Unique Gene Mutations Research Articles

Utility of UV Signature Mutations in the Diagnostic Assessment of Metastatic Head and Neck Carcinomas of Unknown Primary.

Metastatic carcinoma of unknown primary origin to the head and neck lymph nodes (HNCUP) engenders unique diagnostic considerations. In many cases, the detection of a high-risk human papillomavirus (HR-HPV) unearths an occult oropharyngeal squamous cell carcinoma (SCC). In metastatic HR-HPV-independent carcinomas, other primary sites should be considered, including cutaneous malignancies that can mimic HR-HPV-associated SCC. In this context, ultraviolet (UV) signature mutations, defined as ≥ 60% C→T substitutions with ≥ 5% CC→TT substitutions at dipyrimidine sites, identified in tumors arising on sun exposed areas, are an attractive and underused tool in the setting of metastatic HNCUP. A retrospective review of institutional records focused on cases of HR-HPV negative HNCUP was conducted. All cases were subjected to next generation sequencing analysis to assess UV signature mutations. We identified 14HR-HPV negative metastatic HNCUP to either the cervical or parotid gland lymph nodes, of which, 11 (11/14, 79%) had UV signature mutations, including 4 (4/10, 40%) p16 positive cases. All UV signature mutation positive cases had at least one significant TP53 mutation and greater than 20 unique gene mutations. The management of metastatic cutaneous carcinomas significantly differs from other HNCUP especially metastatic HR-HPV-associated SCC; therefore, the observation of a high percentage of C→T with CC →TT substitutions should be routinely incorporated in next generation sequencing reports of HNCUP. UV mutational signatures testing is a robust diagnostic tool that can be utilized in daily clinical practice.

Genomic alterations in oral multiple primary cancers

Oral squamous cell carcinoma (OSCC) is the predominant type of oral cancer, while some patients may develop oral multiple primary cancers (MPCs) with unclear etiology. This study aimed to investigate the clinicopathological characteristics and genomic alterations of oral MPCs. Clinicopathological data from patients with oral single primary carcinoma (SPC, n = 202) and oral MPCs (n = 34) were collected and compared. Copy number alteration (CNA) analysis was conducted to identify chromosomal-instability differences among oral MPCs, recurrent OSCC cases, and OSCC patients with lymph node metastasis. Whole-exome sequencing was employed to identify potential unique gene mutations in oral MPCs patients. Additionally, CNA and phylogenetic tree analyses were used to gain preliminary insights into the molecular characteristics of different primary tumors within individual patients. Our findings revealed that, in contrast to oral SPC, females predominated the oral MPCs (70.59%), while smoking and alcohol use were not frequent in MPCs. Moreover, long-term survival outcomes were poorer in oral MPCs. From a CNA perspective, no significant differences were observed between oral MPCs patients and those with recurrence and lymph node metastasis. In addition to commonly mutated genes such as CASP8, TP53 and MUC16, in oral MPCs we also detected relatively rare mutations, such as HS3ST6 and RFPL4A. Furthermore, this study also demonstrated that most MPCs patients exhibited similarities in certain genomic regions within individuals, and distinct differences of the similarity degree were observed between synchronous and metachronous oral MPCs.

Genomic Alterations Correlated to Trastuzumab Resistance and Clinical Outcomes in HER2+/HR- Breast Cancers of Patients Living in Northwestern China.

Anti-HER2 therapy has significantly improved the survival rates of patients with HER2+ breast cancer. However, a subset of these patients eventually experience treatment failure, and the underlying genetic mechanisms remain largely unexplored. This underscores the need to investigate the genomic heterogeneity of HER2+ breast cancer. In this study, we focus on HER2+/HR- breast cancer, as it differs from HER2+/HR+ breast cancer in terms of genetic and biological characteristics. We performed gene-targeted genome sequencing on 45 HER2+/HR- breast cancer samples and identified 650 mutations across 268 cancer-related genes. TP53 (71.1%) and PIK3CA (35.6%) were the most frequently mutated genes in our sample. Additionally, ERBB2 (77.8%), CDK12 (42.2%), and MYC (11.1%) exhibited a high frequency of copy number amplifications (CNAs). Comparative analysis with two other HER2+/HR- breast cancer cohorts revealed that our cohort had higher genetic variation rates in ARID1A, PKHD1, PTPN13, FANCA, SETD2, BRCA2, BLM, STAG2, FAT1, TOP2A, POLE, ATM, KMT2B, FGFR4, and EPAS1. Notably, in our cohort, NF1 and ATM mutations were more prevalent in trastuzumab-resistant patients (NF1, p=0.016; ATM, p=0.006) and were associated with primary trastuzumab resistance (NF1, p=0.042; ATM, p=0.021). Moreover, patients with NF1 mutations (p=0.009) and high histological grades (p=0.028) were more likely to experience early relapse. Ultimately, we identified a unique cancer-related gene mutation profile and a subset of genes associated with primary resistance to trastuzumab and RFS in patients with HER2+/HR- breast cancer in Northwest China. These findings could lay the groundwork for future studies aimed at elucidating the mechanisms of resistance to trastuzumab and improving HER2-targeted treatment strategies.

Dominant Optic Atrophy With Varied Phenotype in a Family of Eastern India: A Case Report of a Rare Mutation

Background: Autosomal dominant optic atrophy (ADOA) can present with a varied phenotype in the form of pure ADOA, ADOA with sensorineural hearing loss, or the extended spectrum of DOA plus syndrome. Multiple genes have been identified over the years and majority of the cases are attributed to OPA1 gene mutation. Here we present an index case of DOA plus syndrome with affection of multiple family members across 2 generations with a rare mutation detection. Case Presentation: A middle-aged female presented with onset of bilateral progressive visual blurring in the first decade followed by progressive sensorineural hearing loss since the second decade and onset of lower limb predominant cerebellar ataxia since the third decade. Routine parameters, imaging and electrophysiological studies were negative. Next generation clinical exome sequencing (NGCES) revealed an unique OPA1 gene mutation with worldwide evidence of only 3 such cases in database. Conclusion: DOA suspicion should be made in early onset visual defects with multiaxial involvement with a significant family history.

Unique Spectrum of Driver Gene Mutations in Patients with Non-Small Cell Lung Carcinoma (NSCLC) from Coal-manufacturing Districts in Southwest China

IntroductionThe coal-manufacturing districts in the Eastern Yunnan province of Southwest China are the utmost occurrence and death of lung tumors. As previously reported on unique clinical characteristics of non-small cell lung cancer (NSCLC) in patients from these regions without a clear understanding of the aetiology and molecular characteristics. We aim to identify the unique driver gene mutations spectrum.Material and methodsSamples from 1120 NSCLC patients from Eastern Yunnan were gathered for next-generation sequencing. Seventeen gene targets were sequenced. We have compared individuals' medical and genetic features from the coal- and non-coal-manufacturing zones.ResultsThe mutation rates of EGFR (L858R, 19-Del, G719X+L861X, L858R+EGFR amplification) and ERBB2 (20ins) were low in patients from coal-manufacturing regions. Interestingly, EGFR (G719X, S768I, G719X+S768I), KRAS (G12C), TP53 (R158L) and NTRK3 demonstrated a much higher mutation frequency. Furthermore, EGFR compound mutations were linked with the patient's job and TNM staging IIIb-IV. The OncodriverCLUST algorithm authenticated 6 genes (KRAS, EGFR, ROS1, NRAS, BRAF, and ERBB2) as driver genes in patients from coal manufacturing. EGFR with KRAS, BRAF, RET, and TP53 with ALK and KRAS were mutually exclusive. Mutations in the TP53 signaling pathway were the most common in NSCLC patients from the coal-fabricating districts.ConclusionsOur analyses confirmed the unique spectrum of driver genetic mutations and emphasized the potential of future targeted therapy in NSCLC patients from the coal-manufacturing districts of Eastern Yunnan. Our data broaden the view of NSCLC pathogenesis and its relationship with the environmental conditions in coal-producing regions.

Agreement between CBNAAT, liquid culture and line probe assay for detection of Mycobacterium tuberculosis and anti-tubercular drug resistance in extrapulmonary samples

PurposeCartridge based nucleic acid amplification test (CBNAAT) has been endorsed by the WHO as the screening test for diagnosing extrapulmonary tuberculosis (EPTB). In the present study we report the agreement between CBNAAT (Xpert MTB/RIF), liquid culture (LC) and line probe assay (LPA) for diagnosis of Mycobacterium tuberculosis and detection of drug resistance among EPTB cases. MethodsThe EP samples were subjected to CBNAAT (Xpert MTB/RIF, Cepheid, USA) and wherever possible, to LC (MGIT 960, Becton Dickinson, USA) followed sequentially by first line and second line-LPA (FL-LPA, SL-LPA, Hain Lifescience, Germany) on the isolates. ResultsTotal 566/4080 (13.9%) EP samples were detected positive for M. tuberculosis on CBNAAT. Aspirates from lymph nodes were most often positive (11/30; 36.6%), followed by pus (240/873; 27.5%) and CSF samples (166/104; 15.8%). The detection of M. tuberculosis was more in adults than children except in tissue biopsy samples. Rifampicin resistance was also higher among adults except CSF in which resistance was more in children. Total 185 of 566 (32.7%) CBNAAT positive and 770 of 3510 (21.9%) CBNAAT negative samples could be cultured of which 110/185 (59.4%) and 33/770 (4.3%) respectively turned positive. FL-LPA and SL-LPA of 143 culture isolates showed that 27 isolates had drug resistance, of which 3 (2.1%) were XDR, 11 (7.7%) were Pre-XDR (FQ) and 13 (9.1%) were MDR. Of these 27 resistant isolates, 12 were negative by CBNAAT and two were mislabeled as Rifampicin sensitive or indeterminate based on the unique RpoB gene mutation patterns on LPA. The positive and negative agreements between LC and CBNAAT for detection of M. tuberculosis were 67.1% and 92.7% respectively and between LPA and CBNAAT for rifampicin resistance detection were 98.9% and 92.9% respectively. ConclusionsFor EPTB, CBNAAT should be accompanied with LC wherever possible irrespective of the CBNAAT result.

Mutation Analysis of Second Primary Tumors in Oral Cancer in Taiwanese Patients through Next-Generation Sequencing.

Head and neck cancer has poor overall survival. Patients with head and neck cancer more frequently develop second primary tumors than do patients with other cancers, leading to a poor prognosis. In this study, we used next-generation sequencing to analyze and compare mutations between first tumors and second tumors in oral cancer. We retrieved tumor tissues collected from 13 patients who were diagnosed twice as having cancer. We used driver gene and trunk mutations to distinguish between recurrent cancer and primary cancer in oral cancer. We observed unique driver gene mutations in three patients with an initial clinical diagnosis of recurrent cancer; hence, we believe that the corresponding patients had primary cancer. Four patients with an initial clinical diagnosis of primary cancer were found to actually have recurrent cancer according to our results. Genetic testing can be used to enhance the accuracy of clinical diagnosis.

Genome Profiling of SARS-CoV-2 in Indonesia, ASEAN and the Neighbouring East Asian Countries: Features, Challenges and Achievements

Whole-genome sequencing (WGS) has played a significant role in understanding the epidemiology and biology of SARS-CoV-2 virus. Here, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asian countries as a genomic surveillance during the COVID-19 pandemic. Nottingham–Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative has facilitated collaboration between the University of Nottingham and a team in the Research Center for Biotechnology, National Research and Innovation Agency (BRIN), to carry out a small number of SARS-CoV-2 WGS in Indonesia using Oxford Nanopore Technology (ONT). Analyses of SARS- CoV-2 genomes deposited on GISAID reveal the importance of clinical and demographic metadata collection and the importance of open access and data sharing. Lineage and phylogenetic analyses of two periods defined by the Delta variant outbreak reveal that: (1) B.1.466.2 variants were the most predominant in Indonesia before the Delta variant outbreak, having a unique spike gene mutation N439K at more than 98% frequency, (2) Delta variants AY.23 sub-lineage took over after June 2021, and (3) the highest rate of virus transmissions between Indonesia and other countries was through interactions with Singapore and Japan, two neighbouring countries with a high degree of access and travels to and from Indonesia.

Analysis of GJB2 Gene Mutations in 1330 Deafness Cases of Major Ethnic Groups in Northwest China.

Background: The GJB2 gene is the most common deafness gene, and epidemic characteristics have obvious racial specificity. Our study aimed to investigate the prevalence and ethnic specificity of the GJB2 gene in deafness in major ethnic groups in Northwest China, evaluate the value of molecular screening for deafness in minority populations, and explore the strategies and methods for genetic diagnosis. Methods: Ethics approval was obtained to collect 1330 cases of moderate to very severe nonsyndromic sensorineural deafness in northwestern China. The mutation characteristics of ethnic minorities were analyzed and compared with those of 464 patients with nonsyndromic sensorineural deafness among ethnic Han in the northwestern from research group by Sequence Scanner V25.0. Then, we analyzed the ethnic specificity of the mutations. Results: A total of 15 GJB2 sequence changes were detected in 1330 minority patients. The study showed that the allele frequency in Tibetan patients was significantly lower than that in Hui and Dongxiang patients, that in Uygur patients was significantly lower than that in Han and Hui patients, and that in Kazak and Tibetan patients was significantly lower than that in Han patients, and the differences between other ethnic groups were not statistically significant. Each ethnic group has a unique GJB2 gene mutation spectrum, and its hotspot mutation distribution has its own characteristics, with c.235delC, c.109 G > A, c.299-300delAT, and c.35delG being common. Conclusions: It has been confirmed that GJB2 gene mutation has a high prevalence in patients with nonsyndromic sensorineural hearing loss in Northwest China. Each ethnic group has a unique mutation spectrum for the GJB2 gene, which is related to its genetic background. It is necessary to develop a corresponding gene diagnosis strategy according to the hotspot mutations and mutation spectrum of each ethnic group.

The beta-glucuronidase intracisternal A particle insertion model results in similar overall MPSVII phenotype as the single base deletion model when on the same C57BL/6J mouse background

Two unique gene mutations in the enzyme beta-glucuronidase (GUSB) that result in the lysosomal storage disease Mucopolysaccharidosis (MPS) type VII had previously been reported to have differing disease phenotype severities when compared on differing mouse strains. The MPSVII mouse has proven to be a highly efficacious model to study mucopolysaccharidoses and for evaluating potential gene or stem cell therapies for lysosomal storage diseases. We examined the single base pair deletion (MPSVII) and the intracisternal A particle element insertion (MPSVII2J) in GUSB compared with control animals by skeletal measures, electroretinography, auditory-evoked brainstem response and life span on a C57BL/6J background strain. In all measures, both mutations result in either a trend toward or significant changes from the background strain control. In all measures, there is no significant phenotypic difference between the two mutations. The 2J variant is a more easily genotyped and equally affected phenotype, which holds promise for further studies of chimerism and stem cell therapy approaches.

Analysis of tumor heterogeneity at the molecular level: a case report of efficacy separation in metastatic breast cancer

: Although breast cancer frequently metastasizes to the lungs, rarely breast cancer patients develop separation of the efficacy of different lesions in pulmonary metastases. Differentiation at genetic level between drug-resistant lesion and sensitive lesion may be very challenging but is essential for prognostic and therapeutic considerations. In the present case study of a 51-year-old patient who had recurrent disease with pulmonary metastases after a left modified radical mastectomy three years later. Her two lesions of left pulmonary metastatic disease had a separation of therapeutic effects by treatment with albumin paclitaxel in imaging. Histological and immunohistochemical finds suggested that these two lesions were not inconsistent. A total of 230 cancer predisposition genes were analyzed by next generation sequencing and 25 gene mutations were detected, including MUTYH, ALK, MSH6, PIK3CA, TP53, AR, BRAF, EGFR, EPHX1, NRAS, RET, STK11, FBXW7, CD274 and MET were identified. The unique gene mutations of drug-resistant lesion were: CD274, MET, EPHX1, FBXW7 and TP53. The mutation genes specific to sensitive lesions were: KDR, FGFR4, CDH1, FGFR2, PTEN and SLC22A4. Which indicated that there was a difference between the two lesions at the genetic level. Even if there are efficacy separation in different metastatic lesions of the same organ in the same patient, the underlying cause may be genetic differences. Moreover, gene sequencing is also a supplement to traditional pathology. In summary, our results support the usage of next generation sequencing as a supplementary tool, assisting in revealing genetic heterogeneity. This is the first case report, to our knowledge, of difference between lesions in lung metastases, suggesting the existence of a patient with distant metastases who would be different for therapy as a result of carrying heterogeneity.

Inherited Cholestatic Diseases in the Era of Personalized Medicine.

http://aasldpubs.onlinelibrary.wiley.com/hub/journal/10.1002/(ISSN)2046-2484/video/15-3-reading-mack a video presentation of this article.

Mutant U2AF1S34F and U2AF1Q157P Induce Distinct RNA Splicing and Hematopoietic Phenotypes In Vivo

Myelodysplastic syndromes (MDS) are the most common myeloid malignancies among the elderly. Patients present with bone marrow (BM) failure manifested by low peripheral blood (PB) counts and are at increased risk of developing acute myeloid leukemia. Mutations of U2AF1, a gene that encodes a spliceosome protein, are identified in 11% of MDS patients. The two most common U2AF1 mutants, S34F and Q157P, alter the splicing of two distinct sets of pre-mRNA targets in vitro and each co-occur with unique gene mutations in MDS patients, suggesting these mutants may affect MDS pathogenesis differently. In mice, U2AF1S34F expression leads to altered splicing, reduced B-cell counts, and features of MDS. Similar studies have not been performed for U2AF1Q157P. To study the impact of U2AF1Q157P expression on splicing and hematopoiesis in vivo, we created a doxycycline (DOX)-inducible ("Tet-On") transgenic mouse that expresses mutant U2AF1Q157P and is isogenic to our previously reported U2AF1S34F and U2AF1WT transgenic mice. First, we confirmed DOX-inducible expression of the U2AF1Q157P transgene in BM by RT-PCR-seq. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P, we performed non-competitive BM transplants into lethally irradiated congenic recipient mice. Donor BM from U2AF1WT or U2AF1S34F mice was also transplanted for comparison. Six weeks after transplant, mice were maintained on DOX chow to induce U2AF1 transgene (U2AF1WT, U2AF1S34F, or U2AF1Q157P) expression (n = 10 mice per genotype). After six weeks on DOX, there were no significant changes in PB counts for U2AF1Q157P mice compared to U2AF1WT controls. In contrast, white blood cell (WBC) and B-cell counts were significantly reduced in U2AF1S34F mice, as reported previously. Assessment of the BM revealed increased numbers (per five leg bones) of hematopoietic stem and progenitor cells (LSK [Lin− Sca-1+ c-kit+] and LK [Lin− Sca-1− c-kit+]) in U2AF1S34F mice (1.33×105 LSK and 7.13×105 LK cells) compared to U2AF1WT (1.04×105 LSK and 5.69×105 LK cells; p < 0.05 for LSK and LK), as reported previously. In contrast, there was no change in LSK cells (1.03×105, p = 0.9668) and a non-significant increase in LK cells (6.84×105, p = 0.0547) in U2AF1Q157P mice compared to U2AF1WT. Both U2AF1S34F and U2AF1Q157P mice shared a significant increase in the number of common myeloid progenitors (CMP) compared to U2AF1WT (2.43×105 and 2.39×105 vs. 1.66×105 cells; p < 0.001 and p < 0.01, respectively), although CFU-C interrogated by methylcellulose assay were significantly increased only for U2AF1S34F mice. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P on stem cell function, we mixed equal numbers of whole BM test cells (CD45.2+; U2AF1Q157P or U2AF1WT) with congenic control wild-type BM competitor cells (CD45.1+/CD45.2+) and transplanted them into lethally irradiated congenic recipient mice (CD45.1+/CD45.2+ ; n = 6 per genotype). As in non-competitive transplants, DOX chow was administered six weeks after transplant. After six weeks on DOX, we observed a relative multi-lineage competitive disadvantage by analysis of peripheral blood chimerism (%CD45.2+ WBC) for U2AF1Q157P test compared to U2AF1WT test cells (49.5% vs. 71.7%, respectively, p < 0.001). In addition, stem and progenitor cells were all significantly reduced in the BM of U2AF1Q157P competitive transplant mice compared to U2AF1WT after 18 weeks of DOX (LSK, 36.1% vs. 92.2%, respectively, p < 0.001; LK, 53.1% vs. 92.0%, p < 0.001). Lastly, using a Nanostring array, we identified consensus 3' splice sites of cassette exons that were increased or decreased in RNA from c-kit enriched mutant (U2AF1S34F or U2AF1Q157P) BM cells relative to U2AF1WT (FDR < 0.1). As expected, we observed altered consensus 3' splice sites at the −3 position (for U2AF1S34F) and +1 position (for U2AF1Q157P) of differentially spliced exons, indicating altered but different pre-mRNA splicing induced by either U2AF1 mutant. In aggregate, hematopoietic expression of U2AF1Q157P causes a multi-lineage competitive disadvantage of BM stem cells and expanded myeloid progenitors in the non-competitive transplant setting, like U2AF1S34F. However, PB counts and lineage distribution are not affected, indicating that the two common U2AF1 mutants, Q157P and S34F, are associated with different hematopoietic phenotypes and alterations to splicing, and may have different roles in MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

Older Patients with NPM1 Mutated AML Have Distinctive Genomic Mutation Landscape Associated with Enrichment in Immunosuppressive Gene Signature

Background: Mutations in the nucleophosmin (NPM1) gene are associated with better responses to chemotherapy and improved survival among acute myeloid leukemia (AML) patients. However, older AML patients (≥ 60 years old) with NPM1 mutation have worse survival outcomes than younger patients (<60 years old). This may be attributed to more adverse biologic features (frequent complex karyotype, FLT3 mutations) in addition to lower odds to receive intensive curative chemotherapy due to co-morbidities. We sought to compare the outcomes of older NPM1 mutated AML patients with younger NPM1 mutated patients after exclusions of patients with adverse-risk per ELN 2017 criteria. We also compared their genomic mutation profile and gene expression utilizing the Beat AML dataset. Methods: We queried the Beat AML dataset, supported in part by the Leukemia & Lymphoma Society and the OHSU Knight Cancer Institute, for pts with NPM1 gene mutations who did not have adverse-risk ELN 2017 (poor cytogenetic profile or mutations in FLT3, TP53 or ASXL1). Descriptive statistics described baseline characteristics and responses. Kaplan-Meier with log-rank test was used for survival analysis. DNA mutation data were obtained from the exome sequencing and analyzed using the beat AML data viewer (Vizome). RNA exome sequencing data were downloaded. Differential expression of raw count RNA-Seq and gene set enrichment was done using R via limma and ClusterProfiler packages. Results: Among 562 unique patients in the Beat AML umbrella trial, there were 81 patients with newly diagnosed NPM1 mutated AML after exclusion of patients with ELN 2017 adverse-risk category. Among these patients there was 49 older patients (≥ 60 years old) and 32 younger patients (<60 years old). 39 (77.6%) in the older group received intensive induction chemotherapy and 30 patients (93.7%) in the younger group. 29 (59.1%) patients achieved complete morphologic responses in the older patient group compared to 28 (84.4%) in the younger patient group (OR 0.2, P=0.009). Median overall survival in the older patient group was 20.1 months compared to 25.4 months in the younger group (HR 0.52, P=0.08). Exome sequencing data were available for 43 and 30 patients from the older and younger group respectively. There was a median of 6.5 (2-20) and 7 (2-19) mutations in the older and younger group respectively (P=0.78). After exclusion of the benign mutations and variant of unknown significance, the median number of mutations was 4 in both group (P=0.28). Both groups shared only 24 (3.9%) of the gene mutations while there were 334 unique gene mutations in the older group and 262 in the younger group. Most common gene mutations were DNMT3a, TET2, NRAS, WT1, and PTPN11 with frequencies are shown (Figure 1). RNA sequencing data was available for 26 patients from the older group and 18 patients from the younger group. We explored the gene expression profile of the top 1000 differentially expressed genes in both groups after adjustment. There was distinctive clustering of the gene expression profile between the two groups (Figure 2). Gene set enrichment analysis identified multiple immune-related pathways among the highly enriched gene sets in both groups but with different functions in the two groups. There was significant gene set enrichment in the TGFβ signaling in the older patient group which is associated with immune suppression and microenvironment modulation. While the younger group showed significant enrichment in the TNFa, IL17, PI3K-AKT signaling which are associated with inflammation. Conclusion: Older AML patients with NPM1 mutations, and no adverse risk features, had lower rate of complete responses and a trend towards a worse survival compared to younger patients. Whole exome sequencing did not show increased mutational burden. However, 96% of the mutated genes were different between the two groups as were the gene expression profiles. Gene set enrichment analysis showed contrasting enriched immune-related pathways between both groups. The immunosuppressive TGFβ signaling gene set were significantly enriched in the older group while the inflammatory TNFa, IL17, PI3K-AKT signaling gene sets were significantly enriched in the younger group. Older AML patient with NPM1 mutations have distinctive genomic landscape compared to the younger patient which may explain in part the worse clinical outcomes in the absence of other adverse risk features. Disclosures Lin: Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Deciphering Common Gene Fusions and Mutations in Acute Leukemia of Ambiguous Lineage

Background The acute leukemia of ambiguous lineage (ALAL) is high risk and uncommon in acute leukemia, including acute undifferentiated leukemia (AUL) and mixed phenotype acute leukemia (MPAL). On account of the rareness and age-influenced genome landscape, the genetic characterization was always limited and showed discrepancy among different cooperative groups. Methods By integrating the immunophenotype, cytogenetics, and molecular biology of ALAL patients, we found that the molecular profile played an important role in the diagnosis, treatment, and prognosis of ALAL patients. Through an in-depth analysis of the molecular features, we found the genetic differences between different subtypes of ALAL, and studied the unique gene mutation patterns and synergy of genomic abnormalities in different subtypes. Results Most patients in the cohort were children and young adults, and among B/myeloid and T/myeloid MPAL, while rare in AUL. Here we showed that ALAL patients show both genetic commonality and discrepancy among subtypes. Except for the BCR-ABL1 and KMT2A-r, which were mainly in B/myeloid MPAL, we reported that SET-NUP214 was recurrent in T/myeloid MPAL and AUL patients. The PHF6 were also frequently mutated in T/myeloid MPAL with most loss of function and significantly associated with SET-NUP214. Otherwise, there was a big difference between B/myeloid and T/myeloid MPAL in genetic profile. The most mutated genes in B/myeloid MPAL were CEBPA, RUNX1, NRAS, TET2, WT1, PTPN11, and FLT3, while NOTCH1, WT1, RUNX1, FLT3, PHF6, ETV6, and EZH2 were frequently in T/myeloid MPAL. Moreover, the alterations in the genes encoding transcriptional regulation were both recurrently mutated in B/myeloid and T/myeloid MPAL, especially WT1 and RUNX1, which were the most mutated genes and mutually exclusive with each other. Alterations in the genes involved in JAK_STAT signaling, RAS signaling, and epigenetic regulation were also recurrently mutated in B/myeloid and T/myeloid MPAL, despite some differences in incidence rate. However, the deletion of IKZF1 exclusively occurred in B/myeloid MPAL, while genes involved in NOTCH signaling were mainly altered in T/myeloid MPAL. When analyzing the synergy among genetic abnormalities, we found that SET-NUP214 and PHF6 (p = 0.00002), FLT3 and RUNX1 (p = 0.009), BCR-ABL1 and RUNX1 (p = 0.02) were the most significantly co-occurred genes (Figure 1-A, B). Although the association between NRAS and WT1 were not significantly (p = 0.11), they co-occurred in five patients (Figure 1-C). Since this was the first time to report that SET-NUP214 was recurrent in ALAL patients, we retrospectively review the therapy of patients with the aberration. According to the treatment of these ALAL patients, we confirmed that even with transplantation therapy the prognosis of SET-NUP214-positive ALAL patients was still very poor with a high risk of relapse. Discussion We charted the genomic landscape of ALAL to comprehensively reveal the commonality and discrepancy among subtypes of ALAL patients. The founding lesion of SET-NUP214 in early primitive hematopoietic cells was showed to be involved in the block of differentiation. Together with the loss of function in PHF6, which was showed a contribution to the lineage promiscuity and enhance the proliferation of hematopoietic cells, the combination event could make leukemia with ambiguous lineage. After deciphering the molecular characterization of ALAL patients, we proposed several candidate updates to WHO 2016 (Table 1), as these molecular features may help the diagnosis, therapy, and prognosis of the ALAL patients. Disclosures No relevant conflicts of interest to declare.

Abstract 4243: Genetic variants frequently detected in Egyptian breast cancer tumors: Comprehensive cancer panel by ion torrent DNA sequencing technology

Abstract Breast cancer is the leading cause of cancer-related death in women worldwide. In Egypt, it is the most common cancer among females and its incidence is progressively increasing with a great tendency to occur with advanced stages in younger ages. Due to the heterogeneity of breast cancer, it was classified into different subtypes, each exhibits a unique gene mutation profile, based on biological characteristics and on gene expression pattern. Therefore, further research is needed to investigate the unknown genetic mutations involved in the progression of that disease. This study aimed to sequence 409 exons of tumor suppressor genes and oncogenes to identify the frequency of the detected genetic mutations in breast cancer using Ion Comprehensive Cancer Panel. Forty-eight tissue samples of various breast cancer subtypes were collected from National Cancer Institute (NCI) outpatient clinic, Cairo University. Analysis revealed 191 exonic and splicing variants. In this paper, we will address the most frequently detected Egyptian genetic variants (in 31.25 % of cases or more) as well as other deleterious variants commonly associated with breast cancer. Most of the detected genetic variants were checked in 1000g, dbSNP and Exac All databases. Other variants were found at known hotspot sites. We reported fifty-one somatic and germline mutations in thirty-two genes; AKAP9, BUB1B, RPS6KA2, AURKB, FANCA, RNF213, FGFR4, KAT6B, NLRP1, KAT6A, PER1, ERBB4, IL6ST, PIK3CA, P53, AURKA, WRN, PALB2, PTEN, GATA3, AKT1, ERBB2 and KRAS. Only KAT6B incurred non-frameshift deletion and only GATA3 had frameshift insertion while KAT6A, ERBB4 and PTEN had frameshift deletion. All the identified variants were detected with different frequencies in each breast cancer subtype. Each sample harbored at least four mutations and the maximum number of mutations per sample was twelve. The current data showed that gene panels analyzed by Next-Generation Sequencing (NGS) identifies large number of germline and somatic mutations that is crucial for understanding cancer predisposition and developing personalized or combination therapies that efficiently target the individual breast cancer-specific mutations. Key words: Breast cancer, Somatic mutations, Germline mutations, Ion torrent sequencing, Targeted sequencing. Note: This abstract was not presented at the meeting. Citation Format: Abdel-Rahman N. Zekri, Osman Mansour, Samah A. Loutfy, Mohamed M. Hafez, M. Gomaa, Abeer Bahnassy, Mai M. Lotfy, Amira S. Youssef, Ola S. Ahmed, Mohammed Abouelhouda, Auhood Nassar. Genetic variants frequently detected in Egyptian breast cancer tumors: Comprehensive cancer panel by ion torrent DNA sequencing technology [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 4243.

Abstract P3-01-18: In vivo isolation of circulating tumour cells using CellCollector and detection of gene mutations in different metastasis organ sites in breast cancer

Abstract Introduction: Metastasis is thought to result from tumour cell entry into the circulation and migration to distant organs, where the mutation landscape of metastatic breast cancer (MBC) may shift and vary. The genotypic features of circulating tumour cells (CTCs) typically differ from those of primary breast cancer (BC) cells. Gene mutation analysis of CTCs in MBC may benefit patients by identifying those amenable to specific therapies. Currently, CTCs are primarily isolated in vitro from small volumes of blood. The aim of this study was to isolate CTCs in vivo using CellCollector and screen for specific gene mutations in cells from different metastasis organ sites and molecular subtypes in MBC patients. Methods: In this study, we used a novel technology, CellCollector, to collect peripheral CTCs. Thirty MBC patients were enrolled, and 17 were analysed with next-generation sequencing (NGS) methods. Clinical characteristics were analysed along with CTC enumeration and detection rates. Whole-genome amplification (WGA) was used to amplify the CTC genomic DNA of 127 genes. Results: We isolated CTCs in vivo from 20 of 30 MBC patients (66.7%), with a median and mean (range) of 2 (0-15) CTCs. In non-cancer patients, no CTCs were detected. We analysed CTC enumeration and the detection rate in different clinical characteristic subgroups. We found that in their corresponding subgroups, patients younger than 45 years old, with brain metastasis, with three or more metastasis organ sites, or with HER2-positive subtypes had the highest CTC medians and means.As far as clinical characteristics were concerned, the number of CTCs seemed correlated with more advanced clinical characteristics. In the one metastasis organ, two metastasis organs and three or more metastasis organs subgroups, the CTC detection rates were 38.5% (5/13), 77.8% (7/9), and 100.0% (8/8), respectively. The CTC detection rate correlated with the number of metastasis organs; patients with more metastasis organ sites had higher CTC detection rates. We also found that different metastasis organs and molecular subtypes contain high-frequency mutation genes, and also contain unique gene mutations. Conclusions: In MBC, CellCollector can be used to collect intact CTCs, from which we can obtain gene mutation information. Different metastasis organs and molecular subtypes may have corresponding unique mutations, which may provide a basis for future gene therapy. CTCs enumeration data and correlations to clinicopathologic characteristicsPatients' characteristics CTCs enumeration RangemedianmeanTotal0-1522Age (years) <450-422≥45 and <600-1512≥600-212Metastatic location Brain0-1556Lung+liver0-712Bone+local recurrence0-411Number of metastatic locations 10-40120-15133-41-723Molecular subtypes Luminal A000Luminal B0-511HER2 positive0-1523Triple negative0-712HER2, human epidermal growth factor receptor 2 Citation Format: Geng C, Li S, Yang S, Shi J, Ding Y, Gao W, Cheng M, Sun Y, Xie Y, Sang M. In vivo isolation of circulating tumour cells using CellCollector and detection of gene mutations in different metastasis organ sites in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-18.

Correlation between myeloperoxidase expression and gene alterations and prognosis in acute myeloid leukemia

目的观察髓过氧化物酶（MPO）在急性髓系白血病（AML）中的表达，探讨其与AML临床特征、基因突变、疗效和预后的关系。方法回顾性分析233例初诊AML患者骨髓中原始细胞MPO表达的阳性率，根据MPO表达率不同将其分为MPO低表达组（MPO≤70%）与MPO高表达组（MPO>70%），并对两组患者的临床特征、基因突变、疗效和预后进行比较。结果①233例AML患者中MPO低表达组121例（51.9%），MPO高表达组112例（48.1%）。NCCN预后良好组患者多为MPO高表达（χ2=32.773，P<0.001），而MPO低表达与预后不良核型相关（χ2=7.078，P=0.008）。②MPO低表达组DNMT3A基因（χ2=6.905，P=0.009）、RNA剪接复合物相关基因（SF3B1/SRSF2/U2AF1）（χ2=5.246，P=0.022）、RUNX1基因（χ2=4.577，P=0.032）、ASXL1基因（χ2=7.951，P=0.005）及TP53基因(P=0.004）突变发生率明显高于MPO高表达组，而C-KIT基因（χ2=8.936，P=0.003）及CEBPA基因（χ2=12.340，P<0.001）突变更多见于MPO高表达组，尤其CEBPA双位点突变。③MPO低表达组首次诱导治疗缓解（CR1）率为38.8%，MPO高表达组为68.1%，差异有统计学意义（χ2=15.197，P<0.001）。多因素分析显示MPO低表达是影响患者CR1的独立危险因素。④MPO低表达组患者的2年总生存（OS）率及无进展生存（PFS）率均明显低于高表达组（18.0%对89.4%和11.5%对56.7%），差异有统计学意义（χ2分别为15.212和17.016，P值均<0.001）。Cox模型多因素分析显示MPO低表达是影响患者OS及PFS的独立预后不良指标。⑤正常核型AML中，MPO低表达组患者OS和PFS差于MPO高表达组（2年OS率：31.1%对83.7%，χ2=2.895，P=0.089；2年PFS率：18.8%对45.8%，χ2=5.068，P=0.024）。结论不同MPO表达的AML具有独特的基因突变谱；MPO低表达是影响AML患者CR1、OS和PFS的独立危险因素，细胞化学染色检测MPO表达可能为评估AML疗效和预后提供一种简单而有效的手段。

Clinical and Genetic Factors to Inform Reducing Colorectal Cancer Disparitites in African Americans.

Colorectal cancer (CRC) is the third most prevalent and second deadliest cancer in the U.S. with 140,250 cases and 50,630 deaths for 2018. Prevention of CRC through screening is effective. Among categorized races in the U.S., African Americans (AAs) show the highest incidence and death rates per 100,000 when compared to Non-Hispanic Whites (NHWs), American Indian/Alaskan Natives, Hispanics, and Asian/Pacific Islanders, with an overall AA:NHW ratio of 1.13 for incidence and 1.32 for mortality (2010-2014, seer.cancer.gov). The disparity for CRC incidence and worsened mortality among AAs is likely multifactorial and includes environmental (e.g., diet and intestinal microbiome composition, prevalence of obesity, use of aspirin, alcohol, and tobacco use), societal (e.g., socioeconomic status, insurance and access to care, and screening uptake and behaviors), and genetic (e.g., somatic driver mutations, race-specific variants in genes, and inflammation and immunological factors). Some of these parameters have been investigated, and interventions that address specific parameters have proven to be effective in lowering the disparity. For instance, there is strong evidence raising screening utilization rates among AAs to that of NHWs reduces CRC incidence to that of NHWs. Reducing the age to commence CRC screening in AA patients may further address incidence disparity, due to the earlier age onset of CRC. Identified genetic and epigenetic changes such as reduced MLH1 hypermethylation frequency, presence of inflammation-associated microsatellite alterations, and unique driver gene mutations (FLCN and EPHA6) among AA CRCs will afford more precise approaches toward CRC care, including the use of 5-fluorouracil and anti-PD-1.

Gene mutations and clinical phenotypes in Chinese children with Blau syndrome.

The mutations of CARD15 gene and clinical features of Chinese patients with Blau syndrome were analyzed. We identified 10 missense mutations, out of which five were new: R334L, E383D, R471C, C495R and D512F. The rest of them, R334W, R334Q, G481D, M513T and R587C, have been reported previously. Among all the mutations, R334W, R334Q and C495R had the highest frequency. Blau syndrome was found at early age after birth. It began with lepidic rash and symmetric polyarthritis and was phenotypically characterized by typical rash, arthritis, iridocyclitis and arteritis. Cardiac involvement was also found in Blau syndrome. In addition to nerve deafness, renal involvement, osteochondroma and central nervous system involvement were also found in our patients. Therefore, Chinese children with Blau syndrome have unique gene mutations and complicated clinical phenotypes. Pathologic examination and CARD15 mutation testing should be considered for diagnosis as early as possible for suspected patients.