Detection and analysis of tumour biomarkers to strengthen the diagnosis of acute and chronic leukaemias

AbstractMolecular markers in leukaemia are essential to diagnose, establish prognosis factors and determine the correct treatment of patients; therefore, it is imperative to include molecular biology studies, so that, combined with cytomorphology and immunophenotyping studies, they constitute the differential diagnosis of these neoplasias. It is extremely important to implement a panel of molecular markers that allows us to detect oncogenes derived from chromosomal translocations, genes derived from epigenetic alterations and drug-resistant genes.A panel of molecular markers that included 11 genes derived from chromosomal translocations BCR-ABL major and minor breakpoints, E2A-PBX1, MLL-AF4, TEL-AML1, PML-RARα, AML1-ETO was standardised; cancer testis antigens (CTA) derived from NY-ESO1 and MAGE-A3 epigenetic alterations and multi-drug-resistant genes ABCB1 and ABCG2. 30 patients diagnosed with leukaemia from Mexico's General Hospital (Hospital General de Mexico) were included. They suffered from acute lymphoblastic leukaemia (ALL) and acute myeloblastic leukaemia (AML); bone marrow mononuclear cells were used, from which RNA was extracted for the synthesis of cDNA and RT-PCR for each of the markers. In acute lymphoblastic leukaemia (ALL), BCR-ABL biomarkers expressed under 30% (3/10), E2A-PBX1 10% (1/10), ABC-B1 80% (8/10), and ABC-G2 60% (6/10). Patients with acute myeloblastic leukaemia (AML) expressed 30% PML-RARα (3/10), 40% ABC-B1 (4/10), and 10% ABC-G2 (1/10). Lastly, in patients with chronic myeloid leukaemia (CML), BCR-ABL was over 100% (10/10), ABC-B1 20% (2/10), and ABC-G2 50% (5/10). The presence of transcripts from chimeric genes minor BCR-ABL and E2A-PBX1 in ALL; PML-RARα in AML; and major BCR-ABL in CML, confirms the importance that the panel of molecular markers has in strengthening the diagnosis and prognosis of these conditions

Pattern of differential expression of costimulatory molecules in myeloma cell line MM1.R

T cells constantly recognise and eliminate circulating tumour cells. They require two signals in order to be activated: antigen presentation via TCR, and costimulatory interaction with different surface molecules of the B7 family, which may either enhance or attenuate immune response. In several cancers these molecules are deregulated, thereby promoting immune escape and the settlement or migration of cancer cells. In multiple myeloma, the expression of B7 co-inhibitory molecules and their relationship to disease progression have been identified. The MM1.R cell line is a useful cellular model for studying the progression of this disease, and therefore an analysis of the pattern of expression in this cell line helps to cast light on the immune status of this disease. Using a real-time PCR (quantitative RT-PCR) assay, we found that the main molecules expressed were those with an inhibitory function (B7-H1, B7-H3 and B7-H4), which suggests a high level of immune inhibition by these cells. Resumen: El reconocimiento y eliminación constante de células tumorales circulantes se logra principalmente mediante la acción de los linfocitos T, células cuya activación requiere de dos señales para poder llevarse a cabo: la presentación del antígeno a través del TCR y una interacción co-estimuladora liderada por distintas moléculas de superficie de la familia B7 que pueden potenciar o atenuar la respuesta. En varios tipos de cáncer, estas moléculas se encuentran desreguladas, favoreciendo el escape inmunológico y el asentamiento y/o migración de las células neoplásicas. En mieloma múltiple se ha identificado la expresión de moléculas B7 co-inhibitorias y su relación con la progresión de la enfermedad. La línea celular MM.1R es un modelo celular útil para estudiar la progresión de esta enfermedad, por lo que el análisis del patrón de expresión en esta línea celular contribuye a entender el estado inmunológico en la enfermedad. Mediante un ensayo de RT-PCR en tiempo real (qRT-PCR) encontramos que las moléculas mayormente expresadas fueron aquellas con función inhibitoria (B7H1, B7H3 y B7H4), lo cual indica una alta inhibición inmunológica por parte de estas células. Keywords: Multiple myeloma, MM1.R, Costimulatory molecules, B7, PCR, Palabras clave: Mieloma múltiple, MM1.R, Coestimuladoras, B7, PC

Expression of cancer testis antigens in patients with Hodgkin's lymphoma and their clinical correlation

Objective: To determine the frequency of expression of cancer testis antigens and their clinical correlation in patients with Hodgkin lymphoma. Methodology of the study: In this analytical, experimental and ambispective study, the MAGE A-3 and NY-ESO-1 antigen expression was correlated with clinical prognostic variables such as clinical stage, response to treatment, and relapse, in a total of 70 patients diagnosed with Hodgkin's lymphoma at the Hodgkin's Lymphoma Clinic of the General Hospital of Mexico “Dr. Eduardo Liceaga”, from December 2000 to December 2015. Twenty-four patients were evaluated using RT-PCR, following extraction of RNA, to detect MAGE-A3 and NY-ESO1 expression. Cellular RNA was extracted from frozen tissue and controls using trizol (Life Technologies, Paisley, UK). 1 μg of RNA was used for cDNA synthesis by M-MLV reverse transcriptase (Life technologies, Paisley, UK). Results: We studied 24 patients with a median age of 28 years, a minimum age of 16 years and a maximum age of 48 years, mostly male. 50% of patients presented complete response to the first line of treatment and 27% of patients presented relapse, 37.5% in relation to the expression of MAGE-A3. Expression of the NY-ESO-1 gene was not found in the study group. Twelve percent of patients died during the study, 8.33% of whom were also positive for MAGE-A3 (p = 0.264.95% CI). No significant correlation was found between MAGE-A3 expression and major clinical prognostic variables. Conclusion: Although the expression of MAGE-A3 in the study group was 37.5% (higher than reported in international studies), we found no correlation with the main clinical prognostics variables. Considering that the expression of MAGE-A3 in the cases studied does not confer prognostic value, making it impossible to use as a prognostic tool in peripheral blood, we are leaving the doors open to continue with this line of research, possibly increasing the number of patients as well as prolonging the follow-up time

Palliative prognostic index and Charlson comorbidity index as predictors of mortality in acute lymphoblastic leukaemia patients who are candidates for palliative care

Objective: To establish whether the palliative prognostic index (PPI), the Charlson comorbidity index (CCI) or other factors are predictors of survival for patients with ALL undergoing palliative care. Materials and methods: Retrospective cohort study of patients diagnosed with ALL undergoing palliative care. We analysed variables at the time of diagnosis (age, WBC count, and risk type), chemotherapy regimens received, PPI, CCI and transfusion requirements at time palliative care was started. Results: We studied 32 patients with a mean age of 37 (18–75) years. Fourteen cases had a PPI = 0 (43.8%). 62.5% (n = 20) with a CCI > 3 had high odds of dying within 10 years. The median survival was 200 days, unaffected by any of the factors analysed. Discussion: Neither PPI, CCI, nor the other studied factors effectively predicted survival. Scales will have to be adapted or new predictive scales devised specifically for patients with ALL. Resumen: Objetivo: Establecer si el índice pronóstico paliativo (IPP), el índice de comorbilidad de Charlson (ICC) u otros factores son predictores de sobrevida en pacientes con LLA sometidos a terapia paliativa. Material y métodos: Cohorte retrospectiva de pacientes con diagnóstico de LLA sometidos a terapia paliativa. Se analizaron variables al momento del diagnóstico (edad, cifra de leucocitos, tipo de riesgo), esquemas recibidos, IPP e ICC al momento de iniciar tratamiento paliativo, así como los requerimientos transfusionales. Resultados: Se estudiaron 32 pacientes con edad promedio de 37 (18–75) años. Catorce casos obtuvieron un IPP de 0 (43.8%). El 62.5% (n = 20) con ICC >3 tenía altas probabilidades de morir en menos de 10 años. La media de supervivencia fue de 200 días, sin afectarse significativamente por ninguno de los factores analizados. Discusión: IPP, ICC, ni otros factores predijeron efectivamente la sobrevida. Será necesario adecuar estas escalas o idear nuevas específicamente para LLA. Keywords: Palliative care, Prognosis, Precursor cell lymphoblastic leukaemia-lymphoma, Survival, Palabras clave: Cuidados paliativos, Pronóstico, Leucemia-linfoma linfoblástico de células precursoras, Supervivenci

Expression of genes MAGE-A3 MAGE-C1, NY-ESO-1 and SSX1 in patients with multiple myeloma at the General Hospital of Mexico

Multiple myeloma is the most common form of plasma cell cancer. It is a disease of elderly people, with a mean age at diagnosis of 65–70, and represents 10–15% of all blood cancers. It is a heterogeneous disease associated with intrinsic factors and disease characteristics such as genetics, which dictate the clinical course of the disease. Multiple myeloma involves several abnormalities in the IgH variable region. The first oncogenic events in this cancer occur in the germinal centre, apparently during isotype switching and somatic hypermutation of B cells. While these primary mutations have been found in myeloma cells, these events alone are not enough to cause pathogenesis. However, few genes have been identified in this type of disease. The expression of cancer/testis antigens (CTAs) is limited to testis tissue and various types of cancer, in which they are considered as a tumour marker as they are associated with the prognosis and monitoring of the disease, and are involved with overall survival and event-free disease. In view of the above, the objective of this study was to analyse the expression of CTAs (MAGE-A3 and -C1, NY-ESO and SSX1) by RT-PCR in patients diagnosed with de novo multiple myeloma admitted to the Haematology Department of Hospital General de México. Our results proved that there is presence of these genes and that they may be involved in resistance, progression and survival. Resumen: El mieloma múltiple es la forma más común de las neoplasias malignas de células plasmáticas. Es una enfermedad de los adultos mayores, la media al diagnóstico es de 65 a 70 años y representa del 10 al 15% de todas las neoplasias hematológicas. Es una enfermedad heterogénea donde existe una asociación con factores y características intrínsecas de la enfermedad, tales como las genéticas, las cuales dictan el curso clínico del padecimiento. El mieloma múltiple involucra diversas anormalidades en la región variable de la IgH. Los primeros eventos oncogénicos en esta neoplasia ocurren en el centro germinal, al parecer durante el proceso de cambio de clase de isotipo y de hipermutación somática de las células B. Si bien estas mutaciones primarias han sido encontradas en células mielomatosas, estos eventos por sí solos no son suficientes para provocar la patogénesis. Sin embargo, son escasos los genes identificados en este tipo de padecimiento. La expresión de los antígenos testiculares de cáncer (ATC) se limita a tejido testicular y a varios tipos de cánceres, en donde ha sido considerada como marcador tumoral, pues están implicados en el pronóstico y seguimiento de la enfermedad, impactando en la supervivencia global y en la enfermedad libre de eventos. Por lo tanto, el objetivo de este trabajo fue analizar la expresión de los ATC (MAGEA3, C1, NY-ESO y SSX1) en pacientes diagnosticados con mieloma múltiple de novo que ingresaron al Servicio de Hematología del Hospital General de México por medio de RT-PCR. Nuestros resultados comprobaron que existe presencia de dichos genes, por lo que podrían estar participando en la resistencia, progresión y supervivencia. Keywords: Cancer/testis antigens, Multiple myeloma, RT-PCR, Palabras clave: Antígenos testiculares de cáncer, Mieloma múltiple, RT-PC

Frequency and clinical association of NY-ESO-1 gene expression in diffuse large B-cell lymphoma

Objective: Our objective was to evaluate the frequency of expression and determine the expression levels of the NY-ESO-1 gene in patients with DLBCL as well as to examine its relationship with clinical parameters and survival. Methods: We analyzed NY-ESO-1 gene expression levels using real-time quantitative RT-PCR (RT-qPCR) in 112 patients with DLBCL. The associations between the expression of the NY-ESO-1 gene and the clinical variables were evaluated using the Chi-square test and Fisher’s exact test. Overall survival (OS) was determined using the Kaplan–Meier method. Result: The results showed that the NY-ESO-1 gene was expressed in 46.4% (52/112) of patients with DLBCL, and NY-ESO-1 gene expression was associated with clinical parameters such as LDH, clinical stage, and International Prognostic Index (IPI) (p ≤ 0.05). High levels of NY-ESO-1 gene expression were associated with advanced disease stages, and the survival rates after 5.3 years of tracking were lower in the patients expressing the NY-ESO-1 gene (66.4%) than in those not expressing the gene (23.1%). Conclusion: The expression levels of the NY-ESO-1 gene in patients with DLBCL may be of great utility for diagnosing and determining the prognosis of this disease