionality is guaranteed as novel upcoming biomarkers can be easily incorporated in a modular panel design.

Preferentially Paternal Origin of De Novo 11p13 Chromosome Deletions Revealed in Patients with Congenital Aniridia and WAGR Syndrome

The frequency of pathogenic large chromosome rearrangements detected in patients with different Mendelian diseases is truly diverse and can be remarkably high. Chromosome breaks could arise through different known mechanisms. Congenital PAX6-associated aniridia is a hereditary eye disorder caused by mutations or chromosome rearrangements involving the PAX6 gene. In our recent study, we identified 11p13 chromosome deletions in 30 out of 91 probands with congenital aniridia or WAGR syndrome (characterized by Wilms’ tumor, Aniridia, and Genitourinary abnormalities as well as mental Retardation). The loss of heterozygosity analysis (LOH) was performed in 10 families with de novo chromosome deletion in proband. In 7 out of 8 informative families, the analysis revealed that deletions occurred at the paternal allele. If paternal origin is not random, chromosome breaks could arise either (i) during spermiogenesis, which is possible due to specific male chromatin epigenetic program and its vulnerability to the breakage-causing factors, or (ii) in early zygotes at a time when chromosomes transmitted from different parents still carry epigenetic marks of the origin, which is also possible due to diverse and asymmetric epigenetic reprogramming occurring in male and female pronuclei. Some new data is needed to make a well-considered conclusion on the reasons for preferential paternal origin of 11p13 deletions.

Abstract A19: HLA allele-specific loss of heterozygosity detection using augmented exome capture approach

Abstract Loss of human leukocyte antigen (HLA) is of increasing interest as a mechanism of cancer immune evasion and biomarker for cancer immunotherapy response. Each cancer patient has six class I HLA alleles that are capable of presenting a set of tumor-specific neoantigens. However, HLA alleles are often deleted in tumors, resulting in a loss of heterozygosity (LOH). When LOH occurs, neoantigen presentation is significantly impaired, potentially facilitating tumor immune evasion. Given the biologic impact of HLA LOH, there is a need for robust algorithms that can detect allele-specific HLA LOH in tumor samples. Here, we describe a novel computational approach to detect HLA LOH from exome sequencing, demonstrate the robustness of the method, and apply the method to calculate the frequencies of HLA LOH in key cancer types. We performed exome sequencing with augmented HLA region capture on the ImmunoID NeXT platform for tumor and normal samples of 184 patients across several cancer types and identified 430 nonhomozygous HLA genes. Next, we extracted the reads mapping to a custom HLA database and mapped them on to the patient-specific HLA alleles. For each allele, we calculated two key features: 1) the tumor b-allele frequency normalized by the native b-allele frequency and 2) the allele-specific tumor to normal coverage ratio. Using these two features, along with tumor purity and ploidy values, we trained a random forest model on a subset of the HLA genes (n=300). While standard copy-number variant (CNV) tools are unable to detect LOH in the polymorphic HLA genes, they can accurately measure deletions in their flanking regions, which we used to validate the accuracy of our allele-specific HLA LOH algorithm. In our test set (n=130), we found a high concordance between our allele-specific deletion calls and the generic deletion calls (94% accuracy, 0.85 F1 Score). When we constrained our test set to samples with high tumor content (>50%, n=40), we saw even stronger concordance (98% accuracy, 0.95 F1 Score). The only discordant call was a focal deletion within an HLA gene that was detected by our algorithm but missed by the CNV tool. Next, we ran our algorithm on patient samples of different cancer indications. For non-small cell lung cancer, we found a high frequency of patients affected by LOH (35%, 9 of 26), which is similar to frequencies previously reported in the literature. Furthermore, we found a lower frequency of melanoma tumors with LOH (15%, 7 of 48). In conclusion, we developed a novel algorithm to call allele-specific HLA LOH on the ImmunoID NeXT exome sequencing platform that augments coverage in the polymorphic HLA locus and demonstrated overall robust performance. The relatively high frequency of LOH events we detected in the melanoma and lung cancer samples suggests the importance of LOH analysis to inform cancer immunotherapy biomarker studies and personalized cancer therapies that depend on neoantigen presentation. Citation Format: Rachel Marty Pyke, Charles Abbott, Simo V. Zhang, Datta Mellacheruvu, John West, Richard Chen, Sean Michael Boyle. HLA allele-specific loss of heterozygosity detection using augmented exome capture approach [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A19.

Abstract P2-09-04: Reclassification of variant of unknown significance in BRCA1 and BRCA2 genes based on loss of heterozigosity assay

Abstract Variants of unknown significance (VUS) occur in approximately 10% of BRCA1 and BRCA2 genetic testing. Definition of VUS as pathogenic or of benign potential is a crucial step in the reporting and counseling process. Thus limiting the clinical utility of a genetic test and impairs the correct patient management. BRCA1 and 2 are tumor suppressor genes and loss of heterozygosity (LoH) as a second hit for inactivating wildtype allele is a relatively common event in tumor. Previously, in a multi-institutional effort, AC Camargo Cancer Center (Brazil), Barretos Cancer Hospital (Brazil) and UTMD Anderson Cancer Center (USA) compiled a total of 332 BRCA1/2 VUS supposedly from unrelated carriers (46 of BRCA1 and 286 of BRCA2). From them, 256 were distinct VUS (37 in BRCA1 and 219 in BRCA2). Breast cancer sub-classification were collected from medical records and in silico analyzes were performed using four software (SIFT, Polyphen2, CADD and REVEL). Based on this analysis, VUS were categorized into five classes: very high, high, medium, low and very low risk of pathogenicity, according to agreement level among the four in silico software. However, all these analysis are not sufficient for supporting the reclassification of VUS. Therefore we proposed to analyze LoH in matched tumor tissue to assess the pathogenic potential of BRCA1 and 2 VUS. Normal (leucocytes or normal breast tumor tissue) samples, when available, were used for correcting PCR amplification bias between the two alleles. DNA was extracted and subjected to PCR reaction. Amplicons libraries were performed according to manufacturing protocol (Ion Plus Fragment Library Kit) and sequenced on the Ion Proton™ System (ThermoFisher Scientific). Specific variants were called in the TVC program (ThermoFisher Scientific) with standard criteria. A total of 12 VUS were investigated (5 in BRCA1 and 7 in BRCA2). LoH was detected in 7 (60%) tumor samples (3 in BRCA1 and 4 in BRCA2). Of these, 4 VUS were classified as very high or high risk of pathogenicity [2 in BRCA1 (BR1p.Y1703C; BR1p.S1655P) and 2 in BRCA2 (BR2p.S2670L; BR2p.E3002K)]. Variant of allele frequency (VAF) of 94, 65, 70 and 75%, respectively, were detected in tumor tissues. Additionally, recent functional assays described these 4 VUS as non-functional or deleterious variants showing complete agreement with the current LoH results. According to ACMG guidelines considering all available data, LoH, deleterious function and concordant pathogenicity probability. These 4 VUS might be reclassified as pathogenic variants. On the other hand, the same combined analysis did not allow reclassification of 3 other VUS. Although VAF were 74, 73 and 70% respectively, they were previously classified as very or low risk of pathogenicity and there are no functional assays describing them until now. Our results showed that LoH analyses can substantially contribute to VUS reclassification, improving the management and counseling of BRCA1/2 VUS-carriers. Citation Format: Dirce M Carraro, Edenir I Palmero, Carolina M Berra, Giovana T Torrezan, Rafael C Brianese, Gabriela C Fernandes, Angelica M Gutierrez Barrera, Henrique C Galvo, Maria NC Formiga, Banu K Arun. Reclassification of variant of unknown significance in BRCA1 and BRCA2 genes based on loss of heterozigosity assay [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-09-04.

Detection of Loss of Heterozygosity in Tk-Deficient Mutants from L5178Y Tk+/--3.7.2C Mouse Lymphoma Cells.

The mouse lymphoma assay (MLA), a forward mutation assay using the Tk+/--3.7.2C clone of the L5178Y mouse lymphoma cell line and the Thymidine kinase (Tk) gene, has been widely used as an in vitro genetic toxicity assay for more than four decades. The MLA can evaluate the ability of mutagens to induce a wide range of genetic events including both gene mutations and chromosomal mutations and has been recommended as one component of several genotoxicity test batteries. Tk-deficient mutants often exhibit chromosomal abnormalities involving the distal end of chromosome 11 where the Tk gene is located, in mice, and the type of chromosome alteration can be analyzed using a loss of heterozygosity (LOH) approach. LOH has been considered an important event in human tumorigenesis and can result from any of the following several mechanisms: large deletions, mitotic recombination, and chromosome loss. In this chapter, the authors describe the procedures for the detection of LOH in the Tk mutants from the MLA, and apply LOH analysis for understanding the types of genetic damage that is induced by individual chemicals.

Detection of Loss of Heterozygosity in cfDNA of Advanced EGFR- or KRAS-Mutated Non-Small-Cell Lung Cancer Patients.

Liquid biopsy is currently approved for management of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) patients. However, one unanswered question is whether the rate of cell-free DNA (cfDNA)-negative samples is due to technical limitations rather than to tumor genetic characteristics. Using four microsatellite markers that map specific chromosomal loci often lost in lung cancer, we conducted a pilot study to investigate whether other alterations, such as loss of heterozygosity (LOH), could be detected in EGFR-negative cfDNA. We analyzed EGFR-mutated NSCLC patients (n = 24) who were positive or negative for EGFR mutations in cfDNA and compared the results with a second cohort of 24 patients bearing KRAS-mutated cancer, which served as a representative control population not exposed to targeted therapy. The results showed that in EGFR-negative post-tyrosine-kinase-inhibitor (TKI) cfDNAs, LOH frequency was significantly higher than in both pre- and post-TKI EGFR-positive cfDNAs. By contrast, no association between KRAS status in cfDNA and number of LOH events was found. In conclusion, our study indicates the feasibility of detecting LOH events in cfDNA from advanced NSCLC and suggests LOH analysis as a new candidate molecular assay to integrate mutation-specific assays.

INTEROBSERVER AGREEMENT IN ORAL DYSPLASIA GRADING, ASSOCIATED WITH PROTEIN EXPRESSION AND LOSS OF HETEROZIGOSITY

Oral leukoplakia (OL) may precede squamous cell carcinoma and its histologic grading is subjective. Loss of heterozygosity (LOH) has been reported as risk predictor of OL malignant transformation. Objective: Correlate histologic and immunohistochemical characteristics of OL and evaluate molecular aspects, aiming at to demonstrate their malignant potential. Study Design: Ninety-six cases of OL were stratified by the World Health Organization (WHO) and Kujan systems by 3 blinded pathologists and correlated to the immunohistochemical biomarkers (IBs) p53, Ki-67, pRb, and p16. In 25 cases, LOH analysis was performed. Results: A moderate concordance among pathologists in both systems (Kendall correlation) was observed. Weak to moderate correlation among IBs was observed, as well as among IBs and histologic grading (Spearman correlation). Staining for p16 showed agreement with all the others IBs. Ki-67 was the most concordant marker in Kujan's system. The LOH was found in moderate and severe dysplasia for the markers TP53 (17 p13.1) and IFNA (9 p21), in 2 cases each. Conclusions: Greater concordance among pathologists as well as between histologic grading and IBs was demonstrated by Kujan's system. In OL, p16 can express alone the diagnoses obtained by all others IBs together. LOH could be related to the degree of histopathologic severity and to the greater expression of IBs.

Loss of Heterozygosity of FLT3-ITD Is Common in Acute Myeloid Leukemia and May be a More Consistent Prognostic Marker Than FLT3-ITD Allele Frequency

Loss of Heterozygosity of FLT3-ITD Is Common in Acute Myeloid Leukemia and May be a More Consistent Prognostic Marker Than FLT3-ITD Allele Frequency

Loss of heterozygosity and immunoexpression of PTEN in oral epithelial dysplasia and squamous cell carcinoma

IntroductionOral epithelial dysplasia (OED) is a risk factor for developing subsequent oral squamous cell carcinoma (OSCC). Loss of heterozygosity (LOH) profiles have been validated as risk predictors of malignant transformation of OED. It is still unclear if Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) allelic loss also occurs in initial stage malignant lesions and if the allelic loss is involved as one of the mechanisms of oral carcinogenesis. Thus, this study objective investigate LOH of PTEN gene and the immunohistochemical expression of the protein in OED and OSCC samples. Material and methodsFormalin-fixed paraffin-embedded samples of 19 OEDs and 16 OSCCs were included to immunohistochemistry and LOH analysis. Two polymorphic microsatellite markers (AFMA086WG9 and D10S1765) located in chromosome 10 were used in this study for LOH analysis. For immunohistochemical analysis, 5 random fields with 400× magnification were evaluated quantitatively and qualitatively in epithelial and neoplastic cells. ResultsAFMA086WG9 marker only demonstrated LOH in OEDs cases (10.5%). D10S1765 marker demonstrated LOH in 57.2% of OEDs and 50% of OSCCs. Higher nuclear immunostaining was detected in cases of OSCCs when compared to OEDs (p < .001) and there was strong cytoplasmic immunoexpression in OSCCs (p < .045). ConclusionsWe provide evidence that the allelic loss of PTEN is present in premalignant oral lesions and OSCCs, however the LOH of PTEN does not seems to influence its protein expression.

Homologous recombination deficiency (HRD): A biomarker for first-line (1L) platinum in advanced pancreatic ductal adenocarcinoma (PDAC).

4132 Background: HRD is an emerging biomarker for platinum therapy in PDAC. The clinical implications regarding differences in outcome between germline and somatic HRD in advanced PDAC treated with 1L platinum is unexplored. Methods: We evaluated overall survival (OS) for advanced PDAC (stage III/IV) based on their pathogenic germline (gHRD) and somatic HRD (sHRD) using integrated genomic profiling from MSK-IMPACT and 1L platinum use. HRD defined by pathogenic alterations from the following genes: BRCA1/2, PALB2, ARID1A/B/2, ATR, ATRX, ATM, BAP1, RAD50/51C/D, BRIP1, NBN, CHECK1/2, FANCA/C, CDK12, and MRE11. Results: Advanced PDAC patients (n=461) treated at MSK enrolled in a prospective database, were evaluated. Median follow-up was 27.6 months (95% CI, 24.6-30.6). Both germline and somatic profilings were available for n=350 (76%) but only somatic profiling was available for n=111 (24%). We identified n=52 patients with gHRD (11.3%), n=42 patients with sHRD (9.1%), and 48 patients with somatic VUS for HRD genes. From all 461 patients, the OS was not different between 1L non-platinum vs. 1L platinum groups (19 M vs. 19.3 M), regardleess of their HRD status. (Table) The OS was superior for gHRD vs. non-gHRD (28.7 M vs. 18.2 M), regardless of 1L treatment choice. However, similar significant OS superiority was neither observed in sHRD vs. non-sHRD, nor in VUS sHRD vs. non-VUS sHRD. In a subgroup analysis of 1L platinum treated patients, the OS was superior in gHRD vs. non-gHRD (NR vs. 17.9 M); however, there was no OS difference between sHRD and non-sHRD. Conclusions: In advanced PDAC patients, only gHRD predicted better overall survival for first-line platinum chemotherapy. These findings emphasize the importance of germline mutation testing of HRD in PDAC. Biomarker validation and functional definition of HRD such as loss of heterozygosity analysis is underway. [Table: see text]

Limit of quantitation of anti-thyroglobulin antibodies measurement in the new platform alinity i for application in differentiated thyroid cancer follow-up

Barrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma. For this reason, endoscopic-based surveillance protocols have been developed. This prevention program is, however, burdensome for the patients and expensive for the national health systems. Thus, diagnostic strategies with a low invasiveness and a reduced economic impact are required. This study investigated the power of plasma circulating free DNA (cfDNA) in predicting neoplastic transformation in the natural history of two BE patients who progressed to esophageal adenocarcinoma. Longitudinally collected DNAs from plasma and paired formalin fixed paraffin embedded samples were examined for both loss of heterozygosity (LOH) in areas proximal to TP53, FHIT and BRCA2 genes, and mutations in TP53 gene. Results showed that: (i) early BE molecular alterations are mainly localized proximal to, or within, TP53 gene; (ii) LOH events present in cfDNA not only retrace the time-matched biopsy profile but better represent the total alterations of the BE epithelium. In conclusion, our findings suggested that LOH analysis in plasma cfDNA could represent an additional, less invasive, diagnostic tool to monitor neoplastic progression of BE epithelium.

Genetics of aldosterone-producing adenomas with pathogenic KCNJ5 variants.

Somatic variants in genes that regulate intracellular ion homeostasis have been identified in aldosterone-producing adenomas (APA). Although the mechanisms leading to an increased aldosterone production in APA cells has been well studied, the molecular events that cause cell proliferation and tumor formation are poorly understood. In the present study, we have performed whole exome sequencing (WES) to characterize the landscape of somatic alterations in a homogeneous series of APA with pathogenic KCNJ5 variants. In the WES analysis on eleven APA, 84 exonic somatic events were called by 3 different somatic callers. Besides the KCNJ5 gene, only two genes (MED13 and ZNF669) harbored somatic variants in more than one APA. Unlike adrenocortical carcinomas, no chromosomal instability was observed by the somatic copy-number alteration and loss of heterozygosity analyses. The estimated tumor purity ranged from 0.35 to 0.67, suggesting a significant proportion of normal cell infiltration. Based on the results of PureCN analysis, the KCNJ5 variants appear to be clonal. In conclusion, in addition to KCNJ5 somatic pathogenic variant, no significant somatic event that would obviously explain proliferation or tumor growth was observed in our homogeneous cohort of KCNJ5-mutated APA. The molecular mechanisms causing APA growth and tumorigenesis remain to be elucidated.

Whole-Genome Single Nucleotide Polymorphism Microarray for Copy Number and Loss of Heterozygosity Analysis in Tumors.

The basis of cancer biology is built upon two fundamental processes that result in uncontrolled cell proliferation and tumor formation: loss of tumor suppressor gene function and gain of oncogene function. Somatic DNA copy number variants (CNVs), which generally range in size from kilobases to entire chromosomes, facilitate gains and losses of chromosomal material incorporating oncogenes and tumor suppressor genes, respectively. In fact, many cancer types are characterized by DNA copy number changes and relatively few single nucleotide mutations (Ciriello et al. Nat Genet 45:1127-1133, 2013). Currently, the optimal method to detect such somatic copy number changes across the cancer genome is whole-genome single nucleotide polymorphism (SNP) microarray analysis.

Characterization of epithelial oral dysplasia in non-smokers: First steps towards precision medicine

ObjectivesTobacco usage is the strongest risk factor in the development of oral squamous cell carcinoma (OSCC), which mandates careful screening for oral cancers in smokers. However, there are indications that oral potentially malignant lesions, such as oral epithelial dysplasia (OED), in non-smokers (NS) have a higher cancer risk than those in smokers. Without tobacco as an etiology, the development of these lesions in NS may suggest genetic susceptibility. The increasing incidence of OSCC in NS calls for a better understanding of the natural history of OED in NS as compared to that of smokers. Materials and methodsPatients from a population-based longitudinal study with more than 10 years of follow up were analyzed. Of the 455 patients with primary OED (233 mild and 212 moderate dysplasia), 139 were NS and 306 were smokers. Demographic and habit information, clinical information (lesion site, size and appearance; toluidine blue and fluorescent visualization), microsatellite analysis for loss of heterozygosity (LOH) and outcome (progression) were compared between the two groups. Results and conclusionsThe majority of patients with OED were smokers. Of these, more were males, non-Caucasians and heavy drinkers. A significantly higher number of OED in NS were in the tongue, whereas a significantly higher number of OED in smokers were in the floor of mouth (FOM). OED in NS showed a greater than 2-fold increase in cancer progression. Strikingly, OED located in the FOM in NS showed a 38-fold increase in cancer progression as compared to those in smokers.

Loss of heterozygosity (LOH) Analysis in individuals with developmental delay/congenital anomalies previously investigated for 22q11.2 Deletion Syndrome

LLoss of heterozygosity (LOH) also called absence of heterozygosity (AOH) or regions of homozygosity (ROH) can be detected by SNP-array analysis. It is observed in regions identical by descent or occurs because of errors in the mechanisms of recombination between homologous chromosomes, DNA replications or DNA repair. The 22q11.2 deletion syndrome (22q11.2DS) has a prevalence of 1/4000 live births and an extensive clinical variability. About 30% of individuals with 22q11.2DS clinical suspicion have been diagnosed with the typical 22q11.2 deletion and other genomic imbalances are often reported related to similar phenotypes. However, most of patients remain without an etiological diagnosis. Currently, there are few studies about LOH in individuals with developmental delay/congenital anomalies. The aim of this study is to describe the preliminary results of LOH analysis in individuals with clinical suspicion of 22q11.2DS. Inclusion criteria was individuals with clinical suspicion without the typical 22q11.2 deletions, previously tested by Fluorescent in situ Hybridization (FISH) or Multiplex Ligation Probe-dependent Amplification (MLPA). Exclusion criteria was parental consanguinity. A total of 124 individuals were investigated by chromosomal microarray analysis (CMA) using the CytoScan HD or 750K Array (Affymetrix®). Data were compared with a database composed by 114 control individuals from Brazilian general population. We considered only LOH biggest than 3Mb and not frequent (&lt;1%) in control individuals. The presence of LOH was more often in the patients than in the control group (12,1% against 3,5%). In the patient group the LOH size were between 3,3Mb and 46,2Mb (average size 22,7Mb) and in the control group it was between 3,1Mb and 8,5Mb (average size 6,0Mb). It was observed only one large LOH for almost all patients, and for almost all the control group it was observed two small LOHs. Among patients with LOH, six also carried VOUS (variants of uncertain significance) and one had a pathogenic imbalance. Laboratorial confirmations and genotype-phenotype correlation are in progress. Our results clearly show a higher proportion and bigger size of LOHs in individuals with developmental delay/congenital anomalies and a possible applicability of LOH analysis as a diagnostic approach in individuals with the same characteristics of the present sample.Support: Fapesp and CNPq

Microsatellite Alterations and Protein Expression of 5 Major Tumor Suppressor Genes in Gastric Adenocarcinomas

PURPOSE: In gastric adenocarcinoma (GC), the major tumor suppressor genes (TSGs) such as p16, PTEN, Rb, E-cadherin, and p53, may play important roles in various regulatory pathways and in tumor suppression. This study evaluated the loss of heterozygosity (LOH) of microsatellite and protein expression of 5 TSGs and the results were examined for their correlation with clinicopathological factors. METHODS: LOH analysis was carried out using polymerase chain reactions with 15 polymorphic microsatellite markers of 5 chromosomes containing TSGs in 100 surgically resected tumors. Protein expression was evaluated by immunohistochemistry (IHC). RESULTS: LOH was detected in 83% of GCs. LOH of 9p21, 10q23, 13q14, 16q22, and 17p13 were detected in 26%, 31%, 24%, 22%, and 35% of cases, respectively. Protein expression of p16, PTEN, Rb, E-cadherin, and p53 were found to be 31%, 39%, 28%, 32%, and 46% of cases. Advanced GCs showed significantly higher rates of 17p13 LOH and p53 expression. 9p21 LOH and E-cadherin IHC were correlated with higher tumor grade. Lymph node metastasis was correlated with the LOH of 9p21, 16q22, and 17p13 and IHC of the Rb and p53. A higher stage was correlated with 10q23 and 17p13 in LOH and p53 for IHC. CONCLUSION: These results suggest that LOH and protein expression of various TSGs are important in carcinogenesis and tumor invasion. Additionally, LOH and IHC may be useful clinical indicators for determining the prognosis of patients with GCs. In particular, the 17p13 LOH and p53 for IHC can be applied as simple evaluations in the clinic.

Major Histocompatibility Mismatch and Donor Choice for Second Allogeneic Bone Marrow Transplantation

Large alternative donor pools provide the potential for selecting a different donor for a second allogeneic (allo) bone or marrow transplant (BMT). As HLA disparity may contribute to the graft-versus-tumor effect, utilizing new mismatched haplotype donors may potentially improve the antitumor activity for relapsed hematologic malignancies despite a previous alloBMT. Data from patients who received a second alloBMT for relapsed hematologic malignancies at Johns Hopkins were analyzed. Outcomes were compared between patients who received a second allograft with the same MHC composition and those who received an allograft with a new mismatched haplotype. Loss of heterozygosity analysis was performed for patients with acute myeloid leukemia (AML) whose first allograft was haploidentical. Between 2005 and 2015, 40 patients received a second BMT for a relapsed hematologic malignancy. The median follow-up is 750 (range, 26 to 2950) days. The median overall survival (OS) in the cohort is 928 days (95% confidence interval [CI], 602 to not reached [NR]); median event-free survival (EFS) for the cohort is 500 days (95% CI, 355 to NR). The 4-year OS is 40% (95% CI, 25% to 64%), and the 4-year EFS is 36% (95% CI, 24% to 55%). The cumulative incidence of nonrelapsed mortality by 2 years was 27% (95% CI, 13% to 42%). The cumulative incidence of grade 3 to 4 acute graft-versus-host disease (GVHD) at 100 days was 15% (95% CI, 4% to 26%); the cumulative incidence of extensive chronic GVHD at 2 years was 22% (95% CI, 9% to 36%). The median survival was 552 days (95% CI, 376 to 2950+) in the group who underwent transplantation with a second allograft that did not harbor a new mismatched haplotype, while it was not reached in the group whose allograft contained a new mismatched haplotype (hazard ratio [HR], .36; 95% CI, .14 to .9; P = .02). EFS was also longer in the group who received an allograft containing a new mismatched haplotype, (NR versus 401 days; HR, .50; 95% CI, .22 to 1.14; P = .09). Although the allograft for this patient's second BMT contained a new mismatched haplotype, AML nevertheless relapsed a second time. Second BMTs are feasible and provide a reasonable chance of long-term survival. An allograft with a new mismatched haplotype may improve outcomes after second BMTs for relapsed hematologic malignancies.