Marija Balic

Critical Evaluation of Real-Time Reverse Transcriptase-Polymerase Chain Reaction for the Quantitative Detection of Cytokeratin 20 mRNA in Colorectal Cancer Patients

We evaluated the usefulness of cytokeratin 20 (CK20) mRNA expression in the quantitative detection of circulating tumor cells in the blood of patients with colorectal cancer (CRC). Blood samples from healthy volunteers (HVs; n = 37), patients with localized (n = 42) and metastatic colorectal cancer (n = 40), and patients with chronic inflammatory bowel disease (CID; n = 15) were examined. After immunomagnetic enrichment using microbeads against human epithelial antigen, total RNA was extracted, reverse transcribed, and analyzed by real-time reverse transcriptase-polymerase chain reaction using the LightCycler instrument. CK20 expression in peripheral blood was found in 46 of 82 (56%) patients with CRC, 8 of 37 (22%) HVs, and 9 of 15 (60%) patients with CID. Levels of CK20 mRNA were significantly higher in blood samples from CRC patients (median 681) than in blood samples from HVs (median 0) (P = 0.001), whereas no difference could be detected between patients with CRC and CID. Although the present technique could not distinguish CRC from CID, the method warrants further efforts to improve sample preparation and tumor cell enrichment, which may render real-time CK20 reverse transcriptase-polymerase chain reaction a feasible technique in identifying circulating tumor cells in peripheral blood of cancer patients.

Pathological Complete Response to Neoadjuvant Trastuzumab Is Dependent on HER2/CEP17 Ratio in HER2-Amplified Early Breast Cancer.

Purpose: To evaluate whether pathologic complete response (pCR) to neoadjuvant trastuzumab is dependent on the level of HER2 amplification. Experimental Design: 114 HER2-overexpressing early breast cancer patients who had received neoadjuvant trastuzumab were included in this study. Absolute HER2 and chromosome 17 centromere (CEP17) were measured by in situ hybridization analysis, and associations were examined between HER2/CEP17 ratio and tumor pCR status (commonly defined by ypT0 ypN0, ypT0/is ypN0, and ypT0/is). Results: In trastuzumab-treated patients, ypT0 ypN0 was achieved in 69.0% of patients with high-level amplification (HER2/CEP17 ratio > 6), but only in 30.4% of tumors with low-level amplification (ratio ≤ 6; P = 0.001). When pCR was defined by ypT0/is ypN0 or ypTis, 75.9% and 82.8% of tumors with high-level amplification had a complete response, whereas only 39.1%, and 38.3% with low-level amplification achieved pCR (P = 0.002 and P < 0.001, respectively). Logistic regression revealed that tumors with high-level amplification had a significantly higher probability achieving ypT0 ypN0 (OR, 5.08; 95% confidence interval, 1.86–13.90; P = 0.002) than tumors with low-level amplification, whereas no other clinicopathologic parameters were predictive of pCR. The association between high-level HER2 amplification and pCR was almost exclusively confined to hormone receptor (HR)–positive tumors (ypT0 ypN0: 62.5% vs. 24.0%, P = 0.014; ypT0/is ypN0: 75.0% vs. 28.0%, P = 0.005; and ypT0/is: 87.5% vs. 28.0%, P < 0.001), and was largely absent in HR-negative tumors. Conclusions: An HER2/CEP17 ratio of >6 in the pretherapeutic tumor biopsy is associated with a significantly higher pCR rate, particularly in HER2/HR copositive tumors, and can be used as a biomarker to predict response before neoadjuvant trastuzumab is initiated. Clin Cancer Res; 23(14); 3676–83. ©2017 AACR.

Clinical translation of a novel microfilter technology Capture, characterization and culture of circulating tumor cells

The most important determinant of prognosis and management of cancer is the presence or absence of metastasis [1]. The road to metastasis involves tumor cells to become detached from the primary tumor and travel in the blood to distant sites, causing secondary tumors. These tumor cells traveling in blood are termed Circulating tumor cells (CTC). Capture of CTC from whole blood has been a challenging feat. The fact that these CTC are few in number, to effectively and efficiently isolate them from whole blood can be thought of as looking for a needle in a haystack. Our microfilter technology exploits the use of size based capture of the larger CTC from the smaller white blood cells and components of whole blood. The effective capture potential of the microfilter platform has driven the area of CTC analysis into a new age of research in the field of cancer. The ability to finally analyze CTC at a molecular level, leads to a deeper understanding of metastatic process, while providing an opportunity to evaluate, monitor and manage treatment options as well as the adherent possibility of having an “on-chip” drug sensitivity assay for focused treatment options. We have demonstrated through clinical trials the ability to effectively identify, enumerate and characterize CTC based on immunfluorescence and FISH assays and provide a companion endpoint for monitoring and evaluating treatment management. Our work on viable CTC capture has resulted in successfully capturing and culturing CTC from blood in mouse models that have been inoculated with breast cancer cell lines to form primary and secondary metastatic cancer sites. The future potential within the microfilter technology to capture viable CTC for culture, will catapult therapeutic interventions to a new level of personalized medicine in cancer management.

Neue Diagnostik für das Lungenkarzinom

ZusammenfassungDie molekulare Diagnostik des Lungenkarzinoms hat in den letzten Jahren eine rasante Entwicklung erfahren. Neue molekulare Targets, Resistenzmutationen für bestehende Therapien und nicht zuletzt die Immuntherapie rücken die molekulare Diagnostik immer mehr in den Mittelpunkt. Die Entdeckung der zirkulierenden Tumorzellen und der freien zirkulierenden Tumor-Desoxyribonukleinsäure (ctDNA) hat besonders für das Lungenkarzinom aufgrund der eingeschränkten Verfügbarkeit von Gewebeproben hohe Bedeutung. Inzwischen ist der Nachweis von Tumormutationen aus dem Blut gut etabliert und kann zur Beurteilung der Tumorlast und auch für die Detektion von Resistenzmutationen verwendet werden. Trotzdem zeigt sich, dass die Analyse von ctDNA technisch aufwendig und komplex ist und dass nicht in allen Tumorstadien und Tumorentitäten ctDNA im Patienten nachweisbar ist. Die neuesten Entwicklungen in der Diagnostik des Lungenkarzinoms und die Bedeutung der „liquid biopsy“ in diesem Kontext sind Thema dieses Artikels.AbstractDuring the past few years, molecular methods in the diagnosis of lung cancer have undergonexa0rapid development. New molecular targets, resistance mutations in existing therapies, and, not least, immunotherapy, have placed molecular diagnosis center stage. Particularly in the case of lung cancer, because of the limited access to tissue samples, the discovery of circulating tumor cells and circulating tumor DNA (ctDNA) is of great importance. Meanwhile, evidence for tumor mutations from the blood is well-established and can be utilized for evaluation of the tumor burden and for the detection of resistance mutations. Nevertheless, analysis of ctDNA is technically elaborate and complex and it is not detectable in patients at all tumor stages and in all tumor entities. The most recent developments in the diagnosis of lung cancer and the significance of “liquid biopsy” in this context are discussed in this article.