Mycobacterium tuberculosis polyclonal infections through treatment and recurrence.

BACKGROUND: Mixed/polyclonal infections due to different genotypes are reported in Tuberculosis. The current study was designed to understand the fate of mixed infections during the course of treatment and follow-up and its role in disease pathogenesis. METHODS: Sputum samples were collected on 0,1,2,3,6,12 and 24 months from 157 treatment-naïve patients, cultures subjected to Drug-Susceptibility-testing (MGIT 960), spoligotyping, MIRU-VNTR and SNP genotyping. All isolated colonies on thin layer agar (7H11) were subjected to spoligotyping. FINDINGS: One thirty three baseline cultures were positive (133/157, 84.7%), 43(32.3%) had mixture of genotypes. Twenty-four of these patients (55.8%) showed change in genotype while six showed different drug-susceptibility patterns while on treatment. Twenty-three (53.5%) patients with polyclonal infections showed resistance to at least one drug compared to 10/90 (11.1%) monoclonal infections (P<0.0001). Eight patients had recurrent TB, two with a new genotype and two with altered phenotypic DST. CONCLUSIONS: The coexistence of different genotypes and change of genotypes during the same disease episode, while on treatment, confirms constancy of polyclonal infections. The composition of the mixture of genotypes and the relative predominance may be missed by culture due to its limit of detection. Polyclonal infections in TB could be a rule rather than exception and challenges the age-old dogma of reactivation/reinfection.

Effective drug treatment identified by in vivo screening in a transplantable patient-derived xenograft model of chronic myelomonocytic leukemia.

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.

Comparative sequence analysis of patient-matched primary colorectal cancer, metastatic, and recurrent metastatic tumors after adjuvant FOLFOX chemotherapy.

BACKGROUND: In the era of genome-guided personalized cancer treatment, we must understand chemotherapy-induced genomic changes in tumors. This study evaluated whether adjuvant FOLFOX chemotherapy modifies the mutational profile of recurrent colorectal cancer (CRC). METHODS: Whole exome sequencing was performed on samples from primary CRC tumors, untreated metastatic tumors, and recurrent tumors following adjuvant FOLFOX chemotherapy. The samples were resected from four patients. RESULTS: The number of mutations or the mutation spectrum in individual patients was nearly identical. Copy number variants persisted regardless of FOLFOX therapy administration. The genomic signature of oxaliplatin exposure (G > T/C > A, T > A/A > T) was not enriched after FOLFOX chemotherapy. Overlapping single nucleotide variants (SNVs) and indels remained in 26-65% of the patient-matched tumor samples. One patient harbored an AKT1 E17K mutation in the recurrent tumor, whereas PIK3CA E542K and E88Q mutations were detected in the primary and untreated metastatic tumor samples. Genes related to intracellular Ca2+ homeostasis were enriched among the genes uniquely mutated after FOLFOX chemotherapy. CONCLUSIONS: We found that the mutation rates, mutation spectrum, and copy number variants were nearly identical regardless of the administration of FOLFOX therapy in the four CRC cases. The mutational discordance between the patient-matched tumor samples is likely caused by tumor heterogeneity and chemotherapy-induced clonal selection. These findings might be useful as pilot data for larger studies to clarify the changes in the mutational landscape induced by adjuvant FOLFOX chemotherapy.

Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer.

The degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumor evolution is poorly understood. Phenotypic drifts can be transmitted via inheritable transcriptional programs. Cell-type specific transcription is maintained through the activation of epigenetically defined regulatory regions including promoters and enhancers. Here we have annotated the epigenome of 47 primary and metastatic estrogen-receptor (ERα)-positive breast cancer clinical specimens and inferred phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Shared regions contain a unique set of regulatory information including the motif for transcription factor YY1. We identify YY1 as a critical determinant of ERα transcriptional activity promoting tumor growth in most luminal patients. YY1 also contributes to the expression of genes mediating resistance to endocrine treatment. Finally, we used H3K27ac levels at active enhancer elements as a surrogate of intra-tumor phenotypic heterogeneity to track the expansion and contraction of phenotypic subpopulations throughout breast cancer progression. By tracking the clonality of SLC9A3R1-positive cells, a bona fide YY1-ERα-regulated gene, we show that endocrine therapies select for phenotypic clones under-represented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.

Circulating Tumour DNA Analysis for Tumour Genome Characterisation and Monitoring Disease Burden in Extramedullary Multiple Myeloma.

Mutational characterisation in extramedullary multiple myeloma (EM-MM) patients is challenging due to inaccessible EM plasmacytomas, unsafe nature of multiple biopsies and the spatial and temporal genomic heterogeneity apparent in MM (Graphical abstract). Conventional monitoring of disease burden is through serum markers and PET-CT, however these modalities are sometimes inadequate (serum markers), not performed in a timely manner (PET-CT) and uninformative for identifying mutations driving disease progression. DNA released into the blood by tumour cells (ctDNA) contains the predominant clones derived from the multiple disease foci. Blood-derived ctDNA can, therefore, provide a holistic illustration of the major drivers of disease progression. In this report, the utility of ctDNA, as an adjunct to currently available modalities in EM-MM, is presented for a patient with EM and oligosecretory (OS) disease. Whole exome sequencing of contemporaneously acquired tumour tissue and matched ctDNA samples revealed the presence of spatial and temporal genetic heterogeneity and the identification of pathways associated with drug resistance. Longitudinal monitoring of plasma samples revealed that ctDNA can be utilised to define the dynamic clonal evolution co-existent with disease progression and as an adjunct non-invasive marker of tumour burden.

Atypical erythroblastosis in a patient with Diamond-Blackfan anemia who developed del(20q) myelodysplasia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia arising from ribosomal protein (RP) defects. Affected patients present with neonatal anemia, occasional dysmorphism, and cancer predisposition. An anemic newborn was diagnosed with DBA due to RPL5 mutation (c.473_474del, p.K158SfsX26). Refractory anemia required regular transfusions and iron chelation therapy. Pancytopenia occurred at age 16 years. Bone-marrow studies showed myelodysplasia, erythroblastosis, and clonal evolution of del(20)(q11.2q13.3). Severe anemia required transfusions. Del(20q), including the L3MBTL1 gene, is reported to be relevant to the hematological phenotype of Shwachman-Diamond syndrome. A combined defect of RPL5 and L3MBTL1 may contribute to the aberrant erythropoiesis in the present case.

Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy.

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.

Patient-derived xenograft models of breast cancer and their predictive power.

Despite advances in the treatment of patients with early and metastatic breast cancer, mortality remains high due to intrinsic or acquired resistance to therapy. Increased understanding of the genomic landscape through massively parallel sequencing has revealed somatic mutations common to specific subtypes of breast cancer, provided new prognostic and predictive markers, and highlighted potential therapeutic targets. Evaluating new targets using established cell lines is limited by the inexact correlation between responsiveness observed in cell lines versus that elicited in the patient. Patient-derived xenografts (PDXs) generated from fresh tumor specimens recapitulate the diversity of breast cancer and reflect histopathology, tumor behavior, and the metastatic properties of the original tumor. The high degree of genomic preservation evident across primary tumors and their matching PDXs over serial passaging validate them as important preclinical tools. Indeed, there is accumulating evidence that PDXs can recapitulate treatment responses of the parental tumor. The finding that tumor engraftment is an independent and poor prognostic indicator of patient outcome represents the first step towards personalized medicine. Here we review the utility of breast cancer PDX models to study the clonal evolution of tumors and to evaluate novel therapies and drug resistance.

Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

Association between the cytogenetic profile of tumor cells and response to preoperative radiochemotherapy in locally advanced rectal cancer.

Neoadjuvant radiochemotherapy to locally advanced rectal carcinoma patients has proven efficient in a high percentage of cases. Despite this, some patients show nonresponse or even disease progression. Recent studies suggest that different genetic alterations may be associated with sensitivity versus resistance of rectal cancer tumor cells to neoadjuvant therapy. We investigated the relationship between intratumoral pathways of clonal evolution as assessed by interphase fluorescence in situ hybridization (51 different probes) and response to neoadjuvant radiochemotherapy, evaluated by Dworak criteria in 45 rectal cancer tumors before (n = 45) and after (n = 31) treatment. Losses of chromosomes 1p (44%), 8p (53%), 17p (47%), and 18q (38%) and gains of 1q (49%) and 13q (75%) as well as amplification of 8q (38%) and 20q (47%) chromosomal regions were those specific alterations found at higher frequencies. Significant association (P < 0.05) was found between alteration of 1p, 1q, 11p, 12p, and 17p chromosomal regions and degree of response to neoadjuvant therapy. A clear association was observed between cytogenetic profile of the ancestral tumor cell clone and response to radiochemotherapy; cases presenting with del(17p) showed a poor response to neoadjuvant treatment (P = 0.03), whereas presence of del(1p) was more frequently observed in responder patients (P = 0.0002). Moreover, a significantly higher number of copies of chromosomes 8q (P = 0.004), 13q (P = 0.003), and 20q (P = 0.002) were found after therapy versus paired pretreatment rectal cancer samples. Our results point out the existence of an association between tumor cytogenetics and response to neoadjuvant therapy in locally advanced rectal cancer. Further studies in larger series of patients are necessary to confirm our results.

Effects of vegetative propagule pressure on the establishment of an introduced clonal plant, Hydrocotyle vulgaris.

Some introduced clonal plants spread mainly by vegetative (clonal) propagules due to the absence of sexual reproduction in the introduced range. Propagule pressure (i.e. total number of propagules) may affect the establishment and thus invasion success of introduced clonal plants, and such effects may also depend on habitat conditions. A greenhouse experiment with an introduced plant, Hydrocotyle vulgaris was conducted to investigate the role of propagule pressure on its invasion process. High (five ramets) or low (one ramet) propagule pressure was established either in bare soil or in an experimental plant community consisting of four grassland species. H. vulgaris produced more total biomass under high than under low propagule pressure in both habitat conditions. Interestingly, the size of the H. vulgaris individuals was smaller under high than under low propagule pressure in bare soil, whereas it did not differ between the two propagule pressure treatments in the grassland community. The results indicated that high propagule pressure can ensure the successful invasion in either the grass community or bare soil, and the shift in the intraspecific interaction of H. vulgaris from competition in the bare soil to facilitation in the grassland community may be a potential mechanism.

[DNA methylation defects in sporadic and hereditary colorectal cancer]. / Defectos de la metilación del ADN en el cáncer colorrectal esporádico y hereditario.

DNA methylation is a fundamental epigenetic mechanism in regulating the expression of genes controlling crucial cell functions in cancer development. Methylation defects (both global hypomethylation and hypermethylation of CpG islands) are implicated in colorectal carcinogenesis. Some nutrients have a clear effect on methylation, suggesting that some dietary-associated differences in the incidence of colorectal cancer could be due to the effect of diet on methylation. The presence of methylation defects has clear diagnostic and prognostic implications. Thus, several tests are being used for colorectal cancer screening based on methylated gene analysis, whether in feces or blood. In addition, the reversibility of methylation processes allows the development of chemotherapies that regulate this process through their antineoplastic activity.