Plasma DNA microsatellite panel as sensitive and tumor‐specific marker in lung cancer patients

Abstract

The majority of lung cancer patients have tumor-derived genetic alterations in circulating plasma DNA that could be exploited as a diagnostic tool. We used fluorescent microsatellite analysis to detect alterations in plasma and tumor DNA in 34 patients who underwent bronchoscopy for lung cancer, including 11 small cell lung cancer (SCLC) and 23 nonsmall cell lung cancer (NSCLC) (12 adenocarcinomas, 11 squamous cell carcinomas) and 20 controls. Allelotyping was performed with a selected panel of 12 microsatellites from 9 chromosomal regions 3p21, 3p24, 5q, 9p, 9q, 13q, 17p, 17q and 20q. Plasma DNA allelic imbalance (AI) was found in 88% (30 of 34 Key words: plasma DNA alterations; lung cancer; microsatellite analysis; histologic type; prognosisLung cancer is one of the most common tumors in the world. The World Health Organization's (WHO) pathologic description of malignant tumors classifies lung cancer into 4 main groups: 1 small cell lung cancer (SCLC), squamous cell carcinoma (SQC), adenocarcinoma (ADC) and large cell carcinoma (LCC). For prognostic and therapeutic purposes, SQC, ADC and LCC are pooled together in the nonsmall cell lung cancer (NSCLC) group. NSCLC and SCLC represent about 80% and 20% of all primary lung cancers, respectively. 2 Surgery offers the best probability of cure for patients with NSCLC, but surgery cannot be proposed for locally advanced or metastatic stages. In spite of aggressive therapy, however, prognosis of lung cancer patients is generally very poor. Survival at 5 years is 35% for NSCLC operated patients and Ͻ 5% for inoperable NSCLC. Survival is Ͻ 20% at 2 years for limited SCLC and Ͻ 2% for extensive disease. 3-5 Thus, the development of novel diagnostic techniques to identify lung cancer may facilitate earlier diagnosis of primary or recurring cancers and lead to more effective treatments and improved prognosis.As in other cancers, accumulation of genetic alterations is common in lung cancer and can include gene mutations, allelic losses, allelic instabilities and aberrant gene methylation that target oncogenes or tumor suppressor genes (TSG). 6,7 Among microsatellite (MS) alterations, primary lung cancers seldom show microsatellite instability (MSI) but are more often characterized by frequent loss of heterozygosity (LOH). 8 Remarkably, the frequency of MS alterations reported in lung cancers varies substantially and obviously depends on the markers analyzed. For lung cancer, frequent LOH has been reported in regions of chromosomes 3, 5, 9, 13 and 17, with the highest frequency occurring at 3p. 9 -14 Nevertheless, comparison of allelotypes have identified some consistent differences in LOH frequencies such as 50 -80% and 80 -100% at 17p13, 20 -30% and 80 -90% at 13q, 60% and 0 -10% at 9p21, 50% and 90% at 3p in NSCLC and in SCLC, respectively. 14 -17 It has long been known that the concentration of free-circulating DNA in plasma is higher in tumor patients than in healthy people. 18 Microsatellite analysis, mutation analysis in genomic or mitochondrial DNA and gene promot...

Quantitation of circulating DNA in the serum of breast cancer patients by real-time PCR

“…Whether the elevated concentrations of circulating DNA in cancer patients have the same origin or derive from another source of cells, for example the tumour itself, need further investigation. Qualitative studies have shown that genetic alternations such as mutations and LOH were detected in circulating DNA, as well as in the matching tumour cells, suggesting that part of the extra circulating DNA in cancer patients is attributed to DNA released from tumour cells (Gonzalez et al, 2000;Shaw et al, 2000;Beau-Faller et al, 2003). However, our results indicate that the mechanisms of DNA released from tumours are not related to any of the known commonly used major prognostic factors and therefore might highlight different pathways, such as apoptosis, necrosis, hypoxia, that would need to be investigated.…”

Genetic abnormalities in plasma DNA of patients with lung cancer and other respiratory diseases

“…This finding contradicts previous studies and challenges the utility of this test as a diagnostic tool for lung cancer. Some studies have used healthy blood donor controls, 19 and others have used hospital controls, 15 but most have found no genetic alterations in the plasma DNA of patients without cancer. Our controls are unusual in representing the respiratory clinic population from which the lung cancer patients were derived.…”

“…The demonstration that tumour DNA can be detected in the plasma of cancer patients has led to interest in the study of genetic changes in plasma DNA as diagnostic and prognostic markers for several tumour types, including breast, pancreas, colorectal and lung cancers. 5,15 In preliminary studies of plasma DNA in lung cancer, chromosomal deletions, microsatellite alterations and abnormal promoter methylation of tumour suppressor genes have been examined. 16 -18 This has led to the hope that lung cancer might be detected or its prognosis defined using a simple blood test.…”

Generation of blood circulating DNA: the sources, peculiarities of circulation and structure

“…На сегодняшний день много работ посвящено изучению микросателлитных перестроек в цирДНК крови [107][108][109][110][111][112][113]. Микросателлиты -короткие повторяющиеся и высокополиморфные последовательности, состоящие из многократно повторяющегося мотива, например, ди-, три-, тетрануклеотида: (NN-N)n. Микросателлиты более или менее случайно распределены в геноме [114].…”

“…Биологическая функция микросателлитов неясна, однако они часто используются в качестве генетических маркеров для анализа расположения генов-супрессоров опухолей и т.д. Опухолеассоциированные нарушения микросателлитной ДНК были обнаружены в цирДНК плазмы крови при раке лёгких [107], головы и шеи [108], почки [109], мочевого пузыря [110], предстательной железы [111,112] и гепатоцеллюлярной карциноме [113].…”