Molecular and Dynamic Evaluation of Proteins Related to Resistance to Neoadjuvant Treatment with Chemoradiotherapy in Circulating Tumor Cells of Patients with Locally Advanced Rectal Cancer.

The heterogeneity of response to neoadjuvant chemoradiotherapy (NCRT) is still a challenge in locally advanced rectal cancer (LARC). The evaluation of thymidylate synthase (TYMS) and RAD23 homolog B (RAD23B) expression in circulating tumor cells (CTCs) provides complementary clinical information. CTCs were prospectively evaluated in 166 blood samples (63 patients) with LARC undergoing NCRT. The primary objective was to verify if the absence of RAD23B/TYMS in CTCs would correlate with pathological complete response (pCR). Secondary objectives were to correlate CTC kinetics before (C1)/after NCRT (C2), in addition to the expression of transforming growth factor-ß receptor I (TGF-ßRI) with survival rates. CTCs were isolated by ISET and evaluated by immunocytochemistry (protein expression). At C1, RAD23B was detected in 54.1% of patients with no pCR and its absence in 91.7% of patients with pCR (p = 0.014); TYMS- was observed in 90% of patients with pCR and TYMS+ in 51.7% without pCR (p = 0.057). Patients with CTC2 > CTC1 had worse disease-free survival (DFS) (p = 0.00025) and overall survival (OS) (p = 0.0036) compared with those with CTC2 ≤ CTC1. TGF-ßRI expression in any time correlated with worse DFS (p = 0.059). To conclude, RAD23B/TYMS and CTC kinetics may facilitate the personalized treatment of LARC.

Identificação de células tumorais circulantes em pacientes com câncer de canal anal e sua interação com vírus HPV / Identification of circulating tumor cells in patients with anal canal cancer and their interaction with HPV viruses

O câncer tem se tornado um grande problema de saúde pública em países desenvolvidos e em desenvolvimento, sendo responsável por mais de 6 milhões de casos/ óbitos a cada ano, representando cerca de 12% de todas as causas de morte no mundo. O câncer de canal anal corresponde a 4% de todas as neoplasias malignas do trato digestivo baixo e tem como um dos fatores etiológicos, o vírus do papiloma humano (HPV), o que lhe confere forte associação com as doenças sexualmente transmissíveis. A imunossupressão desencadeada pelo HIV também pode acelerar a progressão de lesões precursoras anais associadas ao HPV. Muitos estudos têm demonstrado que as células tumorais circulantes (CTCs) são bons marcadores para avaliação da evolução tumoral. Além disso, essas células são úteis no monitoramento da resposta ao tratamento, contribuindo para avanços na medicina personalizada. O presente estudo teve como objetivos principais: 1) quantificar as CTCs das coletas de primeiro e segundo momento; 2) Correlacionar os achados das análises com evolução clínica dos pacientes. Foram coletadas 24 amostras de sangue total de pacientes com câncer de canal anal localizado e candidatos a tratamento definitivo com quimioradioterapia. Os pacientes tiveram aproximadamente 10 ml de sangue coletados em tubos EDTA em dois momentos distintos: antes do início do tratamento e após seu término. O sangue coletado foi filtrado em uma membrana pelo sistema ISET (Isolation by SizE of Tumor Cells, Rarecells, France). Após a filtração, as células fixadas na membrana de policarbonato foram submetidas à quantificação e à análise por imunocitoquímica (ICC) com os anticorpos p16, CD-45, PD-L1, RAD23B, TYMS e ERCC1 e CISH para detecção da presença do RNAm do vírus HPV. O tempo mediano de seguimento foi de 11 28 meses (1,35- 16,61 meses). Entre as amostras analisadas por ICC, não encontramos nenhuma CTC marcada para p16, houve um paciente cujas CTCs marcaram para PD-L1 na primeira coleta. Dois pacientes tiveram suas CTCs expressando RAD23B na primeira coleta e um na segunda coleta. CTCs expressando TYMS foram encontradas no material de três pacientes na primeira coleta e em um na segunda. ERCC1 estava expresso em CTCs de um paciente na primeira e de três na segunda, onde correlacionou-se com altos níveis de CTCs (p= 0,083) e com ausência de reposta clínica (p= 0,09), porém, sem significância estatística. Nenhuma CTC marcou para CD-45, sendo este anticorpo usado somente para garantir a depleção de leucócitos. Por meio do CISH conseguimos verificar a positividade para HPV nas CTCs em 14 pacientes dos 15 incluídos no estudo na primeira coleta. Os parâmetros obtidos aqui podem ser clinicamente úteis para o melhor monitoramento dos pacientes com câncer de canal anal não metastático, porém, estudos futuros com uma coorte maior precisam ser feitos

Cancer has become a major public health problem in developed and developing countries, accounting for more than 6 million cases of death each year, representing about 12% of all causes of death in the world. Anal canal cancer corresponds to 4% of all malignant neoplasms of the lower digestive tract and has as one of its etiological factors, the human papilloma virus (HPV), which gives it a strong association with sexually transmitted diseases. Immunosuppression triggered by HIV can also accelerate the progression of anal precursor lesions associated with HPV. Many studies have shown that circulating tumor cells (CTCs) are good markers for assessing tumor evolution. In addition, these cells are useful in monitoring the response to treatment, contributing to advances in personalized medicine. The present study had as main objectives: 1) To detect CTCs in patients with cancer of the located anal canal and to identify the presence of the HPV virus in these cells; 2) assess whether there is a relationship between presence of HPV and the number of CTCs, correlating the findings with the clinical evolution of patients through the analysis of medical records. Whole blood samples were collected from patients with cancer of the localized anal canal, candidates for definitive treatment with chemotherapy. The patients had approximately 10 ml of blood collected in EDTA tubes at two different times: before the start of the chemotherapy treatment and after the end of the treatment. The collected blood was filtered on a membrane by the ISET device (Isolation by SizE of Tumor Cells). After filtration, the cells were fixed on the polycarbonate membrane and subjected to quantification and immunocytochemistry (ICC) analysis with antibodies p16, CD-45, PD-L1, RAD23b, TYMS and ERCC1. The median follow-up was 11 28 months (1,35- 16,61 months). There were collected 24 blood samples. Among the samples analyzed by ICC, we did not find any CTC marked for p16. There was one patients whose CTCs stained for PD-L1 in the first collection, two who marked for RAD23B in the first collection and one in the second collection. Three patients had CTCs marked for TYMS in the first collection and one on the second collection. ERCC1 was expressed in CTCs from one patient on the first and in three on the second collection and correlated with higher numbers of CTCs at this moment (p= 0,083) and with absence of clinical response (p= 0,09), although without statistical significance. No CTC stained for CD45, as this antibody was used only to guarantee leukocyte depletion. Through CISH, we verified positivity for HPV in CTCs in 13/15 patients in the first blood collection. The parameters obtained here can be clinically useful for better monitoring of patients with non-metastatic anal canal cancer, however, future studies with a larger cohort need to be done.