Marianna Greco

Aging is associated with increased clonogenic potential in rat liver in vivo

Cancer increases with age and often arises from the selective clonal growth of altered cells. Thus, any environment favoring clonal growth per se poses a higher risk for cancer development. Using a genetically tagged animal model, we investigated whether aging is associated with increased clonogenic potential. Groups of 4‐, 12‐, 18‐, and 24‐month‐old Fischer 344 rats were infused (via the portal vein) with 2 × 106 hepatocytes isolated from a normal syngenic 2‐month‐old donor. Animals deficient in dipeptidyl‐peptidase type IV (DPP‐IV–) enzyme were used as recipients, allowing for the histochemical detection of injected DPP‐IV+ cells. Groups of animals were sacrificed at various times thereafter. No growth of DPP‐IV+ transplanted hepatocytes was present after either 2 or 6 months in the liver of rats transplanted at young age, as expected. In striking contrast, significant expansion of donor‐derived cells was seen in animals transplanted at the age of 18 months: clusters comprising 7–10 DPP‐IV+ hepatocytes/cross‐section were present after 2 months and were markedly enlarged after 6 months (mean of 88 ± 35 cells/cluster/cross‐section). These results indicate that the microenvironment of the aged liver supports the clonal expansion of transplanted normal hepatocytes. Such clonogenic environments can foster the selective growth of pre‐existing altered cells, thereby increasing the overall risk for cancer development associated with aging.

Homozygosity for killer immunoglobin-like receptor haplotype A predicts complete molecular response to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients.

Several recent reports suggest a possible role for killer immunoglobulin-like receptors (KIR) in the onset of chronic myeloid leukemia (CML) and response to therapy with tyrosine kinase inhibitors (TKIs). To explore this hypothesis, we studied KIRs and their human leukocyte antigen class I ligands in 59 consecutive patients with chronic-phase CML (mean age, 53 years; range, 23-81 years) and a group of 121 healthy control participants belonging to the same ethnic group as the patients. The 2-year cumulative incidence of complete molecular response, obtained after a median of 27 months (range, 4-52 months), was 51.2%. An increased frequency of the activating receptor KIR2DS1 (pm = 0.05) and a reduced frequency of the KIR-ligand combination KIR2DS2/2DL2 absent/C1 present (pm = 0.001) were significantly associated with CML. Moreover, KIR repertoires in patients appeared to influence response to TKI therapy. Homozygosity for KIR haplotype A (pm = 0.01), a decreased frequency of the inhibitory KIR gene KIR2DL2 (pm = 0.02), and low numbers of inhibitory KIR genes (pm = 0.05) were all significantly associated with achievement of complete molecular remission. These data suggest that a decrease in properly stimulated and activated NK cells might contribute to the occurrence of CML and indicate homozygosity for KIR haplotype A as a promising immunogenetic marker of complete molecular response that could help clinicians decide whether to withdraw treatment in patients with CML.

Killer immunoglobulin-like receptors can predict TKI treatment-free remission in chronic myeloid leukemia patients

Several factors are predictive of treatment-free remission (TFR) in chronic myeloid leukemia (CML), but few data exist on the role of natural killer (NK) cells and their killer-cell immunoglobulin-like receptors (KIRs). KIR and human leukocyte antigen (HLA) genotypes were investigated in 36 CML patients who discontinued tyrosine kinase inhibitor (TKI) treatment after achieving deep molecular response (MR(4.5)). Cumulative TFR was significantly higher in patients homozygous for KIR A haplotype (85.7% vs. 45.5%; p = 0.029). Younger age, Bx haplotype, and the combination KIR3DS1/KIR3DL1 present/HLA-Bw4 present were significantly associated with relapse. KIR genotypes could prove useful in identifying patients that are likely to maintain MR(4.5) after discontinuing TKI treatment.

One-step detection and genotyping of human papillomavirus in cervical samples by reverse hybridization.

This study describes a nonisotopic polymerase chain reaction-reverse hybridization-based method (PCR-RH) for the one-step detection and genotyping of anogenital human papillomavirus (HPV) in a microwell format. HPV DNA was amplified and labeled by PCR using GP5+/GP6+ primers. Labeled amplicons were hybridized to 20 HPV type-specific capture probes anchored to the surface of plastic microwells and detected by an immunoenzymatic assay. Assay sensitivity was <50 pg labeled amplicon, and no cross-reactivity was observed, as determined by hybridizing serial dilutions of labeled PCR products to either matched or mismatched capture probes. The assay was tested on 66 clinical samples (23 specimens with normal histology, I fibropapilloma, 26 cervical intraepithelial neoplasia grade 1 [CIN1], 9 CIN2, and 7 CIN3) and compared with a method based on restriction fragment length polymorphism (RFLP) of PCR products. PCR-RH and PCR-RFLP performed equally well on clinical samples. The overall HPV detection rate was similar: 65.1% (43/66) for PCR-RH and 57.6% (38/66) for PCR-RFLP. HPV DNA was found in all CIN2 and CIN3 samples by both methods; however, PCR-RH detected more positives among normal biopsy samples and CINI cases. Overall, there was good agreement between the two genotyping methods, but RH yielded fewer cases with undetermined HPV genotype.

Liver repopulation by transplanted hepatocytes and risk of hepatocellular carcinoma.

Background. Transplantation of isolated hepatocytes in rats treated with retrorsine (RS) results in massive repopulation of the host liver. In this study, the long-term fate of hepatocytes transplanted into RS-treated recipients was followed for up to two years. Methods. Dipeptidyl-peptidase type IV-deficient (DPPIV−) Fischer 344 rats were given two injections of RS (30 mg/kg), followed by transplantation of 2 million hepatocytes, isolated from a syngenic, DPPIV+ donor. Results. Extensive (91±7%) liver replacement by transplanted hepatocytes was observed in animals sacrificed 18 months posttransplantation. Similar levels of repopulation persisted at two years (87±5%). No evidence of preneoplastic and/or neoplastic evolution of the transplanted cell population was present in the RS-treated and repopulated livers at any time point considered. Furthermore, serum parameters related to hepatocyte function and integrity were in the normal range. In control groups given cell transplantation in the absence of prior treatment with RS, only small clusters of donor-derived, DPPIV+ hepatocytes were discerned. Conclusions. These results indicate that liver repopulation in this model is largely stable, persisting for up to two years and allowing for a normal liver function. In addition, no increased risk of neoplastic transformation appears to be associated with the process of liver repopulation for as long as over two thirds of the life span of the recipient animal.

HLA-G expression and role in advanced-stage classical Hodgkin lymphoma.

Non-classical human leucocyte antigen (HLA)-G class I molecules have an important role in tumor immune escape mechanisms. We investigated HLA-G expression in lymphonode biopsies taken from 8 controls and 20 patients with advanced-stage classical Hodgkin lymphoma (cHL), in relationship to clinical outcomes and the HLA-G 14-basepair (14-bp) deletion-insertion (del-ins) polymorphism. Lymphnode tissue sections were stained using a specific murine monoclonal HLA-G antibody. HLA-G protein expression was higher in cHL patients than controls. In the group of PET-2 positive (positron emission tomography carried out after 2 cycles of standard chemotherapy) patients with a 2-year progression-free survival rate (PFS) of 40%, we observed high HLA-G protein expression within the tumor microenvironment with low expression on Hodgkin and Reed-Sternberg (HRS) cells. Conversely, PET-2 negative patients with a PFS of 86% had higher HLA-G protein expression levels on HRS cells compared to the microenvironment. Lower expression on HRS cells was significantly associated with the HLA-G 14-bp ins/ins genotype. These preliminary data suggest that the immunohistochemical pattern of HLA-G protein expression may represent a useful tool for a tailored therapy in patients with cHL, based on the modulation of HLA-G expression in relation to achievement of negative PET-2.

Immunological deregulation in classic hodgkin lymphoma.

Classic Hodgkin Lymphoma (cHL) has a unique histology since only a few neoplastic cells are surrounded by inflammatory accessory cells that in the last years have emerged as crucial players in sustaining the course of disease. In addition, recent studies suggest that the abnormal activity of these inflammatory cells (such as deregulation in regulatory T cells signaling, expansion of myeloid derived suppressor cells, HLA-G signaling and natural killer cells dysfunction) may have prognostic significance. This review is focused on summarizing recent advanced in immunological defects in cHL with translational implications.Hodgkin’s Disease (HD) has a unique histology since only a few neoplastic cells are surrounded by inflammatory accessory cells that in the last years have emerged as crucial players in sustaining the course of disease. In addition, recent studies suggest that the abnormal activity of these inflammatory cells (such as deregulation in Tregsignaling, expansion of myeloid derived suppressor cells, HLA-G signaling and NK dysfunction) may have prognostic significance. This review is focused on summarizing recent advanced in immunological defects in HD with translational implications.

Standard therapies versus novel therapies in Hodgkin lymphoma.

The prognostic models in Hodgkin lymphoma (HL) such as the International Prognostic Score (IPS), retrospectively constructed in the last twenty years from different cohorts of patients treated with ABVD or ABVD-equivalent regimens have been shown a limited predictive value on treatment outcome when applied to a prospective cohort of patients. In the turn of millennium a new class of prognostic factors has emerged, aimed to test the chemosensitivity to treatment in a single patient-basis, such as the minimal residual disease (MRD) assessment with molecular biology, or interim PET/CT performed early during treatment. The main challenge in the management of both early and advanced-stage HL is to achieve a durable remission or cure while minimizing therapy toxicity. An adaptive therapy strategy based on interim PET results could distinguish high from low-risk patients: the former with a potential benefit from an intensify regimen, the latter in whom treatment could be de-escalated or abbreviated for minimizing long-term adverse effects. Conversely, chemosensitivity evaluation in early-stage HL has been the underpinning of de-escalation trials aimed at assessing the safety and the efficacy of omitting radiotherapy in interim PET-negative patients. Brentuximab Vedotin (BV) is a novel antibody-drug conjugate targeting CD30 linked to a potent synthetic antitubulin chemotherapeutic agent, monomethyl auristatin E (MME). BV showed an impressive activity against refractory/relapsed HL and now is being incorporated in a modified ABVD schedule in first-line treatment of HL, with promising efficacy and a low toxicity profile. This novel therapeutic strategy will tell us if traditional ABVD or BEACOPP chemotherapy could be abandoned for the brand-new targeted therapy. Despite the brilliant results of HL treatment, which proved able to achieve a long-term disease control in 80-90% of the patients, the search of new prognostic has continued over the last two decades and the progress of the knowledge of the pathobiology of HL has led to a discovery of a series of markers related to microenvironment of HL tissue and molecular mechanisms to escape host immune reaction against the tumor. The strong association between the locus A of the human leukocyte antigen (HLA-A) system and EBV-positive HL suggests that both an abnormal antigen presentation and some specific EBV antigen per se could play a pivotal role in the pathogenesis of cHL. In EBV+HL patients, the susceptibility or the resistance to develop HL depends on the HLA allele A-*01 or A-*02, respectively. Tumor escape mechanism to block the immune response of the host against the neoplastic tissue is one of the property of a recently discovered subset of myeloid-derived suppressor cells (MDSC) that induce tumor vasculogenesis and escape from immunity via T cell suppression. Initially, these myeloid cells were identified as granulocytes or monocytes; however, MDSC derive from multipotent progenitor cells that can differentiate in the tissue as monocyte-derived MDSC (Mo-MDSC) with a phenotype CD14+/dull and granulocyte-derived MDSC (G-MDSC) expressing CD15 on cell surface. The latter were shown to produce arginase. Recent investigations suggest that MDSC present in the bone marrow in patients with several solid and hematological cancers in response to chemokine release in the tumor site are correlated with tumor-associated macrophages (TAM). Both TAM and MDSC have been strongly associated with a significantly worse prognosis in HL. As a consequence, several efforts are ongoing to standardize the methods to assess TAM and MDSC and prospective studies are being planned to validate their prognostic role.

Early Complete Molecular Response to First-Line Nilotinib in Two Patients with Chronic Myeloid Leukemia Carrying the p230 Transcript

Chronic myeloid leukemia (CML) with the rare fusion gene e19a2, encoding a p230 protein, has been described in patients with typical or rather aggressive clinical course. Although tyrosine kinase inhibitors (TKIs) induce a substantial cytogenetic and molecular response in all phases of CML, a minority of p230 positive patients have been treated with TKIs. We report two cases of CML patients carrying the p230 transcript, who achieved fast and deep complete molecular response (CMR) after frontline treatment with nilotinib. Our results suggest the use of nilotinib as frontline agent for the treatment of this CML variant.

The hOCT1 and ABCB1 polymorphisms do not influence the pharmacodynamics of nilotinib in chronic myeloid leukemia

First-line nilotinib in chronic myeloid leukemia is more effective than imatinib to achieve early and deep molecular responses, despite poor tolerability or failure observed in one-third of patients. The toxicity and efficacy of tyrosine kinase inhibitors might depend on the activity of transmembrane transporters. However, the impact of transporters genes polymorphisms in nilotinib setting is still debated. We investigated the possible correlation between single nucleotide polymorphisms of hOCT1 (rs683369 [c.480C>G]) and ABCB1 (rs1128503 [c.1236C>T], rs2032582 [c.2677G>T/A], rs1045642 [c.3435C>T]) and nilotinib efficacy and toxicity in a cohort of 78 patients affected by chronic myeloid leukemia in the context of current clinical practice. The early molecular response was achieved by 81% of patients while 64% of them attained deep molecular response (median time, 26 months). The 36-month event-free survival was 86%, whereas 58% of patients experienced toxicities. Interestingly, hOCT1 and ABCB1 polymorphisms alone or in combination did not influence event-free survival or the adverse events rate. Therefore, in contrast to data obtained in patients treated with imatinib, hOCT1 and ABCB1 polymorphisms do not impact on nilotinib efficacy or toxicity. This could be relevant in the choice of the first-line therapy: patients with polymorphisms that negatively condition imatinib efficacy might thus receive nilotinib as first-line therapy.