Florescence In Situ Hybridization Research Articles

A Case Report of a Complex Constitutional Karyotype Resulting in Three Derivative Chromosomes Characterized by Chromosomal Analysis, Florescent in situ Hybridization (FISH), and Chromosome Microarray

Abstract Introduction/Objective Structurally complex karyotypes are rarely identified in the postnatal constitutional setting. Abnormalities involving multiple chromosomes are likely to have a deleterious effect or be incompatible with life. Herein, we describe a case of a structurally complex karyotype in a newborn male. Methods/Case Report A newborn male presented at birth with microcephaly, severe ventriculomegaly, cerebellar hypoplasia and absent cavum septum pellucidum. Seizures, optic nerve dysplasia, and feeding difficulties were later noted. Microarray testing revealed eight small deletions of unclear significance on chromosome 7, with three located on the p arm and five clustered on the q arm. The deletions were gene poor, and none contained known haploinsufficient genes. Based on this finding a structural abnormality involving chromosome 7 was suspected. Karyotype revealed a structurally abnormal chromosome 7 and a complex rearrangement between chromosomes 11 and 18. Chromosome paint and metaphase FISH studies were performed to further characterize the abnormalities. Based on results from these cytogenetic tests, the observed abnormalities were characterized as an insertion on chromosome 7, and a translocation between chromosome 11 and 18 with an inversion on the derivative 18 [46,XY,ins(7)(p21q22q11.2),der(11)t(11;18)(q14.2;p11.2),der(18)(11qter→11q21::18q11.2→18p11.2::11q14.2→11q21:: 18q11.2→18qter)]. Rapid whole genome sequencing was done at an outside institution which was negative. Results (if a Case Study enter NA) NA Conclusion The exact cause of this individual’s phenotypic findings is unknown. Although the genomic imbalance detected by CMA is small, the complex structural abnormalities likely contribute to the clinical findings. Further supporting a deleterious effect of the karyotype findings is the negative whole genome sequencing results in this individual. Some apparently balance rearrangements have been shown to disrupt genes or important regulator elements. It is possible that a gene was disrupted at the breakpoint of one or more of the detected abnormalities or the rearrangement resulted in abnormal gene regulation. Additional submicroscopic abnormalities may also be present that were not detectable by the methodologies used.

Defining Primary Vs Secondary AML: Correlation between Pathology and NGS/FISH

Introduction: The treatment of acute myelogenous leukemia (AML) is often determined by factors that include an antecedent dysplasia. At presentation it is often unknown whether a patient has had dysplastic bone marrow features or not. We opted to look back at our leukemia patients to determine the frequency of antecedent dysplasia and to determine if next generation sequencing (NGS) or Florescent in situ hybridization (FISH) findings correlate with the pathologic review. Methods: We chart reviewed 43 charts from a period of 9/21/2021 to current from Orlando Health records. We focused on 29 charts that we could gather complete information from. We reviewed initial bone marrow biopsies and NGS/ FISH findings. We determined if a patient had a known history of myelodysplasia from previous records. If there was no known history of myelodysplastic syndrome (MDS) but dysplasia was seen on the bone marrow biopsy, we looked at NGS/FISH. While there can be overlap with AML, findings that suggest MDS are as follows: NGS: TET2, NRAS, U2AF1, RUNX1, TP53, SF3B1 FISH: 5q-, -5 (5p15, 5q31, 5q33), 7q-, -7 (Cen 7, 7q22, 7q31), Trisomy 8 (Cen 8), MLL (11q23), 20q- (20q12, 20qter) We then examined what treatments were used based on these findings. Results: Overall: 11/29 patients had dysplasia mentioned in their initial pathology report. 4/11 had a known preexistent dysplasia. Of the 7 who didn't have known dysplasia, 6/7 had an NGS or FISH suggesting MDS. Age: Of the patient who had dysplastic findings as well as NGS/FISH to support those findings, the patients had an average age of 57. The one patient without MDS/FISH findings has an age of 69. The average age of all patients with dysplastic findings on their bone marrow was 60. Of the patients who did not have any dysplastic findings on the bone marrow, the average age was 58. Treatment: Vyexos (liposomal daunorubicin and cytarabine), a therapy generally used for secondary leukemias was only used in two patients. Age and performance status was cited as a reason to use alternative therapy. Although one patient did receive 7+3 (idarubicin and cytarabine) instead of Vyxeos. One patient also had severe COVID pneumonia at diagnosis as the reason for not getting induction with Vyexos and instead getting venetoclax/azacitidine. Four patients were either treated with hypomethylating agent of either azacitidine or decitabine, again this was normally chosen due to performance status, age, or active COVID pneumonia as above. Conclusion: These results show that pathology findings of underlying dysplastic changes seem to correlate with NGS or FISH findings. Of the 7 patients with these bone marrow changes but no known MDS, 6 of them had NGS or FISH to support an underlying disease. Factors such as age did not help to predict antecedent MDS. Based on this information it may be appropriate to start with induction therapies such as Vyxeos when dysplastic changes are seen on the bone marrow while FISH and NGS are still pending, if their performance status and age allow to do so. Further larger retrospective studies to compare underlying dysplastic findings versus FISH/NGS findings should be done to further support this concept.

Impact of Presence and Amount of Clonal Plasma Cells in Autografts Affect Outcomes in High-Risk Multiple Myeloma Patients Undergoing Autologous Hematopoietic Stem Cell Transplant

Introduction: Autologous hematopoietic stem cell transplantation (autoHCT) is considered standard of care for patients with multiple myeloma (MM). However, most patients eventually relapse, perhaps due to the presence of clonal plasma cells (CPC) in the autograft. The significance of CPC in the autograft has not been clearly established. MM patients with high-risk chromosomal abnormalities (HRMM) by florescent in situ hybridization (FISH) have worse outcomes compared to patients with standard-risk disease. We conducted a single center retrospective analysis to evaluate the impact of CPC in the autograft on the outcomes of patients with HRMM undergoing an autoHCT at our institution. Methods: Eligible patients had HRMM, received an autoHCT between 2008 and 2018 at our institution, and had 8-color next-generation flow cytometry (NGF) performed on the collected apheresis product for CPC. HRMM was defined as having del17p, t(4;14), t(14;16) and 1q21 gain or amplification by FISH. Patients were divided into two groups: CPC+ or CPC- in the autograft by NGF. Since not all collected autograft products are infused at autoHCT, and some bags may be CPC+ or CPC- for the same patient, we also evaluated the impact of infusion of CPC+ autograft products on patient outcomes. Minimal residual disease (MRD) status on the bone marrow biopsy was evaluated using 8-color next generation flow cytometry with sensitivity of 1/10-5 cells. Results: We analyzed the autograft products of 416 patients: 75 CPC+ (18%) and 341 CPC- (82%). 57% patients were male, and median age at autoHCT was 62 (range 32-83) years. Fewer patients in the CPC+ group received the VRD regimen as induction prior to transplant compared to the CPC- group (25% vs. 44%, p=0.003) and fewer achieved ≥VGPR after induction compared to the CPC- group (32% vs. 62%, p<0.001). Patient and clinical characteristics are presented in Table 1. Median follow up for the whole cohort was 35.7 (range 0.3-139.5) months. 100-day and best post-autoHCT CR rates in the CPC+ and CPC- groups were 8% vs. 19% (p<0.001), and 33% vs. 54% (p<0.001), respectively. The CPC+ group was also less likely to achieve MRD-negative CR post-transplant (11% vs. 42%; p<0.001). Median PFS in the CPC+ vs. CPC- group was 12.8 vs. 32.1 months (p<0.001), and median OS was 36.4 vs. 81.2 months (p<0.001). The degree of autograft CPC involvement was inversely associated with post-transplant day 100 response: mean maximal autograft CPC level was 0.02% (SD 0.02) for patients who achieved ≥VGPR vs. 1.04% (SD 2.59) for those who achieved ≤PR at day 100 post-transplant. Similarly, degree of autograft CPC level was inversely associated with best post-transplant response: mean maximal autograft CPC level was 0.06% (SD 0.16) for patients who achieved ≥VGPR vs. vs. 1.55% (SD 3.17) for those who achieved ≤PR. In patients achieving ≥VGPR prior to autoHCT, those with CPC+ autograft had inferior PFS (hazard ratio (HR) [95% CI]: 3.38 [2.05-5.58], p<0.001; Figure 1C) and OS (HR 2.29 [1.20-4.40], p=0.013; Figure 1D) compared to the CPC- group. Also in patients who achieved MRD- negative CR or VGPR prior to autoHCT, those with CPC+ autograft had inferior PFS (HR 4.35 [1.87-10.16], p<0.001) and OS (HR 6.14 [2.10-17.96], p<0.001) compared to the CPC- group. In a multivariable analysis (MVA), the degree of CPC positivity in the autograft was independently predictive of worse PFS (HR 1.50 [1.12-1.99]; p=0.006) and OS (HR 1.35 [1.08-1.67]; p=0.007). The MVA also showed that infusion of CPC+ autograft products was predictive of worse PFS (HR 2.78 [1.85-4.15]; p<0.001) with a trend towards worse OS (HR 1.66 [0.99-2.78]; p=0.054). Conclusions: Our study shows a major impact of CPC in the autograft on post-autoHCT outcomes in HRMM. Both the presence and degree of CPC in the autograft were highly predictive of inferior PFS and OS, even in patients who had achieved ≥VGPR and MRD-negative CR/VGPR prior to autoHCT. Novel strategies for ex-vivo purging of CPC could improve patient outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 5336: Dissecting the molecular mechanism of trastuzumab resistance in stage IV HER2-positive gastric cancer patients

Abstract Introduction: The effect of trastuzumab in targeted therapy for HER2-positive gastric cancer (GC) is limited by the eventual development of resistance within patients. Although numerous studies have been published on this topic, both the mechanism of innate and acquired trastuzumab resistance are still not fully understood. In this study, we compare the mutational landscape of paired solid tumor samples before and after treatment in an effort to identify acquired events associated with resistance. Methods: We performed whole-exome sequencing on 46 tumor and 23 whole blood samples from patients with stage IV gastric cancer were obtained from before treatment (n = 23) and at disease progression (PD) (n = 23). HER2 status was checked using immunohistochemistry and validated with florescence in situ hybridization (FISH). Correlation analysis was then conducted between the mutational profile and clinical data of patients. Results: Comparisons between innate resistance samples and acquired resistance samples at baseline revealed three copy number variation events (CDK12 amplification, ERBB2 amplification, MECOM amplification) that occurs in significantly different proportions (p &amp;lt; 0.05). In addition, for baseline samples, MECOM was shown to be associated with poor progression-free survival (PFS) (Hazard Ratio (HR), 12; 95% CI, 1.6 - 85; p = 0.027). Commonly acquired events (i.e., lacking in before treatment samples and present at PD) within patients include TP53 mutation, MYC amplification, MCL1 amplification, CDKN2A/2B deletion, and CCNE1 amplification. Of these, patients with MCL1 gene amplification had worse survival (HR, 4.8; 95% CI, 1.2 - 20; p = 0.050) than patients with wild-type MCL1. In addition, the NOTCH pathway acquired mutations in 4 of 23 patients and was found to be correlated with a worse PFS (HR, 5.1; 95% CI, 1.4 - 19; p = 0.023), indicating that this pathway is possibly involved with trastuzumab resistance to some degree. Conclusion: Overall, our results show that comparisons of samples obtained before treatment and at PD can give insight about innate and acquired resistance mechanisms to trastuzumab in stage IV, HER2-positive GC patients. Citation Format: Qi Xu, Jingjing Li, Haimeng Tang, Hua Bao, Junrong Yan, Xue Wu, Yang Shao, Jianying Jin, Cong Luo, Haimin Weng, Jieer Ying. Dissecting the molecular mechanism of trastuzumab resistance in stage IV HER2-positive gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5336.

Alveolar soft part sarcoma in children: a clinicopathological study of 13 cases

Objective: To investigate the clinicopathological manifestations, molecular genetic, diagnostic histology and differential diagnosis of alveolar soft part sarcoma (ASPS) in children. Methods: A total of 13 cases of ASPS diagnosed at Beijing Children's Hospital from August 2009 to November 2018 were collected. HE staining, histochemical staining for PAS and D-PAS, immunohistochemical (IHC) staining for TFE3, INI1 and CD68 and florescence in situ hybridization (FISH) for TFE3 gene translocation were performed. Results: There were four males and nine females, age ranged from 1 year and 2 months to 13 years and 8 months (mean 7.8 years); and four patients were under 5 years old. Histologically, the tumors showed a distinctive and characteristic nested or organoid growth pattern (11 cases) or solid, diffuse growth (2 cases). The tumor cells possessed abundant eosinophilic, or glycogen-rich and clear to vacuolated cytoplasm. The chromatin was relatively dispersed, with prominent and pleomorphic nucleoli; mitotic figures were rare. Vascular invasion was frequently seen. IHC staining showed specific nuclear TFE3 staining. The tumor cells were also positive for INI1,CD68 and vimentin; but were negative for MyoD1, Myogenin, CK and S-100 protein. Seven cases showed PAS and D-PAS staining, with fuchsia acicular or rod-shaped crystals in tumor cytoplasm. Nine cases showed TFE3 break-apart signals by FISH. Conclusions: ASPS is a rare soft tissue sarcoma in children. Compared with ASPA in adults, it has both similarities and unique clinicopathologic characteristics. The diagnosis needs to be confirmed by combining clinical, pathologic, IHC and genetic testing.

Abstract P6-17-38: Clinical and pathological features of breast cancer with 'polysomy' of Chromosome 17

Abstract Background: Anti-HER2 therapy is a standard of care for patients with HER2+ breast cancer. HER2 status is routinely evaluated using immunohistochemical stain and/or florescence in situ hybridization (FISH). Interpretation of FISH results may be challenging in tumors with 'polysomy' of chromosome 17 ('polysomy'17), which is defined by increased chromosome enumeration probe 17 (CEP17) signal number. There is evident that 'polysomy'17 might account for anti-HER2 therapy response in breast cancer (BC) with normal HER2/CEP17 ratio. This study aims to study the clinical and pathological features of BC with 'polysomy'17. Method: Primary BC were selected based on elevated CEP17 count (≥3.0) in HER2 FISH performed at MD Anderson Cancer Center between April 2014 and March 2018 (n=385). Patient charts were reviewed for detailed clinical and pathological features. These cases were further divided into four groups according to HER2/CEP17 and HER2 copy number, based on ASCO-CAP guidelines: group 1 (HER2+) – HER2/CEP17≥2.0, HER2 copies≥4.0; group 3 (HER2+) – ratio&amp;lt;2.0, HER2 copies≥ 6.0; group 4 (HER2 equivocal) – ratio &amp;lt;2.0, HER2 copies ≥4.0 and &amp;lt;6.0; group 5 (HER2-) – ratio&amp;lt;2.0, HER2 copies&amp;lt;4.0. Chi-square tests were performed to study the difference of these characters. Results: In comparison with groups 1 and 3, BC in groups 4 and 5 are more commonly seen in elder patients (p=0.001). Also, these tumors show higher pathological category (p&amp;lt;0.001) and higher rate of lymph nodes metastasis (p&amp;lt;0.05). The clinical and pathological features are summarized in [Table 1]. Conclusion: Understanding the clinical and pathological features of BC with 'polysomy'17 may help clinically to choose patients who might be benefit from anti-HER2 therapy. Further study is to follow up the therapy response of those who received anti-HER2 treatment in this cohort of patients. Clinical and pathological characteristics of breast cancer with 'polysomy' of chromosome 17 Total (N=385)Group 1 ( N=131)Group 3 (N=25)Group 4 (N=69)Group 5 (N=160)P valueAge (year)Mean (range)56.8 (25-94)53.9 (25-83)52.8 (26-75)58 (33-92)59.3 (29-94)0.001Race RaceBlack (%)37 (9.6)14 (10.7)4 (16)5 (7.2)14 (8.8)0.873 Hispanic (%)61 (15.8)23 (17.6)3 (12)12 (17.4)23 (14.4) White (%)251 (65.2)82 (62.6)14 (56)44 (63.8)111 (69.4) Other (%)36 (9.4)12 (9.1)4 (16)8 (11.6)12 (7.4) GenderFemale (%)383 (99.5)131 (100)25 (100)68 (98.6)159 (99.4)0.478 Male (%)2 (0.5)0 (0)0 (0)1 (1.4)1 (0.6) Histology typeIDC, NOS (%)350 (90.9)121 (92.4)20 (80)66 (95.7)143 (89.4)0.471 ILC (%)9 (2.3)3 (2.3)1 (4)0 (0)5 (3.1) Others (%)26 (6.8)7 (5.3)4 (16)3 (4.3)12 (7.5) Nuclear gradeI (%)4 (1.2)2 (1.8)0 (0)1 (1.8)1 (0.7)0.044 II (%)122 (37)30 (26.3)12 (50)19 (33.3)61 (45.5) III (%)203 (61.8)82 (71.9)12 (50)37 (64.9)72 (53.8) NA561711226 Pathological tumor categorypT0+Tis (%)61 (21)36 (36.3)3 (20)5 (10.9)17 (13.1)&amp;lt;0.001 pT1 (%)125 (43.2)39 (39.4)7 (46.7)20 (43.5)59 (45.4) pT2+T3+T4 (%)104 (35.8)24 (24.3)5 (33.3)21 (45.6)54 (41.5) NA9532102330 Pathological lymph node categorypN0 (%)187 (65.4)81 (82.7)11 (73.3)22 (47.8)73 (57.5)0.048 pN1+N2+N3 (%)99 (34.6)17 (17.3)4 (26.7)24 (52.2)54 (42.5) pNx (%)9933102333 Citation Format: Sun H, Chen H, Lim B, Sahin AA. Clinical and pathological features of breast cancer with 'polysomy' of Chromosome 17 [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 P6-17-38.

Evaluating HER2 Gene Amplification Using Chromogenic In Situ Hybridization (CISH) Method In Comparison To Immunohistochemistry Method in Breast Carcinoma.

BACKGROUND:In patients with breast cancer, HER2 gene expression is of a great importance in reacting to Herceptin treatment. To evaluate this event, immunohistochemistry (IHC) has been done routinely on the basis of scoring it and so the patients were divided into 4 groups. Lately, as there have been disagreements about how to treat score 2 patients, chromogenic in situ hybridization (CISH) and florescence in situ hybridization (FISH) are introduced. Since CISH method is more convenient than FISH for gene amplification study, FISH has been substituted by CISH.AIM:The current study is conducted in order to investigate whether using CISH is a better method comparison to IHC method for determines HER2 expression in patients with breast cancer in.METHODS:In this cross-sectional descriptive analytical study, information of 44 female patients with invasive ductal breast cancer were gathered from Imam Reza and Omid Hospital in Mashhad. IHC staining was done for all patients in order to determine the level of HER2 expression, and after scoring them into 4 groups of 0, +1, +2 and +3, CISH staining was carried out for all 4 groups. At the end, results from both methods were statistically evaluated using SPSS software V.22.0.RESULTS:The average age of patients was 50.2 with the standard deviation of 10.96. Using IHC method was observed that 2.6% (1 patient), 26.3% (10 patients), 65.8% (25 patients) and 5.3% (2 patients) percentage of patients had scores of 0, +1, +2 and +3. On the other hand, CISH method showed 36 patients (90%) with no amplifications and 4 (10%) with sever amplifications. In a comparative study using Fisher’s exact test (p = 0.000), we found a significant relation between IHC method and CISH method indicating that all patients showing severe amplifications in CISH method, owned scores of +2 and +3 in IHC method.CONCLUSION:According to the present study and comparing the results with similar previous studies, it can be concluded that CISH method works highly effective in determining HER2 expression level in patients with breast cancer. This method is also able to determine the status of patients with score +2 in IHC for their treatment with herceptin.

A case of azoospermia in a non-destructive testing\xa0worker exposed to radiation

BackgroundInterest in radiation-related health problems has been growing with the increase in the number of workers in radiation-related jobs. Although an occupational level of radiation exposure would not likely cause azoospermia, several studies have reported the relation between radiation exposure and azoospermia after accidental or therapeutic radiation exposure. We describe a case of azoospermia in a non-destructive testing (NDT) worker exposed to radiation and discuss the problems of the related monitoring system.Case presentationA 39-year-old man who was childless after 8 years of marriage was diagnosed with azoospermia through medical evaluations, including testicular biopsy. He did not have any abnormal findings on biochemical evaluations, other risk factors, or evidence of congenital azoospermia. He had been working in an NDT facility from 2005 to 2013, attaching and arranging gamma-ray films on the structures and inner spaces of ships. The patient’s thermoluminescent dosimeter (TLD) badge recorded an exposure level of 0.01781 Gy for 80 months, whereas results of his florescence in situ hybridization (FISH) translocation assay showed an exposure level of up to 1.926 Gy of cumulative radiation, which was sufficient to cause azoospermia. Thus, we concluded that his azoospermia was caused by occupational radiation exposure.ConclusionThe difference between the exposure dose records measured through TLD badge and the actual exposure dose implies that the monitor used by the NDT worker did not work properly, and such a difference could threaten the health and safety of workers. Thus, to protect the safety and health of NDT workers, education of workers and strengthening of law enforcement are required to ensure that regulations are strictly followed, and if necessary, random sampling of NDT workers using a cytogenetic dosimeter, such as FISH, should be considered.

Prognostic Significance of Telomere Length in newly diagnosed Chronic Lymphocytic Leukemia patients: measured By FlowFISH technique

Telomeres are specialized nucleoprotein structures present at the ends of human chromosomes, andare constituted of a tandem of repeats TTAGGG. Telomere length measurement has been assessed by many previousstudies In B-Chronic Lymphocytic Leukemia patients (B-CLL). Telomere shortening was found to correlate withdisease progression. The aim of our study was to measure Telomere Length (TL) by Florescence In-SituHybridization coupled with Flow-cytometer (Flow-FISH) technique in newly diagnosed B-CLL patients. Explore itsprognostic significance, through determination of the relationship between TL; with disease stage, other prognosticmarker such as CD38 & ZAP-70 expression and patient response to treatment.

RBM10-TFE3 Renal Cell Carcinoma: A Potential Diagnostic Pitfall Due to Cryptic Intrachromosomal Xp11.2 Inversion Resulting in False-negative TFE3 FISH.

Xp11 translocation renal cell carcinoma (RCC) are defined by chromosome translocations involving the Xp11 breakpoint which results in one of a variety of TFE3 gene fusions. TFE3 break-apart florescence in situ hybridization (FISH) assays are generally preferred to TFE3 immunohistochemistry (IHC) as a means of confirming the diagnosis in archival material, as FISH is less sensitive to the variable fixation which can result in false positive or false negative IHC. Prompted by a case report in the cytogenetics literature, we identify 3 cases of Xp11 translocation RCC characterized by a subtle chromosomal inversion involving the short arm of the X chromosome, resulting in an RBM10-TFE3 gene fusion. TFE3 rearrangement was not detected by conventional TFE3 break-apart FISH, but was suggested by strong diffuse TFE3 immunoreactivity in a clean background. We then developed novel fosmid probes to detect the RBM10-TFE3 gene fusion in archival material. These cases validate RBM10-TFE3 as a recurrent gene fusion in Xp11 translocation RCC, illustrate a source of false-negative TFE3 break-apart FISH, and highlight the complementary role of TFE3 IHC and TFE3 FISH.

NanoString nCounter® Approach in Breast Cancer: A Comparative Analysis with Quantitative Real-Time Polymerase Chain Reaction, In Situ Hybridization, and Immunohistochemistry.

PurposeAccurate testing for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is essential for breast cancer treatment. At present, immunohistochemistry (IHC)/florescence in situ hybridization (FISH) are widely accepted as the standard testing methods. To investigate the value of NanoString nCounter®, we performed its comparative analysis with IHC/FISH and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for the assessment of ER, PR, and HER2.MethodsData on IHC/FISH results for ER, PR, and HER2 in 240 patients from a single tertiary hospital in Korea were collected and compared with NanoString nCounter® and qRT-PCR results at a single institution.ResultsExpression levels for each gene using NanoString nCounter® showed good correlation with the corresponding data for protein expression by IHC (p<0.001) and gene amplification status for HER2 (p<0.001). Comparisons between gene expression and IHC data showed good overall agreement with a high area under the curve (AUC) for ESR1/ER (AUC=0.939), PgR/PR (AUC=0.796), and HER2/HER2 (AUC=0.989) (p<0.001).ConclusionThe quantification of ER, PgR, and HER2 mRNA expression with NanoString nCounter® may be a viable alternative to conventional IHC/FISH methods.

Co-Expression of MYC, BCL2 and PD-L1 in Primary Central Nervous System Diffuse Large B Cell Lymphomas ( PCNSL)

BackgroundIncreased protein expression and gene translocations of MYC and BCL2/BCL6promote increased cell growth and inhibit apoptosis. They are associated with inferior prognosis in systemic diffuse large B cell lymphomas (DLBCLs). Their role in primary PCNSLs however is less clear. The immune checkpoint molecules, programmed death 1 (PD1) and its ligand PD-L1 are involved in inhibitory pathways to maintain immune tolerance and it is now clear that tumor cells interfere with these pathways to promote escape from immune surveillance. Antibody blockade of these immune checkpoints has been shown to enhance antitumor immunity and is emerging as an important target for cancer immunotherapy. We examined the co expression of these proteins on tumor samples from patients with PCNSL .MethodsImmunohistochemistry (IHC) for MYC, BCL2 and PDL-1 and florescence in situ hybridization ( FISH) for MYC (8q24), BCL2 ( 18q21) and BCL6 (3q27) were performed on tumor samples from 14 patients with PCNSL.ResultsEleven patients were positive for PDL-1 (79%) by IHC. Of these eleven patients, six (55%) were also positive for both MYC and BCL2 by IHC and negative by FISH. Eight patients (57%) were positive for MYC and BCL2 by IHC but negative for PD-L1. One patient was positive by FISH for MYC, BCL2, and BCL6 but negative for PDL-1.ConclusionsOur results, although in a small number of patients, demonstrate that a large percentage of patients with PCNSL who express MYC and BCL2 also express PD-L1.It has been suggested that the frequent over expression of MYC, BCL2, and BCL6 noted in PCNSL may underlie the inferior prognosis as compared to systemic DLBCL. We suggest that the co-expression of MYC/ BCL2 with PD-L1 may contribute to this poor prognosis by engaging the complimentary mechanisms of increasing cell growth, inhibiting apoptosis and evading immune surveillance. The use of PD-L1 inhibitors in hematologic malignancies is limited but has demonstrated clinical responses in relapsed/refractory disease following multiple lines of therapy and warrants further investigation in PCNSLs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Serum Albumin in the Prognosis of Myc-Positive Aggressive B-Cell Lymphomas: Potential for a Novel Prognostic Score

Background: MYC positive double hit lymphomas (DHL) (presence of c-myc and BCL2 or BCL6 rearrangements by florescence in situ hybridization (FISH) analysis) and double protein expressor lymphomas (DEL) (protein expression by immunohistochemistry (IHC)) represent a subset of diffuse large B cell lymphomas (DLBCL) with poor prognosis. Several prognostic scores systems have been applied such as the International Prognostic Index (IPI) and Revised IPI (R-IPI) but the most effective score remains to be elucidated. We explored the role of serum albumin (SA) in the prognosis of DHL/DEL as previously shown in de novo DLBCL (Dalia 2013) and developed a simple prognostic model that could predict clinical outcomes.Methods: We included 91 patients from Moffitt Cancer Center from January 2008 to December 2015 diagnosed with DHL/DEL and performed a retrospective chart review after obtaining institutional review board (IRB) approval. Clinical data included SA, hemoglobin (Hb), age, IPI, treatment type, stage, bulky disease (>10 cm), extranodal involvement, cell of origin (COO) by Hans criteria, relapse and vital status. SPSS and Prism GraphPad were used to perform multivariate analysis (MV) and Kaplan-Meier (K-M) to evaluate statistical significance and survival outcome.Results: Ninety-one patients (DHL= 53, DEL= 38) were included in the analysis. The median age was 63 years (18-87) with 36 (39.56%) females. There was stage III/IV disease in 80.22%, bulky disease in 48.35%, extranodal involvement in 39% of the patients and 15.38% patients had CNS involvement. With a median follow-up of 15.2 months, the median overall survival (OS) was 18.2 months (95%CI 11.9-24.5). MV analysis showed that SA ≤ 3.7g/dL (HR 3.85, 95% CI 1.67-8.81, p = 0.001), Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≥2 (HR 2.28, 95% CI 1.00-5.16, p = 0.049) and Ann Arbor Stage III/IV (HR 2.58, 95% CI 1.19-5.62, p = 0.016) were statistically significant associated with poor OS. We devised the SEA score (Stage, ECOG and Albumin) as follows in 3 risk categories: low risk (LR):0-1 points, intermediate risk (IR): 2 points, high risk (HR): 3 points. K-M OS curves are shown in Figure 1. The median OS for LR, IR and HR were 30.6 (95% CI 13.2 - 48.1), 13.2 (95% CI 7.3-19.1), 7.4 (95% CI 4.2-10.6) months, respectively, p < 0.001.We further explored the impact of SEA score separately in DHL and DEL subgroups. The median OS of DHL was significantly lower than DEL (29.40 vs. 13.30 months, respectively, p = 0.027, Figure 2A). SEA score was able to stratify both DEL and DHL subgroups accurately (Figure 2B and Figure 2C). DEL cohort had significantly greater OS compared to DHL in all risk groups (LR: 44.0 vs. 17.6 months, IR: 13.2 vs. 9.5 months, HR: 8.8 vs. 6.05 months; p< 0.001).Conclusions: DHL/DEL is a heterogeneous disease with a poor OS in this study. We demonstrated that SA<3.7 g/dl is independently associated with poor OS. Hypoalbuminemia could represent a surrogate of comorbid status and worse biology in DHL/DEL patients. SA based prognostic system accurately identified three distinct risk categories. SA could be considered in the risk stratification of DHL/DEL. [Display omitted] DisclosuresShah:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Rosetta Genomics: Research Funding; Bayer: Honoraria; Pfizer: Honoraria. Bello:Seattle Genetics: Speakers Bureau; Gilead: Speakers Bureau. Locke:Kite: Membership on an entity's Board of Directors or advisory committees. Chavez:Janssen: Speakers Bureau.

Racial Impact on Cytogenetic Variations and Outcome in Chronic Lymphocytic Leukemia Patients

INTRODUCTIONChronic lymphocytic leukemia (CLL) is the most common form of leukemia in the United States with a higher incidence in Caucasians compared to African Americans (AA). Few studies have looked into the impact of race on the outcome of patients with CLL, with some reporting a more aggressive nature of disease in AA compared to Caucasians and others reporting no difference in outcome between the two groups. In addition, variation in genetic mutations in different races is not well studied in patients with CLL. Hence, we performed a study to evaluate the racial differences in genetic mutations and outcome in patients with CLL.METHODSWe reviewed the charts of patients with CLL treated in our institution between 2004 and 2014. We collected data on age, gender, race, genetic mutations, rai stage, time to first treatment (TTFT), and time to second treatment (TTST). Both conventional cytogenetics and florescence in-situ hybridization (FISH) analysis were done for all patients. Only Caucasian and African American patients were included in our analysis. We used Fisher’s exact test to assess the statistical significance of any difference. P-values <0.05 were considered significant.RESULTSA total of 177 patients with CLL were analyzed, of which 112 patients were Caucasians and 65 patients were AA. For patients with favorable cytogenetics, deletion 13q14 was seen more often in Caucasian patients compared to AA (41.9% versus 18.4%, p= .001), while trisomy 12 was observed more often in AA patients compared to Caucasians (35% versus 17%, p= .009). There was no difference in patients harboring normal cytogenetics between the two groups. For unfavorable cytogenetics, there was no difference between Caucasian and AA patients in deletion 11q23 (8.9% versus 10.9%, p= .68), or deletion 17p/p53 abnormality (11.6% and 9%, p =.62). There was no difference in Rai stage at the time of diagnosis between Caucasians and AA (Rai stage ≤2: 86.6% versus 76.9%, and Rai >2: 13.4% versus 23.1%, p= .1). No difference was observed between the two groups in regards to TTFT (mean TTFT for Caucasians was 32.2 months versus 32.3 months in AA) or TTST (mean TTST for Caucasians was 50.1 months versus 50.6 months in AA).DISCUSSIONRacial differences in CLL including genetic mutations and outcome are poorly studied. Falchi et al has reported that AA patients tended to have a higher risk feature profile at the time of presentation, with an overall worse overall survival. In a SEER database analysis reported by Shenoy et al, authors found that AA had an inferior overall survival compared to non-African Americans.In our analysis, we aimed to assess for any difference in the incidence of prognostic cytogenetics between Caucasians and AA. We also evaluated the difference in outcome in terms of TTFT and TTST. We found that despite the presence of some differences in the favorable cytogenetic profile at time of diagnosis between both races, it did not translate into a difference in rai stage at time of presentation or the overall outcome as measured by TTFT and TTST. Our results confirm the most recent analysis by Nabhan et al, which utilized the Mayo Clinic CLL database, and found no difference in the outcome to either AA or Caucasian CLL patients when treated similarly.ConclusionBased on our analysis, a possible difference in some prognostic cytogenetics might be present between different races which did not affect the overall outcome. Hence, racial difference should not be taken into account when treating patients with newly diagnosed CLL, since there is no evidence that outcomes are different when treated with conventional therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Double Hit Lymphomas: Role of Immunohistochemistry in the Era of Florescent in-Situ Hybridization

Background Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease. A subset of DLBCL patients with translocations involving MYC and BCL2 genes, called "Double-hit" lymphoma (DHL), are being identified to have poor prognosis after standard chemotherapy. Fluorescent in situ hybridization (FISH) technique which can identify these gene mutations is the gold standard for diagnosis of DHL. Immunohistochemistry (IHC) is a rapid and inexpensive test that can identify abnormal protein expression of these mutated genes. Two studies thus far have shown that MYC IHC can be used as a screening test to identify DHL cases with sensitivities of 74% and 100%, both multi-center studies conducted at university hospitals. We conducted this study at a single large community hospital to determine if these results can be generalized to our institution. Objective Our primary objective was to determine the association between protein and genetic expression of MYC and BCL2 in cases of de novo DLBCL by comparing the results of IHC and FISH. Our secondary objectives were to determine if MYC IHC could be used as a screening test to determine DHL status, to determine if the double hit biology is associated with any clinic-pathological features which might allow for risk stratification of patients and to determine the prevalence of DHL in our cohort of DLBCL patients. Methods Twenty-two patients who were diagnosed with primary DLBCL from February 2015 to February 2016 at Abington Hospital (AH) Jefferson Health were identified after applying exclusion and inclusion criteria. Clinico-pathological data was obtained by review of electronic medical records and biopsy specimens for IHC and FISH were obtained from the Department of Pathology at AH. The FISH test was performed by Quest Diagnostics™ and IHC was performed by the Chair of the Department of Pathology at our institution. Statistical analysis was performed using IBM SPSS Statistics for Windows, version 20.0 (Armonk, NY: IBM Corp). Results Double hit gene rearrangements by FISH were detected in 3/22 (13.6%) patients. MYC IHC was positive in 8/22 (36.3%) cases, and 3 of these 8 cases (37.5%) were FISH positive. Sensitivity, specificity and concordance of MYC IHC as a screening tool were 100%,73.6% and 72.27%, respectively. BCL2 staining was positive in 19/22 (86.3%) cases, and only 3 of the 19 cases (15.7%) were FISH positive. 5/22 (22.7%) cases were positive for both MYC and BCL2 by IHC (double protein expresser status) and 3 of these 5 (60%) cases were positive for DHL status by FISH. DHL biology did not show an association with any clinico-pathological parameters including age, elevated serum Lactate dehydrogenase, R-IPI (Revised International Prognostic Index) score, ECOG performance status, extra-nodal disease and cell-of-origin sub-type. Conclusion The association between MYC IHC and FISH in our study was not statistically significant but showed a trend towards association. Given that sensitivity of MYC in our study was 100%, we propose that it can be used as an effective screening test if similar results can be validated in larger studies. Our study supports the practice of routinely testing for MYC and BCL2 status in all cases of DLBCL, irrespective of clinical features of the disease, to identify the high-risk DHL subset of patients, who can be candidates for more intense newer chemotherapy regimens and for prognostic purposes Disclosures No relevant conflicts of interest to declare.

Abstract 3038: Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL)

Abstract Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. Previously, our retrospective review from single institution series revealed that 30 out of 611 DLBCL patients had aberrations in c-MYC and BCL2 or BCL6 by FISH. These patients exhibited inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS), suggesting that newer therapies are in dire need. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL. Materials &amp; Methods: At the pre-clinical level, we studied 3 novel therapeutic agents targeting BCL2 (ABT-199), c-MYC (JQ-1), and various cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT-199 activity (irreversible proteasome inhibitor carfilzomib(CFZ)) using DH lymphoma (DHL) cell lines (Val, DOHH-2, ROS-50). DHL cell lines were exposed to ABT-199 (0-10 uM), JQ-1 (0-100 uM) and carfilzomib (CFZ) (0-50 nM) at 24, 48 and 72 hours. Changes in cell viability were evaluated using Presto Blue assay. Subsequently, DHL cells were exposed to doublet combinations of ABT-199, JQ-1 and CFZ for 48 hours. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1. Conclusion: ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing. Citation Format: Priyank P. Patel, Alison Zeccola, Juan Gu, Cory Mavis, Sheila N. J. Sait, Vishala Neppalli, Francisco J. Hernandez-Ilizaliturri. Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3038.

Ginsenoside Rg3 prevents INS-1 cell death from intermittent high glucose stress

ABSTRACTBackground: Ginsenoside Rg3 has been proposed to mediate anti-diabetic effects, but their direct effect on pancreatic β cell viability and mechanisms are not clearly understood. Recent studies suggest that intermittent high glucose (IHG) could be more harmful to pancreatic β cells than sustained high glucose. There are few reports about the effect of the ginsenosideRg3 to β cell apoptosis and proliferation against IHG.Methods: INS-1 cells were treated with alternative glucose concentration with or without ginsenoside Rg3. Cell apoptosis and viability were detected by Annexin V staining and MTT assay. The activation of mitogen-activated protein kinases (MAPKs) was analyzed by Western blotting using specific antibodies. Quantification of secreted insulin protein was measured using rat/mouse Insulin ELISA kits. Bromodeoxyuridine (BrdU) staining and florescence in situ hybridization (FISH) analysis was performed to compare cell proliferation.Result: INS-1 cell viability was decreased under IHG and increased with Rg3 treatment.Rg3 significantly reduced the apoptotic INS-1 cells against IHG. The quantification of secreted insulin concentration was increased with Rg3. Rg3 increased INS-1 cell proliferation. ERK and p38 MAPK pathways reduced by IHG were activated by the ginsenoside Rg3.Conclusion: Ginsenoside Rg3 protected INS-1 cell death from IHG with reducing apoptosis and increasing proliferation.

Pre-Clinical Investigation of Targeted Therapies for Double Hit Diffuse Large B-Cell Lymphoma (DH-DLBCL)

Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. DH-DLBCL has inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS). DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL.Materials & Methods: At the pre-clinical level, we studied novel therapeutic strategies targeting key-regulatory pathways abnormally enhanced by the dual BCL2/BCL6 and c-MYC gene rearrangements or amplification using DH lymphoma cell lines (Val, DOHH-2, ROS-50). We evaluated the therapeutic value of three novel agents targeting BCL2 (ABT199), c-MYC (JQ-1), and cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT199 activity (carfilzomib (CFZ), an irreversible proteasome inhibitor) in a panel of DH-DLBCL cell lines. We retrospectively evaluated our single institution outcome experience over the last 15 years in DH-DLBCL patients. Using the lymphoma translational database that includes 611 DLBCL patients, we identified 30 patients (M=17/F=13) with aberrations in c-MYC and BCL2 or BCL6 by FISH. Demographic characteristics and clinical data were collected and analyzed.Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1. Subsequently, we studied the outcome of DHL patients treated at our institute. Mean age at diagnosis was 63 years, mostly Caucasians (N=28). Using the Han's algorithm, 21 of the DH-DLBCL were GCB, 8 were non-GCB, and 1 unclassifiable. 22 patients had rearrangement of c-MYC and BCL2/ or BCL-6 by FISH and 8 had gain/amplification/aneuploidy at these loci. At diagnosis, 96% (n=29) patients presented with stage III/IV, 67% (n=20) with High-intermediate/High IPI score, and 77% (n=23) had extra nodal disease. Front-line chemo-immunotherapy included 1) standard doxorubicin-containing regimens: R-CHOP (N=11) or R-EPOCH (N=8), or 2) intensified regimens: R-DHAC (N=3) and R+HyperCVAD/R+HDMTXCytarabine (N=8). Prophylaxis IT chemotherapy was administered to 21 (70%) patients. The complete remission (CR) rate was 67% (n=20) and 37% (n=11) patients underwent HDC-ASCS (N=9)/allo-BMT (N=2) in first (N=9) or second-remission (N=2). The use of intensified regimens was associated with a non-statistically significant improvement is PFS and OS. Similarly, an improvement in OS was observed among patients receiving HDC-ASCT/allo-BMT as consolidation. As previously noted, CNS prophylaxis was associated with an improvement in OS. Mcl-1 and other Bcl-2 family members expression levels are been determined by IHC. These results highlight the poor clinical outcome of this patient population and the need for alternative therapeutic options.Conclusion: Our data suggests that ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing. DisclosuresNo relevant conflicts of interest to declare.

Transfer of Dicamba Tolerance from Sinapis arvensis to Brassica napus via Embryo Rescue and Recurrent Backcross Breeding.

Auxinic herbicides (e.g. dicamba) are extensively used in agriculture to selectively control broadleaf weeds. Although cultivated species of Brassicaceae (e.g. Canola) are susceptible to auxinic herbicides, some biotypes of Sinapis arvensis (wild mustard) were found dicamba resistant in Canada. In this research, dicamba tolerance from wild mustard was introgressed into canola through embryo rescue followed by conventional breeding. Intergeneric hybrids between S. arvensis (2n = 18) and B. napus (2n = 38) were produced through embryo rescue. Embryo formation and hybrid plant regeneration was achieved. Transfer of dicamba tolerance from S. arvensis into the hybrid plants was determined by molecular analysis and at the whole plant level. Dicamba tolerance was introgressed into B. napus by backcrossing for seven generations. Homozygous dicamba-tolerant B. napus lines were identified. The ploidy of the hybrid progeny was assessed by flow cytometry. Finally, introgression of the piece of DNA possibly containing the dicamba tolerance gene into B. napus was confirmed using florescence in situ hybridization (FISH). This research demonstrates for the first time stable introgression of dicamba tolerance from S. arvensis into B. napus via in vitro embryo rescue followed by repeated backcross breeding. Creation of dicamba-tolerant B. napus varieties by this approach may have potential to provide options to growers to choose a desirable herbicide-tolerant technology. Furthermore, adoption of such technology facilitates effective weed control, less tillage, and possibly minimize evolution of herbicide resistant weeds.

Molecular Diagnosis of Lung Cancers.

Recent advances in cancer diagnosis have seen a rapid and accurate diagnostic test. Initially used as research tools the molecular diagnostic tests have been applicable in the clinical scenario. Purpose: Current study focuses on the significant approach of detection of epidermal growth factor receptor (EGFR) mutations in exon 18-21 and anaplastic lymphoma kinase gene (ALK) rearrangement analysis using molecular methods such as pyrosequencing and florescence in-situ hybridization (FISH) on formalin fixed paraffin embedded (FFPE) from tissue biopsy and cell blocks of 267 lung cancer patients. Methods: In this study, FISH using a break-apart probe for the ALK gene was performed on formalin fixed paraffin-embedded tissue to determine the incidence of ALK rearrangements and hybridization patterns in 267 patients with a referred diagnosis of non-small cell lung cancer. EGFR mutations detection was performed on 267 samples by using Pyrosequencing. Results: Among the 267 patients 11 were positive for ALK-1 gene rearrangement. 260 of the 267 cases were confirmed of adenocarcinoma and 7 of the 267 cases were squamous cell carcinoma. EGFR mutations were detected in 55 patients. The most common mutations found were exon 19 (56.36%), exon 21 (27.27%), exon 20 (3.63%) and exon 18 (9.09%). The influence of gender, non-smoking, and histological type on the EGFR mutations showed increase in female group. Conclusions: Mutation detection testing is mandatory in lung carcinomas for the diagnosis and management of NSCLC.