Tumor heterogeneity revealed by KRAS, BRAF, and PIK3CA pyrosequencing: KRAS and PIK3CA intratumor mutation profile differences and their therapeutic implications.

Current clinical problems in colorectal cancer (CRC) diagnostics and therapeutics include the disease complexity, tumor heterogeneity, and resistance to targeted therapeutics. In the present study, we examined 171 CRC adenocarcinomas from Greek patients undergoing surgery for CRC to determine the frequency of KRAS, BRAF, and PIK3CA point mutations from different areas of tumors in heterogeneous specimens. Ninety two out of 171 (53.8%) patients were found to bear a KRAS mutation in codons 12/13. Of the 126 mutations found, 57.9% (73/126) were c.38G>A mutations (p.G13D) and 22.2% (28/126) were c.35G>T (p.G12V). Remarkably, RAS mutations in both codons 12 and 13 were recorded in the same tumor by pyrosequencing. Moreover, differences in KRAS mutations between tumor center and periphery revealed tumor heterogeneity in 50.7% of the specimens. BRAF c.1799T>A (V600E) mutations were moderately detected in 4/171 (2.3%) specimens, whereas most PIK3CA mutations were revealed by pyrosequencing 6/171 (3.5%). Remarkable tumor heterogeneity is revealed, where double mutations of KRAS in the same tumor and different KRAS mutation status between tumor core and margin are detected with high frequency. It is expected that these findings will have a major impact in cancer diagnosis and personalized therapies.

Multiple pituitary hormone abnormalities, fever, behavioral problems, seizures and apnoic spells in a 6-year old girl.

UNLABELLED: A 6-year old girl was examined having two years previously presented a transient Cushing's syndrome, followed by recurrent hyponatremia, attributed to inappropriate ADH secretion (SIADH). The brain MRI showed no abnormalities on repeated examinations, except for a suggestion of empty sella syndrome. During the past two years she also presented recurrent episodes of a prolonged febrile illness of unknown origin. All investigations related to infectious, autoimmune neoplastic diseases, histiocytosis-X or neurosarcoidosis were negative and the fever was characterized as central. The patient also presented episodes of tonic-clonic seizures, myoclonias and behavioral problems (alternating states of irritability, sleepiness and apathy, optic and hearing illusions and phobias) with or without hyponatremia. Her cerebrospinal fluid (CSF) examination was not indicative of encephalitis and the encephalogram (EEG) showed only slowing of background activity. At the age of 4.75 years she, started to have recurrent episodes of hypopnoea/apnoea with severe desaturation and hypercapnia, occasionally requiring intubation and ventilation. She also developed unilateral miosis corneal ulceration and bilateral ptosis (oculo-sympathetic paresis). Repeat brain MRI and CT scans of the mediastinum excluded organic causes of apnoeas and of oculo-sympathetic paresis, such as neuroblastoma or lymphoma. Furthermore, on a 24 hour electrocardiogram recording, using power spectral analysis, significantly reduced heart rate variability was observed, by comparison with age-specific normal ranges. Thus the apnoeas, ptosis, miosis and temperature instability were attributed to autonomic dysfunction. During the same period, the patient presented significant growth retardation and growth hormone (GH) deficiency was confirmed during two provocative tests (peak GH levels: 3.1 and 2.9 ng/ml (normal>10). Moreover, thyrotropin (TSH) deficiency and persistent hyperprolactinemia were detected. She was started on hGH and thyroxine. She was also put on fluid restriction and increased sodium intake for her SIADH. She was advised to use O2 administration by mask in case of apnoeas. The child died at age 6 6/12 years as a result of apnoea during sleep. IN CONCLUSION: Multiple pituitary hormonal abnormalities, together with symptoms of autonomic neuropathy (apnoeas, ptosis, miosis, tachycardia, temperature instability) and encephalopathy (seizures, myoclonias and behavioral problems) developed in a 4-year old girl. The suggested diagnoses were: 1. Neurometabolic disorder, 2. Mitochondrial disorder, 3. Post infectious autoimmune process.