RESUMO
Liquid biopsy is a novel diagnostic approach at first developed to characterize the molecular profile of solid tumors by analyzing body fluids. For cancer patients, it represents a noninvasive way to monitor the status of the solid tumor with respect to representative biomarkers. There is growing interest in the utilization of circulating tumor DNA (ctDNA) analysis also in the diagnostic and prognostic fields of lymphomas. Clonal immunoglobulin (IG) gene rearrangements are fingerprints of the respective lymphoid malignancy and thus are highly suited as specific molecular targets for minimal residual disease (MRD) detection. Tracing of the clonal IG rearrangement patterns in ctDNA pool during treatment can be used for MRD assessment in B-cell lymphomas. Here, we describe a reproducible next-generation sequencing assay to identify and characterize clonal IG gene rearrangements for MRD detection in cell-free DNA.
Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Rearranjo Gênico , Genes de Imunoglobulinas , Linfoma , Neoplasia Residual , Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Células Clonais , Rearranjo Gênico/genética , Genes de Imunoglobulinas/genética , Humanos , Imunoglobulinas/genética , Biópsia Líquida/métodos , Linfoma/sangue , Linfoma/diagnóstico , Linfoma/genética , Linfoma de Células B/sangue , Linfoma de Células B/diagnóstico , Linfoma de Células B/genética , Neoplasia Residual/sangue , Neoplasia Residual/diagnóstico , Neoplasia Residual/genéticaRESUMO
Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.
Assuntos
Anticorpos/metabolismo , Formação de Anticorpos , Linfócitos B/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/metabolismo , Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Galinhas , Conversão Gênica/efeitos dos fármacos , Dosagem de Genes , Variação Genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Pseudogenes , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Here, we describe the immunoglobulin and T cell receptor (Ig/TCR) molecular rearrangements identified as a leukemic clone hallmark for minimal residual disease assessment in relation to TP53 mutational status in 171 Ph-negative Acute Lymphoblastic Leukemia (ALL) adult patients at diagnosis. The presence of a TP53 alterations, which represents a marker of poor prognosis, was strictly correlated with an immature DH/JH rearrangement of the immunoglobulin receptor (p < 0.0001). Furthermore, TP53-mutated patients were classified as pro-B ALL more frequently than their wild-type counterpart (46% vs. 25%, p = 0.05). Although the reasons for the co-presence of immature Ig rearrangements and TP53 mutation need to be clarified, this can suggest that the alteration in TP53 is acquired at an early stage of B-cell maturation or even at the level of pre-leukemic transformation.
Assuntos
Biomarcadores Tumorais/genética , Rearranjo Gênico , Genes de Imunoglobulinas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Idoso , Ensaios Clínicos como Assunto , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Adulto JovemRESUMO
Chronic lymphocytic leukaemia (CLL) is a haematological malignancy for which reliable prognostic markers are needed in view of its clinical heterogeneity. In approximately 50 percent of CLL patients, immunoglobulin (Ig) rearrangements are modified by somatic hypermutation (SHM), a process that represents a reliable prognostic indicator of favourable progression. In this study, we investigated SHM in 37 Brazilian CLL patients and identified the preferential involvement of specific immunoglobulin gene families and segments through PCR-amplified fragments or subcloned fragments. Forty-one rearrangements were observed and 37 of them were functional. A 98 percent homology cut-off with germinal sequences showed 18 patients (48.7 percent) with SHM. Unmutated cases showed a poorer clinical outcome. V H3 was the most frequent V H family, followed by V H4. The V H4-39 segment was the most frequently used, mainly in unmutated cases, while the V H3 family was predominant in mutated cases. The D3 and J H4/J H6 families were the most frequently observed.