Biomarkers of minimal residual disease and treatment.

Minimal residual disease (MRD) has been defined as a very small numbers of cancer cells that remain in the body after curative treatment. Its presence or absence will ultimately determine prognosis. With the introduction of new technologies the presence of MRD in patients with solid tumours can be detected and characterized. As MRD predicts future relapse, be it early or late treatment failure, in an otherwise asymptomatic patient its treatment and when to start treatment remains to be determined. Thus the concepts of personalized medicine using different biomarkers to classify the biological properties of MRD maybe come possible. Based on this determinations it may be possible to use targeted therapies rather than all patients with the same type of cancer receiving a standard treatment. However, it is important to understand the limitations of the different technologies, what these techniques are detecting and how they may help in the treatment of patients with cancer. The majority of published studies are in patients with metastatic cancer and there are few reports in patients with MRD. In this chapter the concept of MRD, the methods used to detect it and what treatments may be effective based on the biological characteristics of the tumour cells as determined by different biomarkers is reviewed. MRD depends on the phenotypic properties of the tumour cells to survive in their new environment and the anti-tumour immune response. This is a dynamic process and changes with time in the wake of immunosuppression caused by the tumour cells and/or the effects of treatment to select resistant tumour cells. With the use of biomarkers to typify the characteristics of MRD and the development of new drugs a personalized treatment can be designed rather than all patients given the same treatment. Patients who are initially negative for MRD may not require further treatment with liquid biopsies used to monitor the patients during follow-up in order to detect those patients who may become MRD positive. The liquid biopsy used during the follow up of MRD positive patients can be used to detect changes in the biological properties of the tumour cells and thus may need treatment changes to overcome tumour cell resistance.

The Possible Role of Matrix Metalloprotienase-2 in the Relapse in Patients with Stage II Colon Cancer Treated by Curative Surgery.

AIMS: To determine the association of micro-metastatic matrix metalloproteinase-2 (MMP-2) expression,  the absolute lymphocyte count (ALC)) and outcome in stage II colon cancer. MATERIALS AND METHODS: A single centre, prospective observational study, one month post-surgery blood for ALC, circulating tumour cell (CTC) detection and a bone marrow biopsy for micro-metastasis detection were obtained.  CTCs were detected using differential gel centrifugation and immunocytochemistry with anti-CEA and anti-MMP-2, the bone marrow biopsy for the detection of micro-metastasis was processed as for CTCs . At each follow-up  ALC and CTC counts were determined. Bone marrow sampling was repeated if the ALC decreased by >10%, at relapse or at the end of the study period. Three MRD subgroups were defined, Group I MRD negative, Group II only positive for micro-metastasis and   Group III in which CTCs were detected. RESULTS: One hundred and eighty one patients  participated; 105 (58%) patients formed Group 1, 36 (20%) formed Group II  and 40 (22%)  formed Group III for a median follow-up of 4 years .  Of Group I 3/105 (3%), Group II 16/36 (44%) and Group III 34/40 (84%) patients relapsed. The ALC was significantly higher in Groups I and II, the expression of MMP-2 and MMP-2 score in Group II was significantly lower than in Group III patients. A low ALC was associated with a higher expression of MMP-2 in the micro-metastasis and presence of CTCs. CONCLUSIONS: Patients with stable ALCs did not relapse; decreasing ALCs were associated with increasing MMP-2 scores, the appearance of CTCs and relapse.

The CAPRA-S Score and Immune Dysfunction as a Guide to Outcome in Men Treated with Prostatectomy Radical as Mono-Therapy for Prostate Cancer.

OBJECTIVE: Incorporate the immune function as determined by the absolute lymphocyte count (ALC) into the CAPRA-S risk stratification score to determine if predictive values could be improved. MATERIALS AND METHODS: The clinical pathological findings in the surgical specimen and total PSA were used to define the three CAPRA-S risk groups. One month after surgery and at each follow up total PSA and the ALC were determined, until biochemical failure (BF) or the end of the study period. A cut off value of <1,000 lymphocytes/mm3 was used to define lymphocytopenia (LCP). Each CAPRA-S group was sub-divided based on the presence or absence of LCP. Kaplan-Meier biochemical failure free survival (BFFS) curves and restricted mean biochemical failure free survival times were calculated for each group. RESULTS: 404 patients participated of whom 103 (25.5%) underwent BF. 270 men were CAPRA-S low risk (LR), 89 intermediate risk (IR) and 45 high risk (HR), of whom LCP was found in 22 (8%) of low risk, 24 (27%) of intermediate risk and 17 (38%) of high risk men. LCP was significantly associated with a higher PSA, higher Gleason and CAPRA-S scores and BF. HRs were 1.76 for IR, 2.49 for HR and 1.29 for LCP. Five-year BFFS for men without LCP, LR 93.5%, IR 61% and HR 36%, for those with LCP, LR 55%, IR 25% and HR 6%. All patients with LCP and IR or HR scores relapsed within 6 years. 10 year BFFS for men without LCP were 71% LR, 43% IR and 23% HR, LR with LCP 16%. Men with BF had increasing LCP approximately 18 months before BF. CONCLUSIONS: The incorporation of the ALC taken one month after surgery with the CAPRA-S improves risk stratification; decreases in the ALC suggest that BF is occuring. These results need to be confirmed with larger studies.

Increasing Immune Dysfunction is Associated with Increasing Matrix-Metalloproteinase-2 Expression and Predicts Biochemical Failure in Men with Bone Marrow Micro-Metastasis Positive Localized Prostate Cancer.

INTRODUCTION: To determine if there was an association of the ALC (absolute lymphocyte count) and LCP (lymphocytopenia) with the expression of MMP-2 in bone marrow micro-metastasis, the changes occurring during follow-up and association with biochemical failure. METHODS AND PATIENTS: One month after surgery blood and bone marrow samples were taken to determine the presence of micro-metastasis, the presence of circulating prostate cells (CPCs) and ALC. CPCs and micro-metastasis were detected using immunocytochemistry and MMP-2 expression determined in micro-metastasis. Only men positive for micro-metastasis participated in the study. At end follow blood was taken for serum PSA, ALC and CPCs, if the ALC decreased by more than 10% bone marrow sampling was repeated and MMP-2 expression determined, similarly for men with BF. Men who had stable ALCs had an end of study evaluation of the bone marrow. RESULTS: 402 men underwent radical prostatectomy, one month post surgery 79 men were positive for only bone marrow micro-metastasis and formed the study group; of whom 36/79 (45%) underwent BF. Clinical pathological findings were not significantly different between men with or without BF. In men with BF the ALC was significantly lower one-month post surgery. The 5 and 10 year Kaplan-Meier survival was 100% at 5-years and 65% at 10-years for the whole cohort. Men without BF had stable ALCs. A decrease of >10% in the ALC was associated with increasing MMP-2 expression in the micro-metastasis and surrounding stromal tissue, the appearance of CPCs 6-12 months later and BF. CONCLUSIONS: the immune host-tumour cell interaction in the microenvironment is dynamic and changes with time. A decreasing ALC may be a valuable marker in identifying men with high risk of BF and changes in immune mediated dormancy before the PSA rises.

Improvement in the Neutrophil-Lymphocyte Ratio after Combined Fluorouracil, Leucovorina and Oxaliplatino based (FOLFOX) Chemotherapy for Stage III Colon Cancer is Associated with Improved Minimal Residual Disease and Outcome.

INTRODUCTION: Minimal residual disease (MRD) is the net result of the biological properties of disseminated tumour cells and the effect of the immune system and treatment to eliminate them. The aim of this study was to analyse the effect of combined chemotherapy on the immune function as determined by the neutrophil-lymphocyte ratio (NLR) and if it was associated with changes in the subtype of minimal residual disease and outcome in stage III colon cancer. METHODS AND PATIENTS: A prospective, single centre observational study; the NLR was determined immediately prior to and one, two and three months after completing chemotherapy. Circulating tumour cells (CTCs) and bone marrow micro-metastasis (mM) using immunocytochemistry with anti-CEA were determined prior to and one month after chemotherapy. The association of changes in the NLR with MRD subtypes classified as Group I (negative for CTCs and mM), Group II (positive for mM) and Group III (positive for CTCs) as a result of chemotherapy and five-year disease free progression (DFS) analysed. RESULTS: One hundred and eighty eight patients participated of whom 83 (44.9%) relapsed. In non-relapsing patients the NLR significantly increased and was higher after chemotherapy compared with relapsing patients. Significant increases in the NLR were associated with changes to a better MRD prognostic subtype and decreases with a worse MRD subtype. Neither baseline NLR nor MRD subtype predicted response to chemotherapy. DFS for MRD subgroups were 88%, 56% and 6% for Groups I to III respectively. CONCLUSIONS: Immune function as measured by the NLR is associated with MRD prognostic subtypes, improvements in the NLR are associated with improvements in MRD post chemotherapy but neither baseline NLR or MRD predicted outcome.

Atypical presentation of COVID-19: Chronic bilateral testicular pain with lower extremity peripheral polyneuropathy, case report.

In context of COVID-19 pandemic, there has been different presentations of the infection. The relationship of testicular pain with COVID-19 has not been extensively studied. We present a 31 years old male, with SARS-COV-2 infection, repeatedly consulting for intermittent bilateral testicular pain. Two months later he reported acute loss sensibility and pain in extremities, being diagnosed with axonal fine fiber polyneuropathy. Although the presence of SARS-COV-2 in testis remains controversial, there is a potential orchiepididymitis risk due to viral binding to ACE2 receptor in testicle, and also could induce systemic vasculitis as another possible cause of orchitis.

Improvement in immune dysfunction after FOLFOX chemotherapy for Stage III colon cancer is associated with improved minimal residual disease prognostic subtype and outcome.

AIM: Minimal residual disease (MRD) is the net result of the biological properties of disseminated tumour cells and the effect of the immune system and treatment to eliminate them. The aim of this work is to report the changes in MRD status and immune function (lymphocyte count) after FOLFOX chemotherapy, and the outcome in Stage III colon cancer patients. METHOD: This study is a prospective, single-centre observational study. Lymphocyte counts were determined prior to and 1, 2 and 3 months after the completion of chemotherapy. Circulating tumour cells (CTCs) and bone marrow micrometastases were determined using immunocytochemistry with anticarcinoembryonic antigen prior to and 1 month after chemotherapy. MRD was classified as negative (Group I), micrometastasis positive only (Group II) and CTC positive (Group III). Changes in lymphocyte counts and MRD subtype following chemotherapy and relapse-free progression were analysed. RESULTS: Of the total of 185 patients, 83 (44.9%) relapsed. The risk of relapse significantly increased from Groups I to III (p < 0.001) and with decreasing lymphocyte count (p < 0.01). The lymphocyte count significantly decreased from Groups I to III (p < 0.001). Multivariance Cox regression analysis showed hazard ratios of 3.58 (Group II), 17.43 (Group III) and 0.39 (lymphocyte count) in predicting relapse. Following chemotherapy, improved lymphocyte count was associated with improved MRD subtype (p < 0.0001). Neither baseline lymphocyte count nor MRD subtype predicted response to chemotherapy. Five-year relapse-free survival for combined lymphocyte-MRD subtypes was 95%, 57% and 5% for Groups I to III, respectively (p < 0.001). CONCLUSION: Following chemotherapy, improvements in immune function were associated with improved MRD subtype and a better relapse-free survival.

Immune Dysfunction as Measured by the Systemic Immune-Inflammation Index is Associated with the Sub-Type of Minimal Residual Disease and Outcome in Stage II Colon Cancer Treated with Surgery alone.

OBJECTIVE: Within 5 years after curative surgery for stage II colon cancer 25% of patients will relapse due to minimal residual disease (MRD). MRD is the net result of the biological properties of subpopulations of primary tumour cells which enable them to disseminate, implant in distant tissues and survive and the immune system's ability to eliminate them. We hypothesize that markers of immune dysfunction such as the systemic inflammation index (SII) are associated with the sub-type of MRD defined by bone marrow micro-metastasis (mM) and circulating tumour cells (CTCs). A higher immune dysfunction being associated with a more aggressive MRD and worse prognosis. METHODS AND PATIENTS: Blood and bone marrow samples were taken to detect CTCs and mM using immunocytochemistry with anti-CEA one month after surgery. The SII, absolute neutrophil, platelet and lymphocyte counts (ANC, APC, ALC) were determined immediately pre-surgery and one month post-surgery. These were compared with the sub-types of MRD; Group I MRD (-); Group II mM positive and Group III CTC positive; cut-off values of SII of >700 and >900 were used. Follow-up was for up to 5 years or relapse and survival curves using Kaplan-Meier (KM) were calculated. RESULTS: One hundred and eighty one patients (99 women) participated, mean age 68 years, median follow up 4.04 years; I: = 105 patients, II: N= 36 patients, III: N=40 patients. The SII significantly decreased post-surgery only in Group I patients. The frequency of SII >700 and >900 was significantly higher in Group III, between Groups I and II there was no significant difference.  The SII was significantly associated with the number of CTCs detected. The 5-year KM was 98% Group I, 68% Group II and 7% Group III. CONCLUSIONS: The results of the study suggest that the severity of immune dysfunction as determined by the SII is associated with differing sub-types of MRD and a worse prognosis; increasing immune dysfunction is associated with a more aggressive CTC positive MRD sub-type; a more severe immune dysfunction is associated with a higher number of CTCs detected.
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La puntuación capra frente a los subtipos de enfermedad residual mínima para predecir la recidiva bioquímica después de la prostatectomía radical / Capra score versus disease subtypes minimal residual to predict biochemical recurrence after radical prostatectomy

Objetivo: Comparar la puntuación CAPRA (en función de los hallazgos clínico-patológicos) yla enfermedad residual mínima (ERM) (en función delas propiedades biológicas) para predecir la recidivabioquímica (RB).Material y método: Los hallazgos clínico-patológicos de biopsias de próstata determinaron la puntuación CAPRA definiendo pacientes de bajo, intermedio yalto riesgo de la RB. Se obtuvieron muestras de sangrey médula ósea para detectar CPCs (Células ProstáticasCirculantes) y micro-metástasis usando inmunocitoquímica. Se clasificaron como positivas si se detectaba ≥1célula en la muestra. Se formaron tres subgrupos: GrupoA (ERM negativo), Grupo B (micro-metástasis positivo,CPC negativo) y Grupo C (CPC positivo). Los pacientesfueron seguidos durante diez años o hasta la RB. Lascurvas de supervivencia libre de recidiva bioquímica(SLRB) se construyeron usando el método de KaplanMeier, un modelo de parámetro flexible (supervivenciapredecida) y el tiempo de supervivencia medio restringido (TSMR) para cada subgrupo.Resultados: 347 hombres participaron; el riesgode RB aumentó proporcionalmente; HR 1,21 riesgo intermedio, 1,64 riesgo alto para CAPRA versus 1,91Grupo B y 4,43 Grupo C para EMR. Después de diezaños, el SLRB y el TSMR fueron 76%, 50%, 17% y 9,7 y 5 años respectivamente para CAPRA versus 94%,57%, 26% y 10, 9 y 6 años respectivamente paraEMR. El acuerdo entre SLRB observada y prevista fueaceptable para CAPRA (Harrell ́s C 0,64) y muy buena(0,92) para EMR.Las curvas SLRB para la EMR no fueron proporcionales;para Grupos A y B fueron similares hasta cinco años,luego hubo una falla creciente en el Grupo B. La puntuación de CAPRA no logró distinguir entre los GruposA y B, un tercio del Grupo C de alto riesgo tenía unapuntuación CAPRA de bajo riesgo.Conclusiones: La clasificación ERM fue superior dela CAPRA, diferenciando entre la RB temprana y tardía.(AU)

Objective: To compare the classificationCAPRA (based on clinical-pathological findings) andminimal residual disease (MRD) (based on biologicalcharacteristics) to predict biochemical failure (BF).Material and method: The clinical-pathologicalfindings of the prostate biopsy were used to determinethe CAPRA score, classifying patients into low, intermediate and high risk. Blood and bone marrow samples to detect circulating prostate cells (CPCs) and micro-metastasis were taken. The samples were classifiedas positive if ≥1 prostate cell was detected, formingthree subgroups; Group A (MRD negative), Group B(micro-metastasis positive, CPC negative) and Group C(CPC positive). Patients were followed-up for 10 yearsor BF. Kaplan-Meier biochemical failure free survival(BFFS) curves, a predictive flexible parameter survivalmodel and mean restricted survival times (MRST) weredetermined.Results: 347 men participated, BF risk increased withincreasing CAPRA score, HR 1.21 intermediate, 1.64high risk; versus MRD HR 1.91 and 4.43 for Groups Band C. After 10 years the BFFS and MRST were 76%,50% and 17% and 9, 7 and 5 years respectively forCAPRA versus 94%, 57% and 26% and 10, 9 and 6years respectively for MRD. The concordance betweenobserved and predicted BFFS was acceptable forCAPRA (Harrell ́s C 0.64) and very good (0.92) forMRD. The BFFS curves for MRD were not proportional with time, they were similar for 5 years for GroupsA and B, with increasing BFFS in Group B thereafter.The CAPRA score did not distinguish between Groups Aand B, one third of low risk CAPRA patients had CPCsdetected.Conclusions: The MRD classification was superiorto CAPRA, differentiating between early and late failure.(AU)

[CAPRA score vs minimal residual disease as predictors of biochemical recurrence after radical prostatectomy.] / La puntuación CAPRA frente a los subtipos de enfermedad residual mínima para predecir la recidiva bioquímica después de la prostatectomía radical.

OBJECTIVE: To compare the classification CAPRA (based on clinical-pathological findings) and minimal residual disease (MRD) (based on biological characteristics) to predict biochemical failure (BF). METHOD AND PATIENTS: The clinical-pathological findings of the prostate biopsy were used to determine the CAPRA score, classifying patients into low, intermediate and high risk. Blood and bone marrow samples to detect circulating prostate cells (CPCs) and micro-metastasis were taken. The samples were classified as positive if ≥1 prostate cell was detected, forming three subgroups; Group A (MRD negative), Group B (micro-metastasis positive, CPC negative) and Group C (CPC positive). Patients were followed-up for 10 yearsor BF. Kaplan-Meier biochemical failure free survival (BFFS) curves, a predictive flexible parameter survival model and mean restricted survival times (MRST) were determined. RESULTS: 347 men participated, BF risk increased with increasing CAPRA score, HR 1.21 intermediate, 1.64 high risk; versus MRD HR 1.91 and 4.43 for Groups Band C. After 10 years the BFFS and MRST were 76%, 50% and 17% and 9, 7 and 5 years respectively for CAPRA versus 94%, 57% and 26% and 10, 9 and 6 years respectively for MRD. The concordance between observed and predicted BFFS was acceptable for CAPRA (Harrell´s C 0.64) and very good (0.92) for MRD. The BFFS curves for MRD were not proportional with time, they were similar for 5 years for Groups A and B, with increasing BFFS in Group B there after.The CAPRA score did not distinguish between Groups A and B, one third of low risk CAPRA patients had CPCs detected. CONCLUSIONS: The MRD classification was superior to CAPRA, differentiating between early and late failure.

OBJETIVO: Comparar la puntuación CAPRA (en función de los hallazgos clínico-patológicos) y la enfermedad residual mínima (ERM) (en función de las propiedades biológicas) para predecir la recidiva bioquímica (RB).MÉTODOS Y PACIENTES: Los hallazgos clínico-patológicos de biopsias de próstata determinaron la puntuación CAPRA definiendo pacientes de bajo, intermedio y alto riesgo de la RB. Se obtuvieron muestras de sangre y médula ósea para detectar CPCs (Células Prostáticas Circulantes) y micro-metástasis usando inmunocitoquímica. Se clasificaron como positivas si se detectaba ≥1 célula en la muestra. Se formaron tres subgrupos: Grupo A (ERM negativo), Grupo B (micro-metástasis positivo, CPC negativo) y Grupo C (CPC positivo). Los pacientes fueron seguidos durante diez años o hasta la RB. Las curvas de supervivencia libre de recidiva bioquímica (SLRB) se construyeron usando el método de Kaplan Meier, un modelo de parámetro flexible (supervivencia predecida) y el tiempo de supervivencia medio restringido (TSMR) para cada subgrupo. RESULTADOS: 347 hombres participaron; el riesgode RB aumentó proporcionalmente; HR 1,21 riesgo intermedio,1,64 riesgo alto para CAPRA versus 1,91 Grupo B y 4,43 Grupo C para EMR. Después de diez años, el SLRB y el TSMR fueron 76%, 50%, 17% y 9,7 y 5 años respectivamente para CAPRA versus 94%, 57%, 26% y 10, 9 y 6 años respectivamente para EMR. El acuerdo entre SLRB observada y prevista fue aceptable para CAPRA (Harrell´s C 0,64) y muy buena (0,92) para EMR. Las curvas SLRB para la EMR no fueron proporcionales; para Grupos A y B fueron similares hasta cinco años, luego hubo una falla creciente en el Grupo B. La puntuación de CAPRA no logró distinguir entre los Grupos A y B, un tercio del Grupo C de alto riesgo tenía una puntuación CAPRA de bajo riesgo. CONCLUSIONES: La clasificación ERM fue superior de la CAPRA, diferenciando entre la RB temprana y tardía.

The association of the neutrophil-lymphocyte ratio with the presence of minimal residual disease and outcome in patients with Stage II colon cancer treated with surgery alone.

AIM: Despite curative surgery, 25% of patients with Stage II colorectal cancer will relapse due to minimal residual disease (MRD). Markers of immune function, such as the neutrophil to lymphocyte ratio (NLR), may be associated with MRD defined by bone marrow micro-metastasis (mM) and circulating tumour cells (CTCs). METHOD: A prospective cohort study of consecutive patients with Stage II colon cancer patients attended at a single centre between 2007 and 2014. Blood and bone marrow samples were taken to detect CTCs and mM using immunocytochemistry with anti-carcinoembryonic antigen 1 month after surgery. The NLR and absolute neutrophil and lymphocyte counts were determined immediately pre-surgery and 1 month post-surgery. These were compared with the sub-types of MRD: group I MRD(-); group II mM positive and group III CTC positive. Cut-off values of the NLR of >3.0 and >5.0 were used. Follow-up was for up to 5 years or relapse and disease-free survival (DFS) was calculated using Kaplan-Meier analysis. RESULTS: In all, 181 patients (99 women) participated. Mean age was 68 years. Median follow-up was 4.04 years: I, N = 105; II, N = 36; III, N = 40. The NLR significantly decreased post-surgery only in group I patients. The frequency of NLR >3.0 and >5.0 was significantly higher in group III; between groups I and II there was no significant difference. 5-year DFS was 98% in group I, 68% in group II and 7% in group III. CONCLUSIONS: Patients with a significantly higher immune dysfunction had a shorter time to disease progression, a worse DFS and the presence of CTCs.

OBJETIVO: En los primeros 5 años tras cirugía curativa para cáncer de colon en estadio II, el 25% de los pacientes tendrá una recidiva tumoral a causa de la presencia de enfermedad mínima residual (EMR). La hipótesis del presente estudio es que los marcadores de la función inmune, como la ratio neutrófilos- linfocitos (RNL), están asociados con el subtipo de EMR, clasificado por la presencia de micro-metástasis de médula ósea (mM) o células tumorales circulantes (CTCs) y con los resultados oncológicos. MÉTODOS Y PACIENTES: Se trata de un estudio prospectivo, observacional, monocéntrico que incluye una serie consecutiva de pacientes con cáncer del colon en estadio II tratados con cirugía curativa entre 2007 y 2014. Se tomaron muestras de sangre y médula ósea para detectar CTCs y mM mediante inmuno-citoquímica con anticuerpos anti-CEA un mes después de la cirugía. El numero de neutrófilos y linfocitos y el RNL se determinaron antes y un mes después de la cirugía y sus valores se compararon entre los diferentes grupos de EMR: grupo I, sin evidencia de EMR; Grupo II con mM positivo; Grupo III con CTCs positivo. El seguimiento oncológico fue de hasta 5 años y se calcularon las curvas de sobrevivencia libre de enfermedad (SLE) utilizando las curvas de Kaplan-Meier. RESULTADOS: se incluyeron en el presente estudio 181 pacientes (99 mujeres), con una edad media de 68 años y una mediana de seguimiento de 4,04 años; de acuerdo con la presencia de EMR se clasificaron los pacientes en Grupo I (n=105), Grupo II (n=36) y Grupo III (n=40). El RNL disminuyó significativamente después de la cirugía solo en el Grupo I. El porcentaje de pacientes con RNL> 3,0 y > 5,0 fue significativamente mayor en el Grupo III, sin diferencias significativas entre los Grupos I y II. La SLE a 5 años fue del 98% en el Grupo I, 68% en el Grupo II y 7% en el Grupo III. CONCLUSIONES: Los pacientes con una peor disfunción inmunológica presentan una recidiva mas precoz, una peor SLE y la presencia de células tumorales circulantes.