Cell-Free DNA as a Diagnostic and Prognostic Biomarker in Dogs With Tumors

Cell-free DNA (cfDNA) is derived from apoptosis/necrosis, active cellular secretion, and lysis of circulating cancer cells or micrometastases. In humans, cfDNA is widely used in cancer diagnosis, but veterinary research has yet to be actively conducted to establish it as a cancer biomarker. This retrospective study analyzed cfDNA levels in samples collected from dogs with neoplastic disease (n = 38), clinically ill dogs without neoplasia (n = 47), and healthy dogs (n = 35). cfDNA levels and clinical data were compared among groups, and prognostic analyses were performed within the neoplastic group. Furthermore, continual cfDNA measurements were performed during the chemotherapy of six dogs with lymphoma. Dogs with neoplasia showed significantly higher cfDNA concentrations than dogs without neoplasm, and the cfDNA oncentration in the lymphoid neoplasia group was significantly elevated among all neoplastic groups. Dogs with neoplasia and a plasma cfDNA concentration above 1,247.5 μg/L had shorter survival rates than those with levels below this threshold (26.5 vs. 86.1%, respectively, P < 0.05). In cases with complete remission in response to chemotherapy, the cfDNA concentration was significantly decreased compared with the first visit, whereas the cfDNA concentration was increased in cases with disease progression or death. Interestingly, a significant correlation was found between lymph node diameter and cfDNA concentration in dogs with multicentric lymphoma (R2 = 0.26, P < 0.01). These data suggest that changes in cfDNA concentration could be used as a diagnostic biomarker for canine neoplasia. Furthermore, increased plasma DNA levels might be associated with shorter survival time, and cfDNA concentrations may reflect the response to chemotherapy.

Molecular Diagnosis of Koala Retrovirus (KoRV) in South Australian Koalas (Phascolarctos cinereus)

Koala retrovirus, a recent discovery in Australian koalas, is endogenised in 100% of northern koalas but has lower prevalence in southern populations, with lower proviral and viral loads, and an undetermined level of endogenisation. KoRV has been associated with lymphoid neoplasia, e.g., lymphoma. Recent studies have revealed high complexity in southern koala retroviral infections, with a need to clarify what constitutes positive and negative cases. This study aimed to define KoRV infection status in Mount Lofty Ranges koalas in South Australia using RNA-seq and proviral analysis (n = 216). The basis for positivity of KoRV was deemed the presence of central regions of the KoRV genome (gag 2, pol, env 1, and env 2) and based on this, 41% (89/216) koalas were positive, 57% (124/216) negative, and 2% inconclusive. These genes showed higher expression in lymph node tissue from KoRV positive koalas with lymphoma compared with other KoRV positive koalas, which showed lower, fragmented expression. Terminal regions (LTRs, partial gag, and partial env) were present in SA koalas regardless of KoRV status, with almost all (99.5%, 215/216) koalas positive for gag 1 by proviral PCR. Further investigation is needed to understand the differences in KoRV infection in southern koala populations.

Real Life Use of Bendamustine in Elderly Patients with Lymphoid Neoplasia

Background. Bendamustine is a cytotoxic alkylating drug with a broad range of indications as a single agent or in combination therapy in lymphoid neoplasia patients. However, its tolerability in elderly patients is still debated. Methods: An observational, retrospective study was carried out; patients with chronic lymphocytic leukemia (CLL) or lymphoma, aged ≥ 65 years old, treated with bendamustine-based regimens in first or subsequent lines between 2010 and 2020 were considered eligible. Results: Overall, 179 patients aged ≥ 65 years were enrolled, 53% between 71 and 79 years old. Cumulative Illness Rating Scale (CIRS) comorbidity score was ≥6 in 54% patients. Overall survival (OS) at 12 months was 95% (95% confidence interval [CI]: 90–97%); after a median follow up of 50 months, median OS was 84 months. The overall response rate was 87%, with 56% complete responses; the median time to progression (TTP) was 61 months. The baseline factors affecting OS by multivariable analysis were sex, histological diagnosis, renal function, and planned bendamustine dose, while only type of lymphoma and bendamustine dose impacted on TTP. Main adverse events were neutropenia (grade ≥ 3: 43%) and infections (any grade: 36%), with 17% of patients requiring hospital admission. Conclusions: The responses to bendamustine, as well as survival, are relevant even in advanced age patients, with a manageable incidence of acute toxicity.

NADPH Oxidase 5 upregulation is associated with lymphoma aggressiveness

SUMMARY OBJECTIVES Lymphomas are a heterogeneous set of malignant neoplasias of lymphoid B and NK/T mature and immature cells at various stages of differentiation. Genetic and molecular biology tools are used to appropriately classify the type and prognosis of the lymphomas, which have implications in therapeutic effectiveness. Among them, the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase (NOX5) enzymes have been explored. This study analyzed the expression of NADPH oxidase 5 in lymphoma tissue according to the degree of tumor aggressiveness. METHODS Slides from 64 patients with lymphoma who had paraffin-embedded tissue available were reviewed by two independent, experienced pathologists. They classified tumors according to the WHO classification (2017). NOX5 expression in tissues was assessed by immunohistochemical staining using a tissue microarray. The assay was interpreted using a scoring system of 0, 1, 2, and 3, for cytoplasmic staining of NOX5 corresponding to negative, weak, intermediate, and strong staining, respectively. We compared the expression of NOX5 in patients with aggressive versus non-aggressive lymphomas. RESULTS NOX5 expression was positive in 100% (27/27) of aggressive lymphomas and in 19% (7/37) of non-aggressive ones. The seven patients with positive expression of NOX5 presented intermediate staining (2); strong staining (3) was observed only in tissues of aggressive lymphomas, and negative and weak staining (0 and 1) were observed only in non-aggressive lymphomas. CONCLUSIONS Aggressive lymphomas overexpress NOX5 protein. The higher NOX5 expression in aggressive lymphomas can suggest an involvement of this enzyme on the acquisition of an aggressive phenotype in lymphoid neoplasia.

SHP2 Is Required for BCR-ABL1-Induced Hematologic Neoplasms

BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized treatment of Philadelphia chromosome-positive (Ph+) hematologic neoplasms. Nevertheless, acquired TKI resistance remains a major problem in chronic myeloid leukemia (CML), and TKIs are less effective against Ph+B-cell acute lymphoblastic leukemia (B-ALL). GAB2, a scaffolding adaptor that binds and activates SHP2, is essential for leukemogenesis by BCR-ABL1, and a GAB2 mutant lacking SHP2 binding cannot mediate leukemogenesis. Using a genetic loss-of-function approach and bone marrow transplantation (BMT) models for CML and BCR-ABL1+B-ALL, we show that SHP2 is required for BCR-ABL1-evoked myeloid and lymphoid neoplasia.Ptpn11deletion impairs initiation and maintenance of CML-like myeloproliferative neoplasm, and compromises induction of BCR-ABL1+B-ALL. SHP2, and specifically, its SH2 domains, PTP activity and C-terminal tyrosines, is essential for BCR-ABL1+, but not WT, pre-B cell proliferation. The MEK/ERK pathway is regulated by SHP2 in WT and BCR-ABL1+pre-B cells, but is only required for the proliferation of BCR-ABL1+cells. SHP2 is required for SRC family kinase (SFK) activation only in BCR-ABL1+pre-B cells. RNAseq reveals distinct SHP2-dependent transcriptional programs in BCR-ABL1+and WT pre-B cells. Our results suggest that SHP2, via SFKs and ERK, repressesMXD3/4to facilitate a MYC-dependent proliferation program in BCR-ABL1-transformed pre-B cells.