Jeannine Lacroix

Sensitive detection of rare cancer cells in sputum and peripheral blood samples of patients with lung cancer by preproGRP-specific RT-PCR

RT‐PCR‐based amplification of transcripts expressed in cancer but not in normal non‐neoplastic cells is increasingly used for the sensitive detection of rare disseminated or exfoliated cancer cells to improve cancer staging and early detection protocols. However, these assays are frequently hampered by false‐positive test results due to low‐level transcription of the marker genes in normal cells. To overcome these limitations, target transcripts have to be identified that are tightly suppressed in normal non‐neoplastic tissues, whereas they should be actively transcribed in the respective cancer cells. Here, we tested RT‐PCR assays for 7 neuroendocrine marker transcripts including NCAM, PGP 9.5, gastrin, gastrin receptor, synaptophysin, preprogastrin‐releasing peptide (preproGRP) and GRP‐receptor to detect rare exfoliated tumor cells in peripheral venous blood and sputum samples from patients with lung cancer. Among these preproGRP RT‐PCR was the only assay with which illegitimate transcription in blood or sputum samples from healthy donors or patients with unrelated diseases did not interfere. However, it reproducibly detected up to 10 small‐cell lung cancer cells diluted in either 10 ml blood or 5 ml sputum samples. Single blood and sputum samples were collected directly before diagnostic bronchoscopy from 175 patients suspected to have lung cancer. Twenty‐six of these had small‐cell lung cancer (SCLC). Thereof, 13 patients (50%) tested positive in the blood sample and 5 of 23 patients (22%) tested positive in the sputum sample. Moreover, among 92 patients with non‐small‐cell lung cancer (NSCLC) 25 patients (27%) had disseminated cancer cells in peripheral blood. Amplification of preproGRP transcripts from clinical samples is a sensitive and specific assay to detect disseminated or exfoliated lung cancer cells either in peripheral blood or sputum samples.

Molecular Pathways: Rodent Parvoviruses—Mechanisms of Oncolysis and Prospects for Clinical Cancer Treatment

Rodent parvoviruses (PV) are recognized for their intrinsic oncotropism and oncolytic activity, which contribute to their natural oncosuppressive effects. Although PV uptake occurs in most host cells, some of the subsequent steps leading to expression and amplification of the viral genome and production of progeny particles are upregulated in malignantly transformed cells. By usurping cellular processes such as DNA replication, DNA damage response, and gene expression, and/or by interfering with cellular signaling cascades involved in cytoskeleton dynamics, vesicular integrity, cell survival, and death, PVs can induce cytostasis and cytotoxicity. Although productive PV infections normally culminate in cytolysis, virus spread to neighboring cells and secondary rounds of infection, even abortive infection or the sole expression of the PV nonstructural protein NS1, is sufficient to cause significant tumor cell death, either directly or indirectly (through activation of host immune responses). This review highlights the molecular pathways involved in tumor cell targeting by PVs and in PV-induced cell death. It concludes with a discussion of the relevance of these pathways to the application of PVs in cancer therapy, linking basic knowledge of PV–host cell interactions to preclinical assessment of PV oncosuppression. Clin Cancer Res; 18(13); 3516–23. ©2012 AACR.

Reduced expression of the neuron restrictive silencer factor permits transcription of glycine receptor α1 subunit in small-cell lung cancer cells

Small-cell lung cancer (SCLC) cells express various markers of neuronal differentiation associated with deficient activity of the neuron-restricted silencer factor (NRSF). Here, we characterize mechanisms by which NRSF target genes are upregulated in SCLC and their functional consequences for cell survival. Since the glycine receptor (GlyR) α1 subunit gene, GLRA1, contains a sequence motif for NRSF binding (NRSE) within its 5′ UTR, it served as a cellular surrogate marker for NRSF activity. Expression of GLRA1 in nontransformed cells is largely restricted to cells in the spinal cord, retina and brain stem. In experiments described here, we detected GLRA1 transcripts in three out of four SCLC-derived cell lines and in three of five biopsy samples obtained from SCLCs. In contrast, no GLRA1 transcripts were found in 10 nonmalignant nor 15 non-small-cell lung cancer biopsies. Consistent with this observation, NRSF-mediated suppression of an expression construct harbouring the NRSE of the GLRA1 (GLRA1 NRSE) gene was impaired in three of four ‘classic’ SCLC cell lines, whereas exogenous overexpression of NRSF in NRSF-deficient SCLC cell lines reconstituted silencing of the reporter plasmid. The level of NRSF transcripts as well as the level of specifically bound NRSF to the NRSE correlated with the level of GLRA1 transcripts in SCLC cell lines. Splice variants encoding truncated NRSF proteins and expressed in some SCLC did not antagonize the repression of NRSE-containing genes. Most interestingly, reconstitution of NRSF expression induced apoptosis in SCLC cells, suggesting that inhibition of NRSF activity is a crucial step in the carcinogenesis of a subgroup of SCLC.

DETECTION OF HEMATOGENOUS MICROMETASTASIS IN PATIENTS WITH TRANSITIONAL CELL CARCINOMA

PURPOSE Cytokeratin 20 (CK 20) is selectively expressed in urothelium, gastric intestinal epithelium, in Merkel cells and in a variety of malignant neoplasms. CK 20 RT-PCR assay has been extensively used to detect isolated cancer cells in peripheral blood, lymph nodes and bone marrow samples of patients with colorectal carcinoma. Since CK-20 is also actively expressed in transitional cell carcinoma (TCC), we analyzed, whether CK 20 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is suitable to detect residual tumor cells in patients with transitional cell carcinoma of the bladder and the upper urinary tract. MATERIALS AND METHODS Nested Reverse Transcriptase Polymerase Chain Reaction assay was used to analyze CK 20 transcripts in peripheral venous blood samples and tumor biopsies of 49 patients with transitional cell carcinoma. Blood samples of 22 healthy volunteers served as negative controls. RESULTS CK 20 mRNA was detectable in blood samples of 12 of 49 patients with TCC. All blood samples of the control group tested negative. The detection rate for CK 20 mRNA significantly correlated (p = 0.0019, Cochran-Armitage Trend Test) to the stage of disease and increased from 0% in stage pTa to 63% in stage pT4. CONCLUSIONS These results suggest that CK 20 is a suitable marker for the detection of disseminated TCC cells in peripheral venous blood samples and may be helpful in the molecular staging of TCC patients. The prognostic relevance has to be evaluated in further followup.

Parvovirus H1 selectively induces cytotoxic effects on human neuroblastoma cells

Despite multimodal therapeutic concepts, advanced localized and high‐risk neuroblastoma remains a therapeutic challenge with a long‐term survival rate below 50%. Consequently, new modalities for the treatment of neuroblastoma, e.g., oncolytic virotherapy are urgently required. H‐1PV is a rodent parvovirus devoid of relevant pathogenic effects in infected adult animals. In contrast, the virus has oncolytic properties and is particularly cytotoxic for transformed or tumor‐derived cells of various species including cells of human origin. Here, a preclinical in vitro assessment of the application of oncolytic H‐1PV for the treatment of neuroblastoma cells was performed. Infection efficiency, viral replication and lytic activity of H‐1PV were analyzed in 11 neuroblastoma cell lines with different MYCN status. Oncoselectivity of the virus was confirmed by the infection of short term cultures of nonmalignant infant cells of different origin. In these nontransformed cells, no effect of H‐1PV on viability or morphology of the cells was observed. In contrast, a lytic infection was induced in all neuroblastoma cell lines examined at MOIs between 0.001 and 10 pfu/cell. H‐1PV actively replicated with virus titres increasing up to 5,000‐fold within 48–96 hr after infection. The lytic effect of H‐1PV was observed independent of MYCN oncogene amplification or differentiation status. Moreover, a significant G2‐arrest and induction of apoptosis could be demonstrated. Infection efficiency, rapid virus replication and exhaustive lytic effects on neuroblastoma cells together with the low toxicity of H‐1PV for nontransformed cells, render this parvovirus a promising candidate for oncolytic virotherapy of neuroblastoma.

Nucleic Acid Based Techniques for the Detection of Rare Cancer Cells in Clinical Samples

Solid tumors evolve from cells which have lost control functions safeguarding their genomic integrity by mutation within specific genes. Consequently, proliferating cells within a tumor differ slightly from generation to generation. This permits the continuous production of new and subsequent selection of the best adapted cells, which retain this capacity of self-evolution. The threat of neoplastic diseases is due to the clinical experience, that surgical resection of all cells with this potential for autonomous evolution is often not achievable, since they were spread to distant non-resectable sites in the organism before diagnosis and surgical removal of the primary tumor and might later grow out as metastatic lesion. Lack of diagnostic techniques to detect small preneoplastic lesions as well as single spread cancer cells often causes delayed diagnosis when curative therapy is no longer achievable. Recent advances in characterizing the molecular basis of genomic instability, identifying specific gatekeeper mutations and their functional consequences in neoplastic cells now permit the development of new highly sensitive tests to identify preneoplastic lesions as well as spread single cancer cells. These techniques carry a tremendous potential for simple cost effective cancer early detection and screening assays as well as for diagnostic applications to identify spread cancer cells. Thus, they most likely will soon guide indication for surgical and adjuvant therapy protocols for patients with preneoplastic or neoplastic lesions. However, due to the complexity of the assays and the great variety of technical aspects involved almost all diagnostic applications are not yet standardized. This poses significant problems for quality control as well as inter-laboratory comparability of the results and underlines the urgent demand for well controlled collaborative efforts to evaluate indications and diagnostic standards for these assays. Here, the theoretical background, basic principles and some diagnostic applications of these new tests are reviewed.

Expression of cytokeratin 20 in thyroid carcinomas and peripheral blood detected by reverse transcription polymerase chain reaction

We investigated a nested reverse transcriptase polymerase chain reaction (RT-PCR) system to detect CK20 mRNA in thyroid carcinomas, benign thyroid diseases and peripheral blood to improve diagnosis of thyroid carcinoma and to detect disseminated tumour cells. Frozen tissue samples of 46 thyroid carcinomas and 30 benign thyroid diseases (14 multinodular goiters, 14 follicular adenomas, two Hashimoto’s hyroiditis) were obtained intraoperatively. Preoperative blood samples were drawn from 31 patients with thyroid cancer, nine patients with benign thyroid disorders and 20 healthy volunteers. Nine out of nine medullary, 9/12 follicular, 7/19 papillary and 2/6 anaplastic carcinomas expressed CK20 transcripts. CK20 mRNA was undetectable in 30 tissue sections of benign thyroid diseases. Circulating tumour cells were found in the blood of 3/8 patients with medullary carcinoma, 2/8 patients with follicular carcinoma, 2/11 patients with papillary carcinoma and 1/4 patients with an anaplastic carcinoma. Nine blood samples of patients with benign thyroid diseases and 20 healthy volunteers tested negative. For the first time CK20 mRNA could be detected in tissue sections of thyroid carcinomas and peripheral blood samples of patients with thyroid cancer. It was not detectable in benign thyroid diseases. Our results therefore strongly suggest that CK20 RT-PCR assays may improve the diagnosis of thyroid carcinoma and is able to detect circulating tumour cells in peripheral blood of thyroid carcinoma patients.

Detection of disseminated medullary thyroid carcinoma cells in cervical lymph nodes by cytokeratin 20 reverse transcription-polymerase chain reaction

Local recurrence in differentiated and medullary thyroid carcinoma develops frequently from metastatic infiltration of cervical lymph nodes. Despite an aggressive surgical approach, postoperative calcitonin levels as biochemical evidence for residual cancer cells remain often elevated in patients with medullary thyroid carcinoma. In the present study, we compared the detection rates of disseminated medullary thyroid carcinoma cells in cervical lymph nodes by histopathology with reverse transcription-polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) transcripts as a more sensitive but still specific molecular parameter for residual thyroid cancer cells. Forty-two cervical lymph nodes obtained from 7 patients with CK20positive medullary thyroid carcinomas were cut into two halves, one used for conventional histology, the other subjected to RNA extraction and subsequent amplification of cytokeratin 20 transcripts. Matching results for CK20 RT-PCR and histopathology were found in 74% (31/42)of the examined lymph nodes (52% positive results, 48% negative results). Positive CK20 RT-PCR pointed to residual thyroid carcinoma cells in another 19% (8/42), in which no thyroid carcinoma cells were identified by histopathology. Histology and immunohistochemistry,however, identified tumor cells in 7% (3/42) of the analyzed lymph nodes, from which no CK20 transcript could be amplified (false-negative results). These data suggest that CK20 RT-PCR might be more sensitive to detect nodal involvement of CK20 positive medullary thyroid carcinomas than conventional histopathology. In combination with histology, it might help to identify patients with residual disease after surgery.

Molecular Tools in the Detection of Micrometastatic Cancer Cells — Technical Aspects and Clinical Relevance

The frequent failure to reduce the mortality due to epithelial cancers by common medical intervention results primarily from early dissemination of cancer cells, which is missed by conventional diagnostic procedures used for tumor staging. Individual carcinoma cells present in regional lymph nodes, blood or distant organs (e.g., bone marrow) can be detected by sensitive immunologic or molecular methods. Here, we review recently developed molecular assays for the detection of individual micrometastatic cancer cells and their clinical application in patients with epithelial tumors.

Oncolytic effects of parvovirus H-1 in medulloblastoma are associated with repression of master regulators of early neurogenesis

Based on extensive pre‐clinical studies, the oncolytic parvovirus H‐1 (H‐1PV) is currently applied to patients with recurrent glioblastoma in a phase I/IIa clinical trial (ParvOryx01, NCT01301430). Cure rates of about 40% in pediatric high‐risk medulloblastoma (MB) patients also indicate the need of new therapeutic approaches. In order to prepare a future application of oncolytic parvovirotherapy to MB, the present study preclinically evaluates the cytotoxic efficacy of H‐1PV on MB cells in vitro and characterizes cellular target genes involved in this effect. Six MB cell lines were analyzed by whole genome oligonucleotide microarrays after treatment and the results were matched to known molecular and cytogenetic risk factors. In contrast to non‐transformed infant astrocytes and neurons, in five out of six MB cell lines lytic H‐1PV infection and efficient viral replication could be demonstrated. The cytotoxic effects induced by H‐1PV were observed at LD50s below 0.05 p. f. u. per cell indicating high susceptibility. Gene expression patterns in the responsive MB cell lines allowed the identification of candidate target genes mediating the cytotoxic effects of H‐1PV. H‐1PV induced down‐regulation of key regulators of early neurogenesis shown to confer poor prognosis in MB such as ZIC1, FOXG1B, MYC, and NFIA. In MB cell lines with genomic amplification of MYC, expression of MYC was the single gene most significantly repressed after H‐1PV infection. H‐1PV virotherapy may be a promising treatment approach for MB since it targets genes of functional relevance and induces cell death at very low titers of input virus.