Proto-oncogene Mutations Research Articles

Targeting BRAF and RAS in Colorectal Cancer.

Simple SummaryIn colorectal cancer, mutations of the KRAS and BRAF genes are quite common and can contribute to the activation of cell signaling pathways that lead to cell proliferation and differentiation. These processes promote cancer growth, and in some cases, they may cause cells to develop resistance to certain types of treatment, notably EGFR inhibitors. We summarize recent knowledge regarding the effects of KRAS and BRAF mutations in the setting of colorectal cancer and discuss the new therapies under development.Colorectal cancer (CRC) is still one of the most frequent forms of cancer in the world in terms of incidence. Around 40% of CRC patients carry a mutation of the Kirsten rat sarcoma (KRAS) gene, while 10% have a mutation in the B-Raf proto-oncogene serine/threonine kinase (BRAF) gene. These mutations are responsible for dysregulation of the mitogen-associated protein kinase (MAPK) pathway, leading to the proliferation, differentiation, angiogenesis, and resistance to apoptosis of cells. Activation of the MAPK pathway results in adaptive therapeutic resistance, rendering EGFR inhibitors ineffective. This review aims to highlight the recent findings that have improved our understanding of KRAS and BRAF mutations in colorectal cancer and to describe new targeted therapies, used alone or in combination.

Presentation of Pheochromocytoma, Papillary Thyroid Cancer and Hyperparathyroidism in Three Family Members

Introduction: Although rare, one of the most common inherited disorders is multiple endocrine neoplasia. It is an autosomal dominant disorder that predisposes individuals to certain endocrine abnormalities depending on which type. The type 2A is a combination of medullary thyroid cancer, hyperparathyroidism, and pheochromocytoma which have been explained to be due to a mutation in the RET proto-oncogene. This abstract present a case of a patient with hyperparathyroidism whom family members also have pheochromocytoma and papillary thyroid cancer. Case description: A 45 year-old Hispanic male came to the endocrinology clinic complaining of constipation and headache. He has a personal history of non-toxic multinodular goiter and underwent right sided thyroidectomy in 2015 with pathology report showing follicular adenoma. He is currently on thyroid replacement therapy. He is clinically and biochemically euthyroid with TSH of 2.29 IU/ml. Physical examination was unremarkable. His labs were pertinent for calcium 11.5mg/dl, parathyroid 245.7pg/ml, creatinine of 1.5mg/dl. Two years ago, parathyroid was 189.5pg/ml and calcium was 11mg/dl, 1.5 year ago parathyroid level was 235.5pg/ml, calcium was 11.4mg/dl, urine calcium 9.3mg/dl, 24hr urine calcium 286.4mg, calcitonin <2pg/ml, vitamin D 23ng/ml, 1,25 vitamin D 53ng/ml. In 2017, Sestambi scan showed equivocal focus of faint parathyroid activity in the region of the mid to lower left thyroid lobe versus faint residual thyroid activity and in 2019, scan showed no definite parathyroid adenoma. Surgical intervention was recently recommended due to patient’s DEXA scan showing osteoporosis of the femoral neck. The family history of this patient is pertinent for two sisters; one with pheochromocytoma and the other with papillary thyroid cancer. One of the sisters is a 60 years old diagnosed with pheochromocytoma at 51. Her free normetanephrine level was 682pg/ml and total metanephrine was 727pg/ml at time of diagnosis. Her MRI report showed right adrenal mass measuring 3.5x2.8cm. Laparoscopic right adrenalectomy was done and pathology confirmed pheochromocytoma which was RET negative. She still follows up with endocrinology and calcitonin, chromogranin A and plasma metanephrines have been normal. The second sister is now 53 years old diagnosed with papillary thyroid cancer at age 27 and had total thyroidectomy with pathology confirming papillary thyroid cancer. Discussion: Based on the clinical presentation of these family members, the most likely explanation is familial inheritance. This pattern of inheritance cannot be explained by MEN 2A or 2B due to the absence of medullary thyroid cancer. It has also been reported that this unusual presentation could be a variant of MEN 2A.[i] Due to the family history, close follow up is required to monitor for the possible development of other endocrinopathies in the future.

Occurrence of Hepatocellular Carcinoma in a 40-Year-Old Male With MEN2A Syndrome

Background: Multiple Endocrine Neoplasia Type 2A (MEN 2A) is a genetic syndrome in which the patient is susceptible to the development of: 1. medullary thyroid carcinoma, 2. pheochromocytoma, and 3. parathyroid adenomas or hyperplasia. Our objective is to report the first occurrence of hepatocellular carcinoma in a young adult male with MEN 2A syndrome without prior liver disease.Clinical Case: A 40-year-old male was screened positive for the C634R point mutation of the RET Proto-Oncogene after his sister with MEN 2A syndrome had tested positive. The patient had no history of alcohol or drug abuse. His family history was remarkable for the deaths of his mother and maternal grandmother from unknown malignancies at the ages of 35 and 45 respectively. His physical exam revealed a BP of 135/85mm Hg, BMI 26 kg/m2 and palpable bilateral thyroid nodules. Lab tests included a calcitonin of 131 pg/ml, (0.0-8.4 pg/ml), calcium 11.4 mg/dL (8.4-10.3 mg/dL), parathyroid hormone (PTH) 1765 pg/ml (12-65 pg/ml), ALK PHOS 1856 U/L (20-120 U/L), ALT 16 U/L (<39 U/L), Albumin 4.1gm/dl (3.4-5.0gm/dl), plasma normetanephrine 0.95 nmol/L (0.00- 0.89 nmol L) and urine metanephrines 291 pg/d (62-207 pg/d). A CT scan demonstrated hyperdense 12 mm right and 5 mm left adrenal nodules, both confirmed MIBG positive. Neck ultrasound showed bilateral thyroid nodules, the largest measuring 1.1 x 0.8 x 1.1 cm in the right lobe and a 3.5 x 2.2 x 2.1 cm heterogeneous mass posterior to the right thyroid lobe. The patient was prepped with doxazosin and underwent a laparoscopic right adrenalectomy then 2 months later a total thyroidectomy with neck lymph node dissection, resection of the right parathyroid tumor mass and bilateral inferior parathyroid glands. Surgical pathology confirmed the right adrenal pheochromocytoma, multifocal bilateral medullary thyroid cancer with 1 of 70 neck lymph nodes positive and a single large parathyroid adenoma. He received levothyroxine to maintain euthyroidism. Within 6 months of his surgery, calcitonin was < 2 pg/ml, CEA 2.3 U/ml (<5.0 U/ml) and ALK PHOS 44 U/L. Urine metanephrines remained mildly elevated 291 pg/d (62-207 pg/d). Serial abdominal CT imaging revealed no change of the left adrenal mass but three new bi-lobar 1-2 cm enhancing liver lesions. CT guided needle biopsies of the liver masses revealed hepatocellular cancer without evidence of cirrhosis. The patient underwent successful thermal ablation of all 3 liver lesions. Laboratory evaluation was negative for Hepatitis A, B and C. His alpha fetal protein level has remained stable in the range of 4.7 to 5.8 ng/ml (<15ng/ml).Conclusions: This is the first reported case of hepatocellular carcinoma in a patient with MEN 2A syndrome who had no predisposing liver disease, raising suspicion that his germline RET mutation contributed to his liver cancer.

Never Forget the Family History: MEN2A Syndrome Presenting as Metastatic Medullary Thyroid Cancer and Bilateral Pheochromocytoma

Abstract Introduction: MEN 2A is an autosomal dominant hereditary syndrome considered part of the medullary thyroid carcinoma (MTC) syndromes. This is characterized by MTC, pheochromocytoma, and parathyroid hyperplasia or adenomas causing primary hyperparathyroidism (PHPT). Clinical Case: A 34 year old female was referred to our clinic for multi-nodular goiter diagnosed during routine gynecologic evaluation. A thyroid ultrasound revealed a heterogeneous right thyroid lobe with a hypoechoic 2.5 cm nodule, associated macro calcifications and increased vascularity; and a left nodule measuring 2.3 cm with the same characteristics. Bilateral thyroid nodule biopsies were performed, resulting in MTC confirmed by positive calcitonin staining. Pre-operative studies revealed serum calcitonin and carcinoembryonic antigen (CEA), both of which are considered serologic markers of MTC activity, at 4,340 pg/ml (n &amp;lt;= 5) and 276.2 ng/mL (n &amp;lt;= 2.5 in non-smokers) respectively. The patient reported father with history of unspecified thyroid cancer, and paternal uncle with history of pheochromocytoma with a p.Cys634Trp mutation in RET proto-oncogene. Due to her family history, pre-operative screening for primary hyperparathyroidism (PHPT) resulted in a calcium 10 mg/dL (n 8.6-10.2), PTH 34 pg/mL (n 14-64). Additionally, screening for pheocromocytoma revealed an elevated 24 hour urine metanephrines of 2,276 (n &amp;lt;= 49-290) ug/24h, plasma metanephrines, including fractionated metanephrine (MN) at 163 (n &amp;lt;= 57) pg/ml, fractionated nor-metanephrine (NMN) at 182 (n &amp;lt;= 148) pg/ml, and total, Free (MN+NMN) metanephrines at 345 (n &amp;lt;= 205) pg/ml. CT abdomen revealed bilateral adrenal nodules, right measuring 1.4 x 3.3 cm and left 2.4 x 3.3 cm. The patient underwent posterior retroperitoneoscopic adrenalectomy with cortex sparing prior to thyroidectomy. Adrenal pathology resulted in bilateral pheochromocytoma with peri-adrenal adipose tissue microscopic involvement, and positive synaptophysin and S-100 stain. Subsequently, she underwent total thyroidectomy with extensive cervical lymph node resection, with pathology resulting in MTC with lymph node metastasis, involving 5/18 cervical lymph nodes. Post-operative labs revealed serum calcitonin &amp;lt;= 2 pg/ml, CEA 26.8 ng/mL, MN &amp;lt; 25 pg/ml, NMN 102 pg/ml, and MN+NMN of 102 pg/ml, which suggested initial surgical success. Post-operative genetic test evaluation revealed abnormal RET oncogene testing compatible with MEN 2A, variant 1: c.1902C&amp;gt;G (p.Cys634Trp). Conclusion: This case illustrates that patients presenting with MTC and reporting family history of thyroid cancer should be screened for familial MTC syndrome. Patients with RET mutation should be screened for pheochromocytoma prior to surgery for MTC to prevent life-threatening hypertensive crisis.

Occurrence of sporadic medullary thyroid carcinoma in Graves’ disease in association with a RET proto-oncogene mutation

ABSTRACT Background Graves’ disease may be associated with thyroid cancer, particularly differentiated thyroid cancer. Medullary thyroid cancer (MTC) is less common. The occurrence of sporadic MTC in Graves’ disease in the presence of a RET proto-oncogene has never been reported. Clinical presentation A 63-year-old woman was referred for Graves’ disease. A thyroid ultrasound disclosed five nodules, one of which was classified as Eu-Tirads 5 with a size of 6.7 × 6.5× 11 mm. Fine needle aspiration was reported as Bethesda class IV follicular neoplasm of a Hürthle cell subtype. Calcitonin level was found to be elevated. A total thyroidectomy confirmed the diagnosis of MTC and a bilateral cervical lymphadenectomy was performed, with four lymph nodes being infiltrated by MTC. Genetic testing revealed a M918T mutation in the RET proto-oncogene. Conclusion MTC may occur in Graves’ disease, especially if a nodule is present. In this case, genetic testing should always be performed even if MTC is sporadic. Increased incidence of thyroid cancer in autoimmune thyroid diseases, as well as the link existing between autoimmunity, inflammation and carcinogenesis, leads us to hypothesize that the association here reported is not coincidental.

Generation and characterization of iPSC lines (BCH001) from a boy with intron 14 mutation in the ret proto-oncogene (RET) gene

Congenital central hypoventilation syndrome (CCHS) is characterized by an alteration of the ventilatory response to hypercapnia and hypoxia, and is classically presented in neonates with abnormalities of the autonomic nervous system. Here, we generated human induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) isolated from a male patient clinically diagnosed with CCHS. These iPSC lines carry a heterozygous RET mutation (c.2608-125C > T), express pluripotency markers, have the capacity to differentiate into the normal teratoma tissue, retain the RET mutation and display the normal karyotype, which will also provide a useful resource to study the pathogenesis of CCHS.

An integrated deep learning and dynamic programming method for predicting tumor suppressor genes, oncogenes, and fusion from PDB structures

Mutations in proto-oncogenes (ONGO) and the loss of regulatory function of tumor suppression genes (TSG) are the common underlying mechanism for uncontrolled tumor growth. While cancer is a heterogeneous complex of distinct diseases, finding the potentiality of the genes related functionality to ONGO or TSG through computational studies can help develop drugs that target the disease. This paper proposes a classification method that starts with a preprocessing stage to extract the feature map sets from the input 3D protein structural information. The next stage is a deep convolutional neural network stage (DCNN) that outputs the probability of functional classification of genes. We explored and tested two approaches: in Approach 1, all filtered and cleaned 3D-protein-structures (PDB) are pooled together, whereas in Approach 2, the primary structures and their corresponding PDBs are separated according to the genes' primary structural information. Following the DCNN stage, a dynamic programming-based method is used to determine the final prediction of the primary structures' functionality. We validated our proposed method using the COSMIC online database. For the ONGO vs TSG classification problem the AUROC of the DCNN stage for Approach 1 and Approach 2 DCNN are 0.978 and 0.765, respectively. The AUROCs of the final genes' primary structure functionality classification for Approach 1 and Approach 2 are 0.989, and 0.879, respectively. For comparison, the current state-of-the-art reported AUROC is 0.924. Our results warrant further study to apply the deep learning models to humans’ (GRCh38) genes, for predicting their corresponding probabilities of functionality in the cancer drivers.

Crude annual incidence rate of medullary thyroid cancer and RET mutation frequency

To determine the frequency and type of RET mutation in Slovenian medullary thyroid cancer (MTC) patients and estimate the crude annual incidence of MTC in Slovenia. This referral-center retrospective analysis involved 186 MTC patients diagnosed between 1995 and 2015 and their relatives who underwent genetic counseling and testing. The crude incidence rate of MTC was estimated with the joinpoint regression analysis. Genomic DNA was isolated, and exons 10, 11, 13, 14, 15, and 16 of the RET proto-oncogene were amplified with polymerase chain reaction. Point mutations of the RET gene were detected by single-strand conformation analysis and DNA sequencing. Detected mutations were confirmed by restriction enzymes. The average crude annual incidence rate of MTC in Slovenia was 0.34/100,000. A germline mutation in the RET proto-oncogene was identified in 25.9% of MTC patients. The most frequently altered codons were codons 634 and 618, followed by codon 790, codon 804, and codon 918. Annual incidence increase and nation-specific frequency of RET mutations justify the future use of genetic counseling and testing of MTC patients in Slovenia.

Familial Hyperparathyroidism.

Regulation of the serum calcium level in humans is achieved by the endocrine action of parathyroid glands working in concert with vitamin D and a set of critical target cells and tissues including osteoblasts, osteoclasts, the renal tubules, and the small intestine. The parathyroid glands, small highly vascularized endocrine organs located behind the thyroid gland, secrete parathyroid hormone (PTH) into the systemic circulation as is needed to keep the serum free calcium concentration within a tight physiologic range. Primary hyperparathyroidism (HPT), a disorder of mineral metabolism usually associated with abnormally elevated serum calcium, results from the uncontrolled release of PTH from one or several abnormal parathyroid glands. Although in the vast majority of cases HPT is a sporadic disease, it can also present as a manifestation of a familial syndrome. Many benign and malignant sporadic parathyroid neoplasms are caused by loss-of-function mutations in tumor suppressor genes that were initially identified by the study of genomic DNA from patients who developed HPT as a manifestation of an inherited syndrome. Somatic and inherited mutations in certain proto-oncogenes can also result in the development of parathyroid tumors. The clinical and genetic investigation of familial HPT in kindreds found to lack germline variants in the already known HPT-predisposition genes represents a promising future direction for the discovery of novel genes relevant to parathyroid tumor development.

AAV Joins the Rank of Genotoxic Vectors

AAV Joins the Rank of Genotoxic Vectors

Significance of chromogranin A and synaptophysin in medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare thyroid carcinoma of C-cell deviation that produces and secretes calcitonin (CT) and chromogranin A (CgA) into the blood. Thus, CT and CgA are immunohistochemical and serum markers for MTCs. MTC occurs in sporadic and inheritable forms. The hallmark of inheritable cases in multiple endocrine neoplasm 2 (MEN2) is MTC. MEN2 cases represent 30% through germline RET proto-oncogene mutation and occur in younger ages involving bilateral thyroid lobes. Sporadic cases are 70% and occur in older ages. CgA and synaptophysin (SPY) are the two most widely used and reliable immunohistochemical markers for neuroendocrine tumors, including MTCs. This study aimed to detect different immunohistochemical staining patterns for CgA and SPY between non-symptomatic small lesions and invading larger aggressive tumors in both MEA2 cases and sporadic cases. There was different CgA and SPY immunostaining in MEA2 cases where small tumors (≤0.3 cm) were lesser immunostained for CgA and SPY, despite intense staining for CT, compared to the larger (≥0.5 cm) tumors, stronger immunostained for CgA. There was also different CgA and SPY immunohistochemical staining in sporadic cases between small lesions (≤0.5 cm) and larger tumors (≥1.0 cm). One small sporadic tumor (0.5 cm × 0.3 cm) was strongly and weakly, patchy stained for CgA and SPY, respectively, while larger sporadic tumors were diffusely and strongly stained for CgA and SPY. Therefore, stronger CgA and SPY immunostaining for larger tumors in both MEA2 and sporadic cases may be used as independent, aggressive immunohistochemical markers for MTCs.

Liver resection for colorectal liver-limited metastases in elderly patients: a propensity score matching analysis

BackgroundFew studies have focused on the role of hepatectomy for colorectal liver-limited metastases in elderly patients compared to matched younger patients.MethodsFrom January 2000 to December 2018, 724 patients underwent hepatectomy for colorectal liver-limited metastases. Based on a 1:2 propensity score matching (PSM) model, 64 elderly patients (≥ 70 years of age) were matched to 128 younger patients (< 70 years of age) to obtain two balanced groups with regard to demographic, therapeutic, and prognostic factors.ResultsThere were 73 elderly and 651 younger patients in the unmatched cohort. Compared with the younger group (YG), the elderly group (EG) had significantly higher proportion of American Society of Anesthesiologists score III and comorbidities and lower proportion of more than 3 liver metastases and postoperative chemotherapy (p < 0.05). After PSM for these factors, rat sarcoma virus proto-oncogene/B-Raf proto-oncogene (RAS/BRAF) mutation status and primary tumor sidedness, the EG had significantly less median intraoperative blood loss than the YG (175 ml vs. 200 ml, p = 0.046), a shorter median postoperative hospital stay (8 days vs. 11 days, p = 0.020), and a higher readmission rate (4.7% vs.0%, p = 0.036). The EG also had longer disease-free survival (DFS), overall survival (OS), and cancer-specific survival (CSS) compared to the YG, but these findings were not statistically significant (p > 0.05). Old age was not an independent factor for DFS, OS, and CSS by Cox multivariate regression analysis (p > 0.05).ConclusionsHepatectomy is safe for colorectal liver-limited metastases in elderly patients, and these patients may subsequently benefit from prolonged DFS, OS, and CSS.

The clinical significance of RET gene fusion among Chinese patients with lung cancer.

BackgroundThe incidence of lung cancer is growing fast in China, however, the prognosis remains dismal due to the limited therapeutic approaches. The “ret proto-oncogene mutation” (RET) fusions have been proven to be the driver gene in lung cancer development and the therapeutic target of several multi-target tyrosine kinase inhibitors.MethodsWe applied formalin-fixed, paraffin-embedded (FFPE) samples of 39 patients with non-small cell lung cancer (NSCLC) using the Lung Plasma panel covering 168 cancer-associated genes and performed capture-based targeted deep sequencing to identify the RET fusion partners and concurrent gene mutation with Miseq. The log-rank test was used to compare the survival difference of patients according to treatment strategies. Statistical analyses and graphs were performed using R language and GraphPad Prism.ResultsMost of the samples were advanced (stage IIIb and IV) lung adenocarcinomas (80.77%). KIF5B-RET fusions were identified in 52% of the samples and K15-E12 was the most common variant. 6 (15%) samples harbored concurrent TP53 mutation and 3 samples were positive with EGFR mutation including a mutation in exon 19. Of these patients included, ten received cabozantinib, two received anlotinib, and one received crizotinib. Two (20%; 0–45) samples achieved stable disease and two were progressed in the cabozantinib treated group. Median progression-free survival (PFS) was 4 months (95% CI: 3.2–4.8) and median overall survival (OS) was 25 months (95% CI: 1.5–48.5). Three (11.54%; 0–24) samples achieved partial response in patients without RET inhibitor treatment and 4 (15.38%; 2–29) were stable disease. The median PFS was 11 months (95% CI: 1.2–20.8). There was no significant difference in PFS and OS between groups with or without RET inhibitors treatment.ConclusionThis study provided insight into the RET fusions patients treatment. The survival benefit of current RET inhibitors was limited. More precise and potent RET inhibitors should be developed in the near future.

Total colonic aganglionosis and cleft palate in a newborn with Janus-cysteine 618 mutation of RET proto-oncogene: a case report

BackgroundHirschsprung disease, the most important congenital colonic dysmotility in children results from neural crest migration, differentiation, proliferation, or apoptosis defects where the rearranged during transfection (RET)-Protooncogene pathway has a central role. Although palatal and retinal anomalies in the context of chromosomopathies and some mono−/oligogenic syndromes are reported associated with Hirschsprung disease the role of inactivating RET mutations in these cases is not clarified.Case presentationWe report on a dysmorphic newborn with cleft palate and palatal synechia, who showed intestinal obstruction after 24 h of life. Transient ileostomy and surgical biopsies were performed to diagnose aganglionosis of the colon and last ileal loop. No chromosomal anomalies or copy number variations were found. We identified a paternal heterozygous germline mutation c.1852 T > C, which results in the substitution of cysteine by arginine in the RET-receptor tyrosine kinase (p.C618R mutation). There was no family history of Hirschsprung disease, but the father underwent surgery for medullary thyroid carcinoma and was affected by retinal dystrophy.ConclusionsThe occurrence of Hirschsprung disease and carcinoma shows how a single mutation may be responsible for adverse effects: gain and loss of function of the same receptor. Furthermore, it would be interesting to study its dual role in face and retina embryology, and to extend targeted investigations of RET hotspots in these developmental abnormalities to facilitate counselling, follow-up, and tumor prevention. Complex surgical procedures and genetic testing as well as socio-economic impact are a challenge for familiar compliance.

Does Pemetrexed Work in Targetable, Nonsquamous Non-Small-Cell Lung Cancer? A Narrative Review.

Simple SummaryThe chemotherapy agent pemetrexed is currently considered in combination with other therapies for the treatment of advanced nonsquamous non-small-cell lung cancer (NSCLC) in patients negative for gene mutations/rearrangements. The aim of this review was to highlight data from clinical studies with pemetrexed in patients with advanced nonsquamous NSCLC positive for gene mutations/rearrangements. The results of the review suggest that pemetrexed could be a treatment option in patients with advanced nonsquamous NSCLC positive for certain gene mutations/rearrangements.Pemetrexed is currently mainly considered for the treatment of advanced nonsquamous non-small-cell lung cancer (NSCLC) negative for gene mutations/rearrangements (wild-type disease (WTD)). This narrative review aimed to highlight the role of pemetrexed in the treatment of onco-driven nonsquamous advanced NSCLC by reviewing published clinical studies. For epidermal growth factor receptor (EGFR) mutations, patient survival following first-line pemetrexed–platinum was longer than for WTD. Later-line pemetrexed-based treatment after tyrosine kinase inhibitor (TKI) failure provided greater benefits than non-pemetrexed regimens. First- and later-line pemetrexed-based therapy also provided survival benefits in patients with anaplastic lymphoma kinase (ALK) or ROS proto-oncogene 1 (ROS1) rearrangements. In patients with rearranged during transfection (RET) proto-oncogene rearrangements, survival with pemetrexed was similar to that in ALK- and ROS1-positive patients and longer than that in patients with Kirsten rat sarcoma (KRAS) virus proto-oncogene mutations or WTD, although the available studies were limited. For Erb-b2 receptor tyrosine kinase 2 (ERRB2) mutations, first-line pemetrexed showed outcomes similar to those for EGFR and KRAS alterations. Data on pemetrexed in patients with KRAS mutations or MNNG HOS-transforming (MET) expression were limited. Pemetrexed could be an option for first- and second-line treatment for TKI failure in nonsquamous advanced NSCLC with select targetable driver mutations.

Abstract 3828: Tumor promoting effects of sulforaphane on diethylnitrosamine-induced murine hepatocarcinogenesis

Abstract Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor involved in protection against initiation of carcinogenesis in normal cells. Notably, recent studies have demonstrated that mutation of the gene encoding this transcription factor causes its overactivation in various types of human malignancies. The aberrant activation of NRF2 is considered to accelerate the proliferation and progression of cancer cells. However, the exact function of NRF2 in the stage of pre-malignancy remains still largely unresolved. The differential effects of NRF2 on multi-stage carcinogenesis have raised a concern about the validity of NRF2 activators for chemoprevention. In the present study, a well-known NRF2 activating chemopreventive phytochemical, sulforaphane (SFN), was daily injected intraperitonally (25 mg/kg) for 3 months to male C57BL/6 mice at 6 months after single intraperitoneal administration of a hepatocarcinogen, diethylnitrosamine (DEN). The relative fold changes of liver weight, tumor growth, and the number and the size of hepatomas measured at 9 months after DEN administration were significantly higher in SFN-treated mice than that in vehicle-treated mice. Moreover, the expression of NRF2, its target protein NAD(P)H:quinone oxidoreductase 1 (NQO1), and the cell proliferation marker proliferating cell nuclear antigen (PCNA) and the DNA damage marker gamma-histone 2A family member X (γH2AX) was markedly elevated in SFN-treated mice compared with that in vehicle-treated mice as assessed by the Western blot assay. Immunohistochemistry and Immunofluorescence staining data further verified higher expression of NRF2 and PCNA in SFN-treated mice. These results suggest that once HCC is initiated, SFN stimulates the progression of DEN-induced HCC via activation of NRF2, rather than exerting a cancer chemopreventive effect. RNA sequencing analysis with liver tumors from DEN-treated mice revealed the B-raf V637E mutation (4/7). We speculate that mutation of B-raf proto-oncogene may contribute, at least in part, to activation of oncogenic NRF2 in DEN-induced murine carcinogenesis. The effects of SFN on DEN-induced mutation spectrum is under investigation. This study was supported by the Global Core Research Center (GCRC) Grant (No. 2011-003-0001) from the National Research Foundation (NRF) of Republic of Korea. Citation Format: Jie Zheng, Do-Hee Kim, Xi Zhu Fang, Seong Hoon Kim, Soma Saeidi, Su-Jung Kim, Young-Joon Surh. Tumor promoting effects of sulforaphane on diethylnitrosamine-induced murine hepatocarcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3828.

CD133 Expression in Medullary Thyroid Cancer Cells Identifies Patients with Poor Prognosis.

The identification of markers able to determine medullary thyroid cancer (MTC) patients at high-risk of disease progression is critical to improve their clinical management and outcome. Previous studies have suggested that expression of the stem cell marker CD133 is associated with MTC aggressiveness. To evaluate CD133 impact on disease progression in MTC and explore the regulatory mechanisms leading to the upregulation of this protein in aggressive tumors. We compiled a series of 74 MTCs with associated clinical data and characterized them for mutations in RET and RAS proto-oncogenes, presumed to be related with disease clinical behavior. We found that CD133 immunohistochemical expression was associated with adverse clinicopathological features and predicted a reduction in time to disease progression even when only RET-mutated cases were considered in the analysis (log-rank test P < 0.003). Univariate analysis for progression-free survival revealed CD133 expression and presence of tumor emboli in peritumoral blood vessels as the most significant prognostic covariates among others such as age, gender, and prognostic stage. Multivariate analysis identified both variables as independent factors of poor prognosis (hazard ratio = 16.6 and 2; P = 0.001 and 0.010, respectively). Finally, we defined hsa-miR-30a-5p, a miRNA downregulated in aggressive MTCs, as a CD133 expression regulator. Ectopic expression of hsa-miR-30a-5p in MZ-CRC-1 (RETM918T) cells significantly reduced CD133 mRNA expression. Our results suggest that CD133 expression may be a useful tool to identify MTC patients with poor prognosis, who may benefit from a more extensive primary surgical management and follow-up.

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials.

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

RET gene mutation analysis and long-term clinical outcomes of medullary thyroid cancer patients.

Medullary thyroid carcinoma is a rare, potentially aggressive tumour, with relatively worse prognosis than well-differentiated thyroid cancer. We evaluated the long-term outcomes and prognosis of medullary thyroid carcinoma patients at a single institution in India and compared outcomes based on results of RET protooncogene mutation analysis. Data were retrieved through a prospectively maintained thyroid cancer database from 1998 to June 2019, and medullary thyroid carcinoma patients were recruited. RET gene mutation status (exon 10-16) was assessed. Patient with a minimum follow-up of 12 months was eligible to be part of the long-term outcome analysis. Out of 149 peripheral blood samples, 42 were positive for RET gene mutation (prevalence of 28.1%). The median follow-up duration was 48 months, ranging from 12 to 240 months. Long-term clinical outcomes of 113 patients were assessed. Two deaths were noted in this series. Both 5- and 10-year survival was cent per cent. Overall survival was 98.2% (97.3% in RET positive and 98.7% in RET negative group). Progression-free survival was 55.4% in total (60% in RET positive and 53.3% in RET negative group). No statistically significant difference was found between RET positive and RET negative groups concerning overall survival (P = 0.6011) and progression-free survival (P = 0.5140). Univariate analysis revealed high calcitonin (>10 pg/mL), stage IV disease, and presence of lymph nodal metastasis to be significant predictors of disease recurrence, however, multivariate analysis demonstrated the presence of lymph node metastases as the only significant predictor of recurrence (P = 0.0005). Medullary thyroid carcinoma patients had relatively favourable long-term outcomes. Long-term survival was similar irrespective of RET mutation status. Presence of lymph node metastases appeared to be the strongest predictor of overall and progression-free survival, followed by Calcitonin level and stage of the disease.

HEREDITARY ENDOCRINE TUMOURS: CURRENT STATE-OF-THE-ART AND RESEARCH OPPORTUNITIES: Early thyroidectomy in multiple endocrine neoplasia: a four decade experience.

Forty years ago, physicians caring for the J-kindred, a 100+ member family with multiple endocrine neoplasia type 2A (MEN2A), hypothesized that early thyroidectomy based on measurement of the biomarker calcitonin could cure patients at risk for development of medullary thyroid carcinoma (MTC). We re-evaluated 22 family members with proven RET proto-oncogene mutations (C634G) who underwent thyroidectomy and central lymphadenectomy between 1972 and 1994 based on stimulated calcitonin abnormalities. Current disease status was evaluated by serum calcitonin measurement and neck ultrasound in 18 of the 22 prospectively screened patients. The median age of the cohort at thyroidectomy was 16.5 years (range 9-24). The median duration of follow-up at the time of examination was 40 years (range 21-43) with a median current age of 52 years (range 34-65). Fifteen of the 18 patients had no detectable serum calcitonin (<2 pg/mL). Three had detectable serum calcitonin measurements, inappropriately elevated following total thyroidectomy. None of the 16 patients imaged had an abnormal ultrasound. Survival analysis shows no MTC-related deaths in the prospectively screened patients, whereas there were many in prior generations. Early thyroidectomy based on biomarker testing has rendered 15 of 18 MEN2A patients (83%) calcitonin-free with a median follow-up period of 40 years. There have been no deaths in the prospectively screened and thyroidectomized group. We conclude that early thyroidectomy and central lymph node dissection is an effective prophylactic treatment for hereditary MTC.