A dualistic model of primary anal canal adenocarcinoma with distinct cellular origins, etiologies, inflammatory microenvironments and mutational signatures: implications for personalised medicine.

BACKGROUND: Primary adenocarcinoma of the anal canal is a rare and aggressive gastrointestinal disease with unclear pathogenesis. Because of its rarity, no clear clinical practice guideline has been defined and a targeted therapeutic armamentarium has yet to be developed. The present article aimed at addressing this information gap by in-depth characterising the anal glandular neoplasms at the histologic, immunologic, genomic and epidemiologic levels. METHODS: In this multi-institutional study, we first examined the histological features displayed by each collected tumour (n = 74) and analysed their etiological relationship with human papillomavirus (HPV) infection. The intratumoural immune cell subsets (CD4, CD8, Foxp3), the expression of immune checkpoints (PD-1, PD-L1), the defect in mismatch repair proteins and the mutation analysis of multiple clinically relevant genes in the gastrointestinal cancer setting were also determined. Finally, the prognostic significance of each clinicopathological variable was assessed. RESULTS: Phenotypic analysis revealed two region-specific subtypes of anal canal adenocarcinoma. The significant differences in the HPV status, density of tumour-infiltrating lymphocytes, expression of immune checkpoints and mutational profile of several targetable genes further supported the separation of these latter neoplasms into two distinct entities. Importantly, anal gland/transitional-type cancers, which poorly respond to standard treatments, displayed less mutations in downstream effectors of the EGFR signalling pathway (i.e., KRAS and NRAS) and demonstrated a significantly higher expression of the immune inhibitory ligand-receptor pair PD-1/PD-L1 compared to their counterparts arising from the colorectal mucosa. CONCLUSIONS: Taken together, the findings reported in the present article reveal, for the first time, that glandular neoplasms of the anal canal arise by HPV-dependent or independent pathways. These etiological differences leads to both individual immune profiles and mutational landscapes that can be targeted for therapeutic benefits.

FDG PET/CT radiomics for predicting the outcome of locally advanced rectal cancer.

PURPOSE: The aim of this study was to investigate the prognostic value of baseline 18F-FDG PET/CT textural analysis in locally-advanced rectal cancer (LARC). METHODS: Eighty-six patients with LARC underwent 18F-FDG PET/CT before treatment. Maximum and mean standard uptake values (SUVmax and SUVmean), metabolic tumoral volume (MTV), total lesion glycolysis (TLG), histogram-intensity features, as well as 11 local and regional textural features, were evaluated. The relationships of clinical, pathological and PET-derived metabolic parameters with disease-specific survival (DSS), disease-free survival (DFS) and overall survival (OS) were assessed by Cox regression analysis. Logistic regression was used to predict the pathological response by the Dworak tumor regression grade (TRG) in the 66 patients treated with neoadjuvant chemoradiotherapy (nCRT). RESULTS: The median follow-up of patients was 41 months. Seventeen patients (19.7%) had recurrent disease and 18 (20.9 %) died, either due to cancer progression (n = 10) or from another cause while in complete remission (n = 8). DSS was 95% at 1 year, 93% at 2 years and 87% at 4 years. Weight loss, surgery and the texture parameter coarseness were significantly associated with DSS in multivariate analyses. DFS was 94 % at 1 year, 86 % at 2 years and 79 % at 4 years. From a multivariate standpoint, tumoral differentiation and the texture parameters homogeneity and coarseness were significantly associated with DFS. OS was 93% at 1 year, 87% at 2 years and 79% after 4 years. cT, surgery, SUVmean, dissimilarity and contrast from the neighborhood intensity-difference matrix (contrastNGTDM) were significantly and independently associated with OS. Finally, RAS-mutational status (KRAS and NRAS mutations) and TLG were significant predictors of pathological response to nCRT (TRG 3-4). CONCLUSION: Textural analysis of baseline 18F-FDG PET/CT provides strong independent predictors of survival in patients with LARC, with better predictive power than intensity- and volume-based parameters. The utility of such features, especially coarseness, should be confirmed by larger clinical studies before considering their potential integration into decisional algorithms aimed at personalized medicine.

Severe Prolonged Cough as Presenting Manifestation of FIP1L1-PDGFRA+ Chronic Eosinophilic Leukaemia: A Widely Ignored Association.

Chronic eosinophilic leukaemia associated with the FIP1L1-PDGFRA fusion gene (F/P+ CEL) is a rare cause of marked persistent hypereosinophilia, arising almost exclusively in male patients. Clinical presentations are heterogeneous with a higher incidence of eosinophil-mediated cardiomyopathy than in other hypereosinophilic syndrome variants. Features of chronic myeloproliferative disease are often present, including splenomegaly and elevated serum vitamin B12 levels. The diagnosis is made by fluorescence in situ hybridization (FISH) showing the deletion of the CHIC2 locus and/or RT-PCR showing the FIP1L1-PDGFRA fusion transcript. Treatment with imatinib mesylate, a tyrosine kinase inhibitor, results in rapid and complete resolution of hypereosinophilia and associated symptoms, except for those related to sub-endocardial fibrosis that may be irreversible. We report the case of a male patient in whom isolated intractable cough remained the only clinical manifestation of F/P+ CEL for 4 years. Furthermore, eosinophil autofluorescence, an as yet unreported artefact in this setting, precluded the detection of the CHIC2 deletion and further delayed diagnosis, underlining that both FISH and RT-PCR should be performed when this disease is suspected.

Duplication of the MYB oncogene in T cell acute lymphoblastic leukemia.

We identified a duplication of the MYB oncogene in 8.4% of individuals with T cell acute lymphoblastic leukemia (T-ALL) and in five T-ALL cell lines. The duplication is associated with a threefold increase in MYB expression, and knockdown of MYB expression initiates T cell differentiation. Our results identify duplication of MYB as an oncogenic event and suggest that MYB could be a therapeutic target in human T-ALL.

Genetic basis of congenital erythrocytosis: mutation update and online databases.

Congenital erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3-bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1, and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive Internet-based database focusing on the registration of clinical history, hematological, biochemical, and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database.

Transient leukemia in a newborn without Down syndrome: case report and review of the literature.

UNLABELLED: Transient neonatal leukemia occurs almost exclusively in Down syndrome babies. We report here the unusual case of a newborn without Down syndrome who presented neonatal transient leukemia and who achieved spontaneously complete remission. Trisomy 21 and GATA1 mutation were both present in leukemic cells. While close follow-up is advised since true leukemia may develop later, the patient is still in remission for 2.5 years. We performed a literature review of 15 other similar cases. CONCLUSION: Our case of transient leukemia without Down syndrome and the literature review highlight the important role of trisomy 21 and GATA1 mutation in the development of transient neonatal leukemia.

In vivo vulnerabilities to GPX4 and HDAC inhibitors in drug-persistent versus drug-resistant BRAFV600E lung adenocarcinoma.

The current targeted therapy for BRAFV600E-mutant lung cancer consists of a dual blockade of RAF/MEK kinases often combining dabrafenib/trametinib (D/T). This regimen extends survival when compared to single-agent treatments, but disease progression is unavoidable. By using whole-genome CRISPR screening and RNA sequencing, we characterize the vulnerabilities of both persister and D/T-resistant cellular models. Oxidative stress together with concomitant induction of antioxidant responses is boosted by D/T treatment. However, the nature of the oxidative damage, the choice of redox detoxification systems, and the resulting therapeutic vulnerabilities display stage-specific differences. Persister cells suffer from lipid peroxidation and are sensitive to ferroptosis upon GPX4 inhibition in vivo. Biomarkers of lipid peroxidation are detected in clinical samples following D/T treatment. Acquired alterations leading to mitogen-activated protein kinase (MAPK) reactivation enhance cystine transport to boost GPX4-independent antioxidant responses. Similarly to BRAFV600E-mutant melanoma, histone deacetylase (HDAC) inhibitors decrease D/T-resistant cell viability and extend therapeutic response in vivo.

Germline heterozygous SH2B3-mutations and (idiopathic) erythrocytosis: Detection of a previously undescribed mutation.

Erythrocytosis or polycythemia refers to a true or apparent increase in hemoglobin or hematocrit. When no etiology of erythrocytosis is identified, people are diagnosed with "idiopathic erythrocytosis" (IE). The identification of new contributing genes has recently improved the diagnostic workup of IE. As such mutations within the SH2B3 gene, which codes for the LNK protein and negatively regulates the JAK-STAT pathway, have been identified in cases diagnosed as IE. This reports describes the presence of a previously undescribed germline SH2B3 variant p.(Thr335ArgfsTer4) within IE and emphasizes the advantages of gene panel sequencing as second step in the diagnostic work-up.

Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine. We observed that human PDAC tumors expressing elevated levels of glycolytic enzymes together with high levels of glyoxalase 1 (GLO1), the major MG-detoxifying enzyme, present with a poor prognosis. Next, we showed that glycolysis and subsequent MG stress are triggered in PDAC cells rendered resistant to gemcitabine when compared with parental cells. In fact, acquired resistance, following short and long-term gemcitabine challenges, correlated with the upregulation of GLUT1, LDHA, GLO1, and the accumulation of MG protein adducts. We showed that MG-mediated activation of heat shock response is, at least in part, the molecular mechanism underlying survival in gemcitabine-treated PDAC cells. This novel adverse effect of gemcitabine, i.e., induction of MG stress and HSR activation, is efficiently reversed using potent MG scavengers such as metformin and aminoguanidine. We propose that the MG blockade could be exploited to resensitize resistant PDAC tumors and to improve patient outcomes using gemcitabine therapy.

Risk Assessment of Infectious Endogenous Banana Streak Viruses in Guadeloupe.

Infectious alleles of endogenous banana streak viruses (eBSVs) are present in the genome of all banana interspecific cultivars, including plantains and cooking types. Activation of these infectious eBSV alleles by biotic and abiotic stresses leads to spontaneous infections by cognate viruses and raises concerns about their ability to promote outbreaks of banana streak viruses under field cultivation conditions. We undertook a comprehensive risk assessment study of infectious eBSV alleles of species BSOLV, BSGFV and BSIMV in banana interspecific cultivars in Guadeloupe, a tropical island of the Caribbean where bananas are grown for export and local markets. We carried out a prevalence survey of BSOLV, BSGFV and BSIMV species in a range of cultivars grown in Guadeloupe. Our results suggest that BSOLV and BSGFV infections arise from the activation of infectious eBSVs rather than vector-borne transmission and point to a correlation between altitude and infection rates in interspecific hybrids with AAB genotypes. We studied the dynamics of activation of infectious eBSOLV and eBSGFV alleles by tissue culture and field cultivation in a range of cultivars. We showed that tissue culture and field cultivation trigger distinct activation pathways, resulting in distinct activation patterns. We also showed that activation decreased over time during cell culture and field cultivation and that BSV infections arising from the activation of infectious eBSV alleles cause symptomless infections in the most cultivated plantain in Guadeloupe, French Clair. Overall, our study shows that the risk of BSV outbreaks resulting from the activation of infectious eBSVs in plantain originating from vegetative multiplication is negligible in Guadeloupe.

EVI1-mediated down regulation of MIR449A is essential for the survival of EVI1 positive leukaemic cells.

Chromosomal rearrangements involving the MECOM (MDS1 and EVI1 complex) locus are recurrent genetic events in myeloid leukaemia and are associated with poor prognosis. In this study, we assessed the role of MECOM locus protein EVI1 in the transcriptional regulation of microRNAs (miRNAs) involved in the leukaemic phenotype. For this, we profiled expression of 366 miRNAs in 38 MECOM-rearranged patient samples, normal bone marrow controls and MECOM (EVI1) knock down/re-expression models. Cross-comparison of these miRNA expression profiling data showed that MECOM rearranged leukaemias are characterized by down regulation of MIR449A. Reconstitution of MIR449A expression in MECOM-rearranged cell line models induced apoptosis resulting in a strong decrease in cell viability. These effects might be mediated in part by MIR449A regulation of NOTCH1 and BCL2, which are shown here to be bona fide MIR449A targets. Finally, we confirmed that MIR449A repression is mediated through direct promoter occupation of the EVI1 transcriptional repressor. In conclusion, this study reveals MIR449A as a crucial direct target of the MECOM locus protein EVI1 involved in the pathogenesis of MECOM-rearranged leukaemias and unravels NOTCH1 and BCL2 as important novel targets of MIR449A. This EVI1-MIR449A-NOTCH1/BCL2 regulatory axis might open new possibilities for the development of therapeutic strategies in this poor prognostic leukaemia subgroup.

Angioimmunoblastic T-Cell Lymphoma and Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Novel Form of Composite Lymphoma Potentially Mimicking Richter Syndrome.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is an indolent small B-cell neoplasm that may transform into a clinically aggressive disease, namely Richter syndrome, usually as diffuse large B-cell lymphoma. Besides, CLL/SLL encompasses an increased risk of developing other secondary cancers, including a variety of T-cell lymphomas, often of the anaplastic large-cell type or with a cytotoxic phenotype. Here, we report a small series of patients with composite lymphomas consisting of CLL/SLL and angioimmunoblastic T-cell lymphoma (AITL), a hitherto unrecognized association. The 3 patients (1 male/2 females, 68 to 83 y) presented with high-grade-type symptoms. One patient was clinically suspicious for Richter syndrome, in the others CLL/SLL and AITL were concomitant de novo diagnoses. CLL/SLL and AITL were admixed in the same lymph nodes (3/3 cases) and in the bone marrow (1/2 cases). In all cases, the AITL comprised prominent clear cells with a strong T follicular helper immunophenotype and similar mutations consisting of TET2 or DNMT3A alterations, IDH2 R172K/M, and RHOA G17V. The 3 patients received chemotherapy. One died of early AITL relapse. The other 2 remained in complete remission of AITL, 1 died with recurrent CLL, and 1 of acute myeloid leukemia. These observations expand the spectrum of T-cell lymphoma entities that occur in association with CLL/SLL, adding AITL to the rare variants of aggressive neoplasms manifesting as Richter syndrome. Given that disturbances of T-cell homeostasis in CLL/SLL affect not only cytotoxic but also helper T-cell subsets, these may contribute to the emergence of neoplasms of T follicular helper derivation.

Methylglyoxal Scavengers Resensitize KRAS-Mutated Colorectal Tumors to Cetuximab.

The use of cetuximab anti-epidermal growth factor receptor (anti-EGFR) antibodies has opened the era of targeted and personalized therapy in colorectal cancer (CRC). Poor response rates have been unequivocally shown in mutant KRAS and are even observed in a majority of wild-type KRAS tumors. Therefore, patient selection based on mutational profiling remains problematic. We previously identified methylglyoxal (MGO), a by-product of glycolysis, as a metabolite promoting tumor growth and metastasis. Mutant KRAS cells under MGO stress show AKT-dependent survival when compared with wild-type KRAS isogenic CRC cells. MGO induces AKT activation through phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin 2 (mTORC2) and Hsp27 regulation. Importantly, the sole induction of MGO stress in sensitive wild-type KRAS cells renders them resistant to cetuximab. MGO scavengers inhibit AKT and resensitize KRAS-mutated CRC cells to cetuximab in vivo. This study establishes a link between MGO and AKT activation and pinpoints this oncometabolite as a potential target to tackle EGFR-targeted therapy resistance in CRC.

A case of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia with a rare FIP1L1 breakpoint.

The idiopathic hypereosinophilic syndrome (HES) has remained for a long time a diagnosis of exclusion. Differential diagnosis between the HES and the related chronic eosinophilic leukemia (CEL) relied on the identification of signs of clonality that allowed, when present, the reclassification of patients as CEL. Recently, a new acquired mutation was described in approximately 50% of the HES/CEL patients: a cryptic deletion on chromosome band 4q12 generating a FIP1L1-PDGFRA fusion gene. According to the World Health Organization classification, this clonal abnormality has been proposed as a new surrogate marker for chronic eosinophilic leukemia diagnosis. Fluorescence in situ hybridization and reverse transcriptase-polymerase chain reaction protocols were developed for an accurate del(4)(q12q12) and FIP1L1-PDGFRA fusion gene detection. Here, we report a patient with a rare FIP1L1 intron 16 breakpoint located outside of the reported FIP1L1 breakpoint region (ie, from FIP1L1 introns 9 to 13). This case illustrates the risk of false-negative results with diagnostic procedures that do not take into account the occurrence of rare FIP1L1 breakpoints. As targeted therapy with tyrosine kinase inhibitors has dramatically changed the prognosis of FIP1L1-PDGFRA (+) CEL, false-negative results could hamper accurate diagnosis and treatment.

Sulfasalazine unveils a contact-independent HSV-TK/ganciclovir gene therapy bystander effect in malignant gliomas.

The efficacy of HSV-TK/ganciclovir-based gene therapy on malignant gliomas largely relies on the amplitude of the bystander effect. In these experiments, the anti-inflammatory drug Sulfasalazine increased the HSV-TK/ganciclovir bystander effect in C6, 9L and LN18 cells but not in U87 glioma cells. Using bi-compartmental culture devices and conditioned medium transfer experiments, we showed that in C6, 9L and LN18 cells but not in U87 cells, Sulfasalazine also unveiled a new, contact-independent mechanism of HSV-TK/ganciclovir bystander effect. Upon treatment with ganciclovir, human LN18-TK but not U87-TK cells synthetized and released TNF-alpha in the culture medium. Sulfasalazine sensitized glioma cells to the toxic effect of TNF-alpha and enhanced its secretion in LN18-TK cells in response to GCV treatment. The caspase-8 inhibitor Z-IETD-FMK and a blocking antibody to TNF-alpha both inhibited the contact-independent bystander effect in LN18 cells. Taken together, these results suggest that TNF-alpha mediates the contact-independent bystander effect in LN18 cells. The treatment with GCV and/or Sulfasalazine of tumor xenografts consisting of a mix of 98% C6 and 2% C6-TK cells shows that Sulfasalazine is also a potent adjunct to the in vivo treatment of gliomas.

Human papillomavirus testing with a liquid-based system: feasibility and comparison with reference diagnoses.

OBJECTIVE: To validate the utilization of cervical specimens collected in the fixative liquid used in the CYTO-screen System (SEROA, Monaco) for oncogenic human papillomavirus (HPV) DNA detection by the Hybrid Capture II technique (HCII) (Digene, Gaithersburg, Maryland, U.S.A) by reference to cytologic and/or histologic results. STUDY DESIGN: A technical feasibility study was conducted on 3 modalities of sample preparation before HCII technique, 1 with a proteinase digestion, I with PBS washing and I using the Digene sample conversion kit recommended for ThinPrep medium preparation (Cytyc Corp., Boxborough, Massachusetts, U.S.A.). The stability of cells after storage at days 28, 60 and 90 was tested on 26 positive samples (13 with high initial indices and 13 with low initial indices). Results of HPV testing were compared to cytologic and histologic results on a sample of 98 smears already identified as high grade squamous intraepithelial lesion (HSIL) (48) or low grade squamous intraepithelial lesion (LSIL) (50). A retrospective analysis was then performed on 995 HPV tests perfornmed routinely in 2003 in terms of comparison with the corresponding cytologic and/or histologic results. RESULTS: The HCII technique after direct treatment by proteinase K appeared to be as effective as the Digene sample conversion kit. By using the first technique, all 26 positive cases remained positive at 60 days, but 4 of 13 (30%) with low indices became negative at 90 days. The sensitivity of HPV testing for detecting biopsy- proven cervical intraepithelial neoplasia (CIN) 2 or worse was 100% in the 50 LSIL and 98% in the 48 HSIL samples. In the retrospective study (n = 995), the cytologic diagnoses of atypical squamous cells of undetermined significance (ASC-US) (n=278), LSIL (n = 137) and HSIL (n = 28) were associated with a positive HPVtest in 44%, 75% and 96% of cases, respectively. On a subsample of 156 patients among 278 with a diagnosis of ASC- US, the sensitivity of HPV testingfor detecting CIN 2 or worse was 88%, specificity 57%, positive predictive value 10% and negative predictive value 99%. Performing HPV testing by the HCII technique for cervical specimens collected in the fixative liquid used in the CYTO-screen System is feasible in the context of an ASC-US cytologic diagnosis.

(18)F-FDG PET/CT imaging in rectal cancer: relationship with the RAS mutational status.

OBJECTIVE: Treating metastatic colorectal cancer with anti-EGFR monoclonal antibodies is recommended only for patients whose tumour does not harbour mutations of KRAS or NRAS. The aim of this study was to investigate the biology of rectal cancers and specifically to evaluate the relationship between fluorine-18 fludeoxyglucose ((18)F-FDG) positron emission tomography (PET) intensity and heterogeneity parameters and their mutational status. METHODS: 151 patients with newly diagnosed rectal cancer were included in this retrospective study. All patients underwent a baseline (18)F-FDG PET/CT within a median time interval of 27 days of tumour tissue sampling, which was performed before any treatment. Standardized uptake values (SUVs), volume-based parameters and texture analysis were studied. We retrospectively performed KRAS genotyping on codons 12, 13, 61, 117 and 146, NRAS genotyping on codons 12, 13 and 61 and BRAF on codon 600. Associations between PET/CT parameters and the mutational status were assessed using univariate and multivariate analysis. RESULTS: 83 (55%) patients had an RAS mutation: 74 KRAS and 9 NRAS, while 68 patients had no mutation (wild-type tumours). No patient had BRAF mutation. First-order features based on intensity histogram analysis were significantly associated with RAS mutations: maximum SUV (SUVmax) (p-value = 0.002), mean SUV (p-value = 0.006), skewness (p-value = 0.049), SUV standard deviation (p-value = 0.001) and SUV coefficient of variation (SUVcov) (p-value = 0.001). Both SUVcov and SUVmax showed an area under the curve of 0.65 with sensitivity of 56% and 69%, respectively, and specificity of 64% and 52%, respectively. None of the volume-based (metabolic tumour volume and total lesion glycolysis), nor local or regional textural features were associated with the presence of RAS mutations. CONCLUSION: Although rectal cancers with KRAS or NRAS mutations display a significantly higher glucose metabolism than wild-type cancers, the accuracy of the currently proposed quantitative metrics extracted from (18)F-FDG PET/CT is not sufficiently high for playing a meaningful clinical role. ADVANCES IN KNOWLEDGE: RAS-mutated rectal cancers have a significantly higher glucose metabolism. However, the accuracy of (18)F-FDG PET/CT quantitative metrics is not as such as the technique could play a clinical role.

Activity of telithromycin against thirteen new isolates of C. burnetii including three resistant to doxycycline.

In this study we have evaluated the in vitro activity of antibiotics against 13 new isolates of Coxiella burnetii using a real-time quantitative PCR assay. MICs against doxycycline ranged from 1 to 8 microg/mL, telithromycin from 0.5 to 2 microg/mL, and all strains had MICs > or = 8 microg/mL for erythromycin. We report that strains resistant to doxycycline exist either in humans or animals.