EGFR mutation status yield from bronchoalveolar lavage in patients with primary pulmonary adenocarcinoma compared to a venous blood sample and tissue biopsy

Background In recent years, there has been a revolution in the genomic profiling and molecular typing of lung cancer. A key oncogene is the epidermal growth factor receptor (EGFR). The gold standard for determining EGFR mutation status is tissue biopsy, where a histological specimen is taken by a bronchoscopic or surgical method (transbronchial biopsy, forceps biopsy, etc.). However, in clinical practice the tissue sample is often insufficient for morphological and molecular analysis. Bronchoalveolar lavage is a validated diagnostic method for pathogenic infections in the lower respiratory tract, yet its diagnostic value for oncogenic mutation testing in lung cancer has not been extensively investigated. This study aims to compare the prevalence of EGFR mutation status in bronchoalveolar lavage and peripheral blood referring to the gold standard - tissue biopsy in patients with primary lung adenocarcinoma. Methods Twenty-six patients with adenocarcinoma were examined for EGFR mutation from tissue biopsy, peripheral blood sample and bronchoalveolar lavage. Results Thirteen patients had wild type EGFR and the other 13 had EGFR mutation. EGFR mutation from a peripheral blood sample was identified in 38.5% (5/13) of patients, whereas EGFR mutation obtained from bronchoalveolar lavage (BAL) was identified in 92.3% (12/13). This study demonstrates that a liquid biopsy sample for EGFR status from BAL has a higher sensitivity compared to a venous blood sample.

A Novel Missense Variant of TP63 Heterozygously Present in Split-Hand/Foot Malformation

Background. Split-hand/foot malformation (SHFM) is a severe congenital disability mainly characterized by the absence or hypoplasia of the central ray of the hand/foot. To date, several candidate genes associated with SHFM have been identified, including TP63, DLX5, DLX6, FGFR1, and WNT10B. Herein, we report a novel variant of TP63 heterozygously present in affected members of a family with SHFM. Methods. This study investigated a Chinese family, in which the proband and his son suffered from SHFM. The peripheral blood sample of the proband was used to perform whole-exome sequencing (WES) to explore the possible genetic causes of this disease. Postsequencing bioinformatic analyses and Sanger sequencing were conducted to verify the identified variants and parental origins on all family members in the pedigree. Results. By postsequencing bioinformatic analyses and Sanger sequencing, we identified a novel missense variant (NM_003722.4:c.948G>A; p.Met316Ile) of TP63 in this family that results in a substitution of methionine with isoleucine, which is probably associated with the occurrence of SHFM. Conclusion. A novel missense variant (NM_003722.4:c.948G>A; p.Met316Ile) of TP63 in SHFM was thus identified, which may enlarge the spectrum of known TP63 variants and also provide new approaches for genetic counselling of families with SHFM.

Pediatric Patient With Neurological And Leukemic Peripheral Blood Involvement By Small Cell Variant Of ALK- Positive Anaplastic Large Cell Lymphoma (ALCL): Case Study

Casestudy Anaplastic large cell lymphoma (ALCL), is a T-cell lymphoma typically consisting of large lymphoid cells including characteristic horseshoe- shaped hallmark cells. The rare small cell morphological variant of ALCL may pose a challenge in diagnosis, especially when the initial presentation is unusual. Results Our patient, a 7-year-old girl presented with a headache. A complete blood count showed leukocytosis and anemia. The smear was reported to have segmented neutrophils, reactive lymphocytes, and monocytes. A spinal tap was performed and flow analysis identified a minute aberrant T cell population (0.3% of total), positive for CD3, CD4, bright CD7; negative CD5, CD8 in the CSF sample. The peripheral blood sample was reviewed again; some small- medium atypical lymphocytes, with irregular nuclear contours and cytoplasmic azurophilic granules were noted. Flow immunophenotyping displayed an aberrant T cell population positive for CD45, CD2, CD3, bright CD7, CD4, CD13; negative CD30, TdT CD5, CD8, CD117, CD34; consistent with T cell lymphoma/leukemia. A cell block prepared from peripheral blood sample showed blood with numerous atypical cells with irregular nuclei positive for ALK, CD30, CD3, CD4, CD7; negative CD5 and CD8. A diagnosis of leukemic ALK(+) ALCL was rendered, though classic hallmark cells were difficult to see. A marrow biopsy showed interstitial and sinusoidal pattern of mainly small to medium-sized cells with irregular nuclei. Molecular study revealed ALK gene alteration showing characteristic NPM1-ALK fusion. The patient underwent a bone bone marrow transplantation but recently relapsed with a submandibular lymph hode biopsy showing the presence of many larger ALCL cells. Conclusion Correct clinical diagnosis of the small-cell variant of ALCL is often challenging as the scarce “hallmark cells” are scattered and difficult to recognize. While leukemic peripheral blood involvement is rare in ALCL, an association has been reported with small-cell variants, which may be a potential explanation for the poor prognosis and aggressive nature of small-cell variant ALCL. A meticulous examination of peripheral blood smears, comprehensive immunophenotypic studies, and early bone marrow and lymph node/tissue biopsy are needed to facilitate diagnosis.

Uric Acid and Nutrient Intake of Adolescent Soccer Players in Yogyakarta

The aim of this study was to view uric acid levels and nutrient intake of soccer players. This study examined the uric acid level differences based on nutrient intake of adolescent soccer players. Indeed, nutritional status monitoring is needed to evaluate health status which can contribute to athletes’ performance. This was an observational study conducted at SSB Real Madrid in April-September 2017. Subjects involved in this study were 27 active joined U-13 or U-16 in SSB Read Madrid UNY, Yogyakarta. Uric acid data were obtained by measuring peripheral blood sample. Nutrient intake data were obtained by using SQFFQ form. The differences in uric acid levels based on nutritional intake category were tested using ANOVA test. The average of uric acid levels were 6.72±1.21 mg/dl with average protein intake of 87.38±27.41 grams. The average of energy, fat, and carbohydrate intake was 2,189.02±464.87 kcal, 46.92±15.75 grams, and 365.61±82.32 grams. There was no difference in uric acid levels based on energy, protein, fat, and carbohydrates intake (p=0.216; p=0.489; p=0.587; and p=0.845) of adolescent soccer players. The clearance of uric acid, the amount and type of fluid intake, the type of animal protein, and the purine-source food probably contributed to athletes’ uric acid levels.

A First Step to a Biomarker of Curative Surgery in Colorectal Cancer by Liquid Biopsy of Methylated Septin 9 Gene

Objectives. To confirm that patients affected by colorectal cancer have the V2 region of Septin 9 (SEPT9) gene hypermethylated in the circulating free DNA from a peripheral blood sample before surgery and to determine if this hypermethylated DNA disappears from the patients after complete resection of the tumour. Methods. Plasma from 10 patients with colorectal cancer was collected preoperative and three months after surgery. The analysis of the methylation status of the promoter region of the SEPT9 gene was performed using a 7500 Fast Real-Time PCR System. Results. Hypermethylation of SEPT9 gene was detected in 8 out of 10 preoperative samples (one negative result was probed to be a Lynch syndrome) and in 4 out of 10 postoperative samples matching with the cases of recurrence or persistence of disease. This means that, in this sample, the preoperative sensitivity and specificity of the test were 88.9% and 100%, respectively, and there is 100% correlation between the positive results of the SEPT9 test and a recurrence/persistence of the disease in patients after surgical resection. Conclusions. Our study shows that circulating hypermethylated SEPT9 is a specific colorectal cancer biomarker. This hypermethylated SEPT9 DNA disappears around three months after surgery and that circulating hypermethylated SEPT9 may be the first noninvasive marker for postsurgical diagnosis; this conclusion must be confirmed with a more significant number of patients.

Combination of Novel c.3484G> T/p.Glu162Ter Variant in ABCB11 and c.208G> A/p.Asp70Asn Variant in ATP8B1 Are Associated with Severe Symptoms in Progressive Family Intrahepatic Cholestasis

Progressive family intrahepatic cholestasis (PFIC) is an autosomal recessive disease that causes chronic cholestasis. It is associated with pathogenic variants in genes that encode proteins involved in bile secretion to canaliculus from hepatocytes. In this study, we present a 16-year-old boy who presented with severe pruritus and cholestatic jaundice. All possible infectious etiologies were negative. A liver biopsy was consistent with intrahepatic cholestasis and portal fibrosis. DNA was isolated from a peripheral blood sample, and whole exome sequencing was performed. A novel c.3484G > T/p.Glu162Ter variant in the ABCB11 gene and a c.208G> A/p.Asp70Asn variant in the ATP8B1 gene were detected. Despite traditional treatment, the patient's recurrent severe symptoms did not improve. The patient was referred for a liver transplantation. This novel c.3484G > T/p.Glu162Ter variant is associated with a severe and recurrent presentation, and the two compound variants could explain the severity of PFIC.

Clinical and Cytogenomic Features of Lymphoblastic Leukemia with Intrachromosomal Amplification of Chromosome 21 (iAMP21) in the Context of Constitutional Ring Chromosome 21

Constitutional ring chromosome 21, r(21)c, is a rare chromosome abnormality associated with variable clinical features that range from mild dysmorphism to severe congenital anomalies and intellectual disability. Recently, r(21)c has been reported to predispose to B-cell acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21), a distinct subgroup of high-risk pediatric B-ALL. Only a few iAMP21-B-ALL cases with r(21)c have been reported to date. The mechanism of leukemogenesis of r(21)c has not been entirely elucidated. Here we report an 11-year-old boy with iAMP21-B-ALL carrying an atypical r(21)c. The patient has a history of attention-deficit/hyperactivity disorder, sensorineural hearing loss s/p cochlear implant, intellectual disability and scoliosis. Three months before admission he developed a soft tissue nodule on the occipital scalp deemed to possibly represent an enlarged lymph node. Subsequently, he presented with spontaneous bruising followed by severe epistaxis. The initial CBC showed a white blood cell count of 4.3K/uL with circulating blasts, absolute neutropenia, profound normocytic normochromic anemia (hemoglobin of 5.2 g/dL), and marked thrombocytopenia (platelets, 12K/uL ). Peripheral blood flow cytometry showed 17.9% phenotypically abnormal B lymphoblasts which were negative for CD45, and positive for CD34, nuclear TdT, CD19, CD22, CD79a, CD10, HLA-DR , as well as CD20 (21% positive). The bone marrow aspirate contained 98% blasts. CNS status was 2a (RBC 0, WBC 0, 8% blasts) and clear after the second lumbar puncture. Fine-needle aspiration of the scalp mass demonstrated B-lymphoblastic leukemia/lymphoma. The patient was treated per COG protocol AALL1131 and was assigned to the very high-risk arm when bone marrow interphase FISH showed iAMP21. The chromosome analysis failed to yield metaphase cells on the diagnostic bone marrow sample. A concurrent genomic microarray showed chromothripsis with multiple non-contiguous losses and high copy gains on 21q involving the RUNX1 gene, as well as mosaic deletions within 7q22.3q36.3, 9p24.3p24.1 (including JAK2), 12q12 (several exons of ARID2), 13q14.2q21.2 (RB1, DLEU1/2/7, miR-15a/miR-16-1 cluster), 14q32.33 (IGH locus), 19p13.2 (several exons of the SMARCA4), and mosaic gains within 3q22.3q29 and Xp22.33p11.3. Day 29 end of induction bone marrow examination was positive for minimal residual disease (MRD) at 0.13% of nucleated mononuclear cells, but FISH was negative for iAMP21. On day 57 of consolidation, the bone marrow was negative for both MRD and iAMP21. However, chromosome analysis on both of these follow-up studies showed an abnormal chromosome 21. Chromosome analysis on peripheral blood lymphocytes confirmed the presence of a constitutional r(21). A subsequent genomic microarray analysis on peripheral blood sample did not show chromothripsis observed in the diagnostic bone marrow sample, but showed a 4.7 Mb terminal deletion and two interstitial deletions (3.0 Mb and 5.5 Mb) on the long arm of chromosome 21. These findings are consistent with a r(21) with interstitial deletions, which is likely responsible for the congenital anomalies reported in this patient. iAMP21 is associated with a poor outcome in B-ALL. Accurate detection of iAMP21 is critical for risk stratification and treatment in B-ALL. The strong association between iAMP21 and r(21)c has been proposed based on previous studies on r(21)c carriers with iAMP21-ALL. Our data further support an increased risk of developing iAMP21-ALL in carriers of constitutional r(21) and demonstrate the value of intensive treatment on iAMP21-B-ALL. The r(21) observed in this patient contains a relatively larger (4.7 Mb) terminal deletion along with two additional interstitial deletions. Due to the scarcity of r(21)c, the pathogenetic mechanisms of this leukemic process is not fully understood, and the clinical significance of loss of additional genetic content is unknown. More case reports are needed to generate more comprehensive clinical and genetic profile for this high risk ALL. Figure 1. Genomic microarray findings on diagnostic bone marrow sample (top) and the follow up peripheral blood sample (bottom). Chromothripsis and amplification were observed only in the diagnostic bone marrow specimen, whereas the peripheral blood sample in remission showed two interstitial and a terminal deletion. Figure 1 Disclosures No relevant conflicts of interest to declare.

The Association Between Affective Temperament Traits and Dopamine Genes in Obese Population

Studies indicate the heritable nature of affective temperament, which shows personality traits predisposing to the development of mental disorders. Dopaminergic gene polymorphisms such as DRD4, COMTVal158Met, and DAT1 have been linked to affective disorders in obesity. Due to possible correlation between the aforementioned polymorphisms and the affective temperament, the aim of our research was to investigate this connection in an obese population. The study enrolled 245 obese patients (178 females; 67 males). The affective temperament was assessed using the Temperament Evaluation of Memphis, Pisa, Paris, and San Diego autoquestionnaire (TEMPS-A). Genetic polymorphisms of DAT1, COMTVal158Met and DRD4 were collected from peripheral blood sample and determined using a polymerase chain reaction (PCR). Only in COMT polymorphisms, the cyclothymic and irritable dimensions were significantly associated with Met/Val carriers (p = 0.04; p = 0.01). Another interesting finding was the correlation between the affective temperament and age in men and women. We assume that dopamine transmission in heterozygotes of COMT may determine the role of the affective temperament in obese persons. Dopaminergic transmission modulated by COMT may be responsible for a greater temperament expression in obese individuals. To our knowledge, this is the first study describing the role of affective temperament in the obese population, but more research is needed in this regard.

Case Report: Detection and quantification of tumor cells in peripheral blood and ascitic fluid from a metastatic esophageal cancer patient using the CellSearch® technology

Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch® technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch® technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch® technology.