Comparação entre expressão de receptores hormonais e de HER-2 entre células tumorais circulantes e metástases de câncer de mama / Comparison of hormonal receptor expression and HER2 status in circulating tumor cells and breast cancer metastases

Introdução: O câncer de mama é a neoplasia mais comum em mulheres. A maioria deles é diagnosticada em estágios iniciais, quando o tratamento visa a cura. Mas apesar dos avanços no tratamento, metástases à distância podem ocorrer. A biópsia das lesões metastáticas é recomendada para confirmar o status do receptor de estrogênio (RE), receptor de progesterona (RP) e receptor do fator de crescimento epidérmico humano 2 (HER2), por ocorrerem discrepâncias nesses padrões entre tumores primários e metástases em até 40% dos casos. As células tumorais circulantes (CTCs) estão relacionadas às evoluções clínicas do câncer de mama e podem potencialmente desempenhar um papel substituto aos procedimentos invasivos de rebiópsia de metástase. A tecnologia ISET® (Isolation by SizE of Tumor Cells, Rarecells-Diagnostics, Paris, França) não é usualmente empregada para detectar CTCs em pacientes com câncer de mama, embora seja reconhecida como uma ferramenta útil em alguns outros tumores. Existem dados emergentes de que a caracterização da expressão proteica das CTC pode refinar seu valor prognóstico. Sabe-se que o fator de transformação de crescimento (TGF-ß) desempenha um papel na progressão e invasividade do câncer de mama. Objetivos: Comparar a expressão de RE, RP e HER2 em tumores primários, CTCs, metástases e avaliar a expressão do receptor TGF-ß tipo 1 (TGF-ß RI) em CTCs como fator prognóstico para sobrevida global. Metodologia: Estudo realizado no A.C.Camargo Cancer Center, Brasil. As amostras de sangue foram coletadas antes da biópsia guiada por tomografia computadorizada de lesões metastáticas suspeitas e processadas pela metodologia ISET®. Os níveis de expressão proteica das CTCs foram comparados aos de tumores primários e metástases e correlacionados aos resultados clínicos. Todos os dados clínicopatológicos foram obtidos dos prontuários médicos. Resultados: Dos 39 pacientes inicialmente incluídos, 27 tiveram tanto a biópsia de metástases quanto a coleta de sangue e foram considerados para análise. As taxas de concordância para a expressão de RE, RP e HER2 entre tumores primários e metástases foram altas. Não foi observada nenhuma perda de expressão de HER2 nas metástases e os tumores triplo negativos mantiveram o mesmo padrão em todas as metástases (p <0,0001). Quando as metástases e CTCs foram classificadas como triplo negativo (TN) ou não ­ TN, as CTCs determinaram alta especificidade (93%), acurácia (84,2%) e valor preditivo negativo (88%). A sobrevida global mediana de pacientes sem expressão de TGF-ß RI em CTCs foi de 42,6 x 20,8 meses para os positivos, clinicamente relevante, porém sem significância estatística (p> 0,05). Conclusões: No câncer de mama, o papel das CTCs detectadas pelo ISET® ainda não está estabelecido. Com este estudo, sugerimos que esta metodologia possa ser útil para avaliar metástases em casos de tumores não TN, assim como a expressão de TGF-ß RI em CTCs, o que pode impactar a sobrevida. Devido à limitação da amostra, estudos futuros devem se concentrar em subtipos específicos de câncer de mama, ampliando a coorte.

Introduction: Breast cancer (BC) is the most common neoplasm in women. Most of BC are diagnosed in early stages, when treatment aims cure. Despite advances in BC treatment, distant metastases may develop. Biopsy of metastatic lesions is recommended to confirm estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, due to discrepancies in these patterns between primary tumors/metastasis in up to 40% of cases. Circulating Tumor Cells (CTCs) are related to breast cancer outcomes and could potentially play a role surrogating invasive procedures of metastasis rebiopsy. ISET® (Isolation by SizE of Tumor Cells, Rarecells-Diagnostics, Paris, France) technology is not currently employed to detect CTCs in breast cancer patients, although recognized as a useful tool in some other tumors. There are emerging data that characterization of CTC protein expression can refine its prognostic value. Transforming growth factor (TGF)-ß play a role in progression/invasiveness of BC. Objectives: To compare ER, PR and HER2 expression in primary tumors, CTCs, metastases and to evaluate TGF-ß type 1 receptor (TGF- ß RI) expression in CTCs as prognostic factor for overall survival. Methods: Study conducted at the A.C.Camargo Cancer Center, Brazil. Blood samples were processed in ISET® before computed tomography­guided biopsy of suspected metastatic lesions. Protein expression levels in CTCs were compared to those in primary tumors/metastases and correlated to clinical outcomes. All clinicopathological data were obtained from medical records. Results: From the 39 patients initially included, 27 had both biopsy of metastases and blood collection and were considered for analysis. Concordance rates for ER, PR and HER2 expression between primary tumors/metastases were high. No loss of HER2 expression at any metastasis site and retention of the same pattern in all triplenegative (TN) tumors (p <0.0001) were observed. When metastases/CTCs were classified as TN/non­TN, CTCs showed high specificity (93%), accuracy (84.2%) and negative predictive value (88%). The median overall survival of patients with no TGF-ß RI expression in CTCs was 42.6 x 20.8 months for positive ones, clinically relevant but not statistically significant (p>0.05). Conclusions: In BC, the role of CTCs detected by ISET® is not yet established. Here, we could suggest that this methodology may be useful to evaluate metastasis in non-TN cases as also TGF-ß RI expression in CTCs, which may impact survival. Due to sample limitation, future studies must focus on specific subtypes of BC, expanding the cohort.

Circulating Tumor Cells: Liquid Biopsy for Early Detection of Cancer

Cancer is a complex, heterogeneic, and dynamic disease involving multiple gene-environment interactions, and affecting numerous biological pathways. As such, the development of reliable and robust non-invasive platforms constitutes a vital step toward realizing the potential of precision medicine. Distant metastases harbor unique genomic characteristics that are not detectable in the corresponding primary tumor of the same patient, and metastases located at different sites show considerable intra-patient heterogeneity. Thus, the analysis of the resected primary tumor alone or, if possible, re-evaluation of tumor characteristics based on the biopsy of the most accessible metastasis, may not reveal sufficient information for treatment decisions. Here, we propose that this dilemma can be solved by a new diagnostic concept: liquid biopsy, that is, the analysis of therapeutic targets and drug resistance-conferring gene mutations in or on circulating tumor cells (CTCs). Finally, the analysis of the resected primary tumor alone may provide misleading information with regard to the characteristics of metastases, the key target for systemic anticancer therapy. Liquid biopsies are noninvasive tests using blood or fluids that detect CTCs or the products of tumors, such as fragments of nucleotides or proteins that are shed into biological fluids from the primary or metastatic tumors. Such biopsies are expected to be informative or easily accessible tools to provide comprehensive information regarding cancers beyond conventional biopsies. Thus, this review addresses the use of CTCs in cancer detection, diagnosis and monitoring and discusses the direction of its clinical application in cancer patient care.

Embolia pulmonar tumoral: una manifestación inicial infrecuente del cáncer / Pulmonary tumour embolism: An infrequent initial manifestation of cancer

Resumen Generalmente la embolia pulmonar puede ser resultado de una trombosis, sin embargo, existen otros tipos de embolismos pulmonares. Se describe el caso de una paciente de 44 años de edad con artritis reumatoide, uso de terapia biológica y sin diagnóstico previo de cáncer, que presentó embolismo pulmonar en el contexto de trombosis venosa en miembros inferiores sin respuesta a la anticoagulación por lo que se estudiaron otras posibles etiologías, documentando finalmente embolismo tumoral por adenocarcinoma de origen desconocido posiblemente de mama. La posibilidad de embolia pulmonar tumoral se debe considerar ante la no respuesta al tratamiento con anticoagulación.

Abstract Pulmonary embolism is usually caused by thrombosis; however there are other types of pulmonary embolisms. The case is presented of a 44 year-old patient with rheumatoid arthritis on biological therapy, with no previous diagnosis of cancer. The patient had a pulmonary embolism in the context of venous thrombosis in the lower limbs, with no response to anticoagulation treatment. It was finally documented as a tumour embolism due to adenocarcinoma of unknown origin, possibly of the breast. The possibility of a pulmonary tumour embolism should be considered in the absence of a response to anticoagulation therapy.

Role of β₁-Integrin in Colorectal Cancer: Case-Control Study / 이화의대지

OBJECTIVES: In the metastatic process, interactions between circulating tumor cells (CTCs) and the extracellular matrix or surrounding cells are required. β1-integrin may mediate these interactions. The aim of this study was to investigate whether β1-integrin is associated with the detection of CTCs in colorectal cancer. METHODS: We enrolled 30 patients with colorectal cancer (experimental group) and 30 patients with benign diseases (control group). Blood samples were obtained from each group, carcinoembryonic antigen (CEA) mRNA for CTCs marker and β1-integrin mRNA levels were estimated by using reverse transcription-polymerase chain reaction, and the results were compared between the two groups. RESULTS: CEA mRNA was detected more frequently in colorectal cancer patients than in control patients (P=0.008). CEA mRNA was significantly reduced after surgery in the colorectal cancer patients (P=0.032). β1-integrin mRNA was detected more in colorectal cancer patients than in the patients with benign diseases (P<0.001). In colorectal cancer patients, expression of β1-integrin mRNA was detected more for advanced-stage cancer than for early-stage cancer (P=0.033) and was significantly decreased after surgery (P<0.001). In addition, expression of β1-integrin mRNA was significantly associated with that of CEA mRNA in colorectal cancer patients (P=0.001). CONCLUSION: In conclusion, β1-integrin is a potential prognostic factor following surgical resection in colorectal cancer patients. β1-integrin may be a candidate for use as a marker for early detection of micrometastatic tumor cells and for monitoring the therapeutic response in colorectal cancer patients.

Circulating Aneuploid Cells Detected in the Blood of Patients with Infectious Lung Diseases

The identification of circulating tumor cells (CTCs) is clinically important for diagnosing cancer. We have previously developed a size-based filtration platform followed by epithelial cell adhesion molecule immunofluorescence staining for detecting CTCs. To characterize CTCs independently of cell surface protein expression, we incorporated a chromosomal fluorescence in situ hybridization (FISH) assay to detect abnormal copy numbers of chromosomes in cells collected from peripheral blood samples by the size-based filtration platform. Aneuploid cells were detected in the peripheral blood of patients with lung cancer. Unexpectedly, aneuploid cells were also detected in the control group, which consisted of peripheral blood samples from patients with benign lung diseases, such as empyema necessitatis and non-tuberculous mycobacterial lung disease. These findings suggest that chromosomal abnormalities are observed not only in tumor cells, but also in benign infectious diseases. Thus, our findings present new considerations and bring into light the possibility of false positives when using FISH for cancer diagnosis.

A New Cell Block Method for Multiple Immunohistochemical Analysis of Circulating Tumor Cells in Patients with Liver Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We developed a new method of detecting circulating tumor cells (CTCs) in liver cancer patients by constructing cell blocks from peripheral blood cells, including CTCs, followed by multiple immunohistochemical analysis. MATERIALS AND METHODS: Cell blockswere constructed from the nucleated cell pellets of peripheral blood afterremoval of red blood cells. The blood cell blocks were obtained from 29 patients with liver cancer, and from healthy donor blood spikedwith seven cell lines. The cell blocks and corresponding tumor tissues were immunostained with antibodies to seven markers: cytokeratin (CK), epithelial cell adhesion molecule (EpCAM), epithelial membrane antigen (EMA), CK18, α-fetoprotein (AFP), Glypican 3, and HepPar1. RESULTS: The average recovery rate of spiked SW620 cells from blood cell blocks was 91%. CTCs were detected in 14 out of 29 patients (48.3%); 11/23 hepatocellular carcinomas (HCC), 1/2 cholangiocarcinomas (CC), 1/1 combined HCC-CC, and 1/3 metastatic cancers. CTCs from 14 patients were positive for EpCAM (57.1%), EMA (42.9%), AFP (21.4%), CK18 (14.3%), Gypican3 and CK (7.1%, each), and HepPar1 (0%). Patients with HCC expressed EpCAM, EMA, CK18, and AFP in tissue and/or CTCs, whereas CK, HepPar1, and Glypican3 were expressed only in tissue. Only EMA was significantly associated with the expressions in CTC and tissue. CTC detection was associated with higher T stage and portal vein invasion in HCC patients. CONCLUSION: This cell block method allows cytologic detection and multiple immunohistochemical analysis of CTCs. Our results show that tissue biomarkers of HCC may not be useful for the detection of CTC. EpCAM could be a candidate marker for CTCs in patients with HCC.

Simultaneous Detection of Disseminated and Circulating Tumor Cells in Primary Breast Cancer Patients / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Disseminated tumor cells (DTCs) from bone marrow (BM) are a surrogate of minimal residual disease (MRD) in primary breast cancer (PBC) patients and associated with an adverse prognosis. However, BM sampling is an invasive procedure. Although there is growing evidence that circulating tumor cells (CTCs) from the blood are also suitable for monitoring MRD, data on the simultaneous detection of DTCs and CTCs are limited. MATERIALS AND METHODS: We determined the presence of DTCs using immunocytochemistry and the pan-cytokeratin antibody A45-B/B3. CTCs were determined simultaneously using a reverse transcription-polymerase chain reaction-based assay (AdnaTest Breast Cancer) and CellSearch (at least one CTC per 7.5 mL blood). We compared the detection of DTCs and CTCs and evaluated their impact on disease-free and overall survival. RESULTS: Of 585 patients, 131 (22%) were positive for DTCs; 19 of 202 (9%) and 18 of 383 (5%) patients were positive for CTCs, as shown by AdnaTest and CellSearch, respectively. No significant association was observed between DTCs and CTCs (p=0.248 and p=0.146 as shown by AdnaTest and CellSearch, respectively). The presence of DTCs (p=0.046) and the presence of CTCs as shown by CellSearch (p=0.007) were predictive of disease-free survival. CONCLUSION: Our data confirm the prognostic relevance of DTCs and CTCs in patients with PBC. As we found no significant relationship between DTCs and CTCs, prospective trials should include their simultaneous detection. Within those trials, the question of whether or not DTCs and CTCs are independent subpopulations of malignant cell clones should be determined by molecular characterization.

Circulating Tumor Cells in Lung Cancer

Circulating Tumour Cells (CTCs) can be released from the primary lung tumour into the bloodstream and they may colonize distant organs and give rise to metastasis. The presence of CTCs in the blood has been documented more than a century ago, and ultrasensitive methods have been recently developed to detect circulating tumour cells (CTCs) in the peripheral blood of lung cancer patients. Most CTCs require an initial enrichment step, since CTCs are a very rare event. The different technologies and also the differences among the screened populations make the clinical significance of detecting CTCs difficult to interpret. Peripheral blood analyses are more convenient for patients than invasive BM sampling and many research groups are currently assessing the clinical utility of CTCs for assessing the prognosis and monitoring the response to systemic therapies in lung cancer patients. Here we will review the different assays that are currently available for CTC detection and analysis of lung cancer. Moreover, molecular analyses of CTCs have provided new insights into the biology of metastasis of lung cancer with important implications for the clinical management of lung cancer patients.

Circulating Tumor Cells: Detection Methods and Potential Clinical Application in Breast Cancer / 한국유방암학회지

Circulating tumor cells (CTCs) are defined as tumor cells circulating in the peripheral blood of patients. CTCs have long been regarded as an attractive research topic. Because of recent technological advances, it is now possible to detect CTCs in the bloodstream. Interestingly, CTCs are present in both of patients with metastatic disease and early stage localized disease in patients with breast cancer. An assay detecting CTCs seems to have significant future potential value in the clinical management of breast cancer as a prognostic marker, monitoring treatment response and selecting target therapy. This review addresses the technical overview of detection methods, possible clinical application and future direction of CTCs research.