Minimal residual disease-driven treatment intensification with sequential addition of ibrutinib to venetoclax in R/R CLL.

Undetectable measurable residual disease (uMRD) is achievable in patients with chronic lymphocytic leukemia (CLL) with the BCL2-inhibitor venetoclax alone or combined with the Bruton's tyrosine kinase inhibitor ibrutinib. This phase 2, multicenter, MRD-driven study was designed to discontinue treatment upon reaching uMRD4 (<10-4) in patients with relapsed/refractory CLL receiving venetoclax monotherapy or after the addition of ibrutinib. Primary end point of the study was proportion of uMRD4 with venetoclax ± ibrutinib. Secondary end points were overall response rate, partial response, complete response, progression-free survival, duration of response, overall survival, and safety of venetoclax ± ibrutinib. Patients with uMRD4 at Cycle 12 Day 1 discontinued venetoclax. MRD+ patients added ibrutinib and continued both drugs up to Cycle 24 Day 28/uMRD4/progression/toxicity. After Cycle 24 Day 28, MRD+ patients continued ibrutinib. Thirty-eight patients (29% with TP53 aberrations; 79% with unmutated IGHV) started venetoclax. Overall response rate with venetoclax was 36 (95%) of 38 patients (20 complete; 16 partial response). Seventeen patients (45%) with uMRD4 at Cycle 12 Day 1 discontinued venetoclax. Nineteen (55%) MRD+ subjects added ibrutinib. After a median of 7 months (range, 3-10 months) of combined treatment, 16 (84%) of 19 achieved uMRD4, thus stopping both drugs. Two MRD+ patients at Cycle 24 Day 28 continued ibrutinib until progression/toxicity. After a median follow-up of 36.5 months, median progression-free survival was not reached; 10 patients progressed (4 restarted venetoclax, 3 without treatment need, 2 developed Richter transformation, and 1 dropped out). Seven (22%) of 32 patients remain uMRD4 after 3 years of follow-up. Neutropenia was the most frequent grade 3 to 4 adverse event; no grade 5 events occurred on study. This sequential MRD-guided approach led to uMRD4 in 33 (87%) of 38 patients, with venetoclax monotherapy or combined with ibrutinib, delivering treatment combination only in a fraction, and ultimately identifying the few patients benefiting from continuous therapy. This trial was registered at www.clinicaltrials.gov as # NCT04754035.

Relevance of Minimal Residual Disease in the Era of Targeted Agents.

The evaluation of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) has evolved in parallel with the enormous progresses in the therapeutic armamentarium and the application of cutting-edge diagnostic techniques the CLL community witnessed in the past few years. Minimal residual disease is considered an objective measure of disease status defined by the number of residual leukemic cells detected in a sample of peripheral blood and/or bone marrow as proportion of the total white blood cells and defined undetectable if fewer than 1 CLL cell among 10,000 white blood cells (10 or 0.01%) is detected. In this review, we aim at shedding light on how to evaluate MRD, what we already know about MRD from the experience with chemoimmunotherapy, and why MRD evaluation remains still relevant in the era of targeted agents.

The IL-1/IL-1 receptor axis and tumor cell released inflammasome adaptor ASC are key regulators of TSLP secretion by cancer associated fibroblasts in pancreatic cancer.

BACKGROUND: The thymic stromal lymphopoietin (TSLP), a key cytokine for development of Th2 immunity, is produced by cancer associated fibroblasts (CAFs) in pancreatic cancer where predominant tumor infiltrating Th2 over Th1 cells correlates with reduced patients' survival. Which cells and molecules are mostly relevant in driving TSLP secretion by CAFs in pancreatic cancer is not defined. METHODS: We performed in vitro, in vivo and ex-vivo analyses. For in vitro studies we used pancreatic cancer cell lines, primary CAFs cultures, and THP1 cells. TSLP secretion by CAFs was used as a read-out system to identify in vitro relevant tumor-derived inflammatory cytokines and molecules. For in vivo studies human pancreatic cancer cells and CAFs were orthotopically injected in immunodeficient mice. For ex-vivo studies immunohistochemistry was performed to detect ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression in surgical samples. Bioinformatics was applied to interrogate published data sets. RESULTS: We show in vitro that IL-1α and IL-1ß released by pancreatic cancer cells and tumor cell-conditioned macrophages are crucial for TSLP secretion by CAFs. Treatment of immunodeficient mice orthotopically injected with human IL-1 positive pancreatic cancer cells plus CAFs using the IL-1R antagonist anakinra significantly reduced TSLP expression in the tumor. Importantly, we found that pancreatic cancer cells release alarmins, among which ASC, able to induce IL-1ß secretion in macrophages. The relevance of ASC was confirmed ex-vivo by its expression in both tumor cells and tumor associated macrophages in pancreatic cancer surgical samples and survival data analyses showing statistically significant inverse correlation between ASC expression and survival in pancreatic cancer patients. CONCLUSIONS: Our findings indicate that tumor released IL-1α and IL-1ß and ASC are key regulators of TSLP secretion by CAFs and their targeting should ultimately dampen Th2 inflammation and improve overall survival in pancreatic cancer.

Modeling multiple myeloma-bone marrow interactions and response to drugs in a 3D surrogate microenvironment.

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions.

Immunomodulatory Drugs in the Context of Autologous Hematopoietic Stem Cell Transplantation Associate With Reduced Pro-tumor T Cell Subsets in Multiple Myeloma.

Immunomodulatory drugs (IMiDs) are effective therapeutics for multiple myeloma (MM), where in different clinical settings they exert their function both directly on MM cells and indirectly by modulating immune cell subsets, although with not completely defined mechanisms. Here we studied the role of IMiDs in the context of autologous hematopoietic stem cell transplantation on the T cell subset distribution in the bone marrow of newly diagnosed MM patients. We found that after transplantation pro-tumor Th17-Th1 and Th22 cells and their related cytokines were lower in patients treated with IMiDs during induction chemotherapy compared to untreated patients. Of note, lower levels of IL-17, IL-22, and related IL-6, TNF-α, IL-1ß, and IL-23 in the bone marrow sera correlated with treatment with IMiDs and favorable clinical outcome. Collectively, our results suggest a novel anti-inflammatory role for IMiDs in MM.

Basophil Recruitment into Tumor-Draining Lymph Nodes Correlates with Th2 Inflammation and Reduced Survival in Pancreatic Cancer Patients.

In pancreatic ductal adenocarcinomas (PDAC), lymphoid infiltrates, comprised mainly of Th2 cells, predict a poor survival outcome in patients. IL4 signaling has been suggested to stabilize the Th2 phenotype in this setting, but the cellular source of IL4 in PDAC is unclear. Here, we show that basophils expressing IL4 are enriched in tumor-draining lymph nodes (TDLN) of PDAC patients. Basophils present in TDLNs correlated significantly with the Th2/Th1 cell ratio in tumors, where they served as an independent prognostic biomarker of patient survival after surgery. Investigations in mouse models of pancreatic cancer confirmed a functional role for basophils during tumor progression. The recruitment of basophils into TDLN relied partly upon the release of chemokine CCL7/MCP3 by "alternatively activated" monocytes, whereas basophil activation was induced by T-cell-derived IL3. Our results show how basophils recruited and activated in TDLNs under the influence of the tumor microenvironment regulate tumor-promoting Th2 inflammation in PDAC, helping in illuminating a key element of the immune milieu of pancreatic cancer. Cancer Res; 76(7); 1792-803. ©2016 AACR.

Th22 cells increase in poor prognosis multiple myeloma and promote tumor cell growth and survival.

There is increased production of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) of multiple myeloma (MM) patients and these favor Th22 cell differentiation. Here, we found that the frequency of interleukin (IL)-22+IL-17-IL-13+ T cells is significantly increased in peripheral blood (PB) and BM of stage III and relapsed/refractory MM patients compared with healthy donors and patients with asymptomatic or stage I/II disease. Th22 cells cloned from the BM of MM patients were CCR6+CXCR4+CCR4+CCR10- and produced IL-22 and IL-13 but not IL-17. Furthermore, polyfunctional Th22-Th2 and Th22-Th1 clones were identified based on the co-expression of additional chemokine receptors and cytokines (CRTh2 or CXCR3 and IL-5 or interferon gamma [IFNγ], respectively). A fraction of MM cell lines and primary tumors aberrantly expressed the IL-22RA1 and IL-22 induced STAT-3 phosphorylation, cell growth, and resistance to drug-induced cell death in MM cells. IL-13 treatment of normal BM mesenchymal stromal cells (MSCs) induced STAT-6 phosphorylation, adhesion molecule upregulation, and increased IL-6 production and significantly favored MM cell growth compared with untreated BM MSCs. Collectively, our data show that increased frequency of IL-22+IL-17-IL-13+ T cells correlates with poor prognosis in MM through IL-22 and IL-13 protumor activity and suggest that interference with IL-22 and IL-13 signaling pathways could be exploited for therapeutic intervention.

Spontaneous control of HIV-1 viremia in a subject with protective HLA-B plus HLA-C alleles and HLA-C associated single nucleotide polymorphisms.

INTRODUCTION: Understanding the mechanisms by which some individuals are able to naturally control HIV-1 infection is an important goal of AIDS research. We here describe the case of an HIV-1(+) woman, CASE1, who has spontaneously controlled her viremia for the last 14 of her 20 years of infection. METHODS: CASE1 has been clinically monitored since 1993. Detailed immunological, virological and histological analyses were performed on samples obtained between 2009 and 2011. RESULTS: As for other Elite Controllers, CASE1 is characterized by low to undetectable levels of plasma HIV-1 RNA, peripheral blood mononuclear cell (PBMC) associated HIV-1 DNA and reduced in vitro susceptibility of target cells to HIV-1 infection. Furthermore, a slow rate of virus evolution was demonstrated in spite the lack of assumption of any antiretroviral agent. CASE1 failed to transmit HIV-1 to either her sexual male partner or to her child born by vaginal delivery. Normal values and ratios of T and B cells were observed, along with normal histology of the intestinal mucosa. Attempts to isolate HIV-1 from her PBMC and gut-derived cells were unsuccessful, despite expression of normal cell surface levels of CD4, CCRC5 and CXCR4. CASE1 did not produce detectable anti-HIV neutralizing antibodies in her serum or genital mucosal fluid although she displayed potent T cell responses against HIV-1 Gag and Nef. CASE1 also possessed multiple genetic polymorphisms, including HLA alleles (B*14, B*57, C*06 and C*08.02) and HLA-C single nucleotide polymorphisms (SNPs, rs9264942 C/C and rs67384697 del/del), that have been previously individually associated with spontaneous control of plasma viremia, maintenance of high CD4(+) T cell counts and delayed disease progression. CONCLUSIONS: CASE1 has controlled her HIV-1 viremia below the limit of detection in the absence of antiretroviral therapy for more than 14 years and has not shown any sign of immunologic deterioration or disease progression. Co-expression of multiple protective HLA alleles, HLA-C SNPs and strong T cell responses against HIV-1 proteins are the most likely explanation of this very benign case of spontaneous control of HIV-1 disease progression.

A new antigen scanning strategy for monitoring HIV-1 specific T-cell immune responses.

Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensitive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast array of database information. This new system, termed Variable Overlapping Peptide Scanning Design (VOPSD), was used for preparing two peptide sets encompassing the candidate HIV-1 vaccine antigens Tat and Nef. Validation of the VOPSD strategy was obtained by direct comparison with 15mer or 20mer peptide sets in a trial involving six laboratories of the AVIP consortium. Cross-reactive background responses were measured in 80 HIV seronegative donors (HIV-), while sensitivity and magnitude of Tat and Nef-specific T-cell responses were assessed on 90 HIV+ individuals. In HIV-, VOPSD peptides generated background responses comparable with those of the standard sets. In HIV-1+ individuals the VOPSD pools showed a higher sensitivity in detecting individual responses (Tat VOPSD vs. Tat 15mers or 20mers: p≤0.01) as well as in generating stronger responses (Nef VOPSD vs. Nef 20mers: p<0.001) than standard sets, enhancing both CD4 and CD8 T-cell responses. Moreover, this peptide design allowed a marked reduction of the peptides number, representing a powerful tool for investigating novel HIV-1 candidate vaccine antigens in cohorts of HIV-seronegative and seropositive individuals.

Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma are associated with nonprogressive HIV-1 infection.

BACKGROUND: Long-term survival of HIV-1 infected individuals is usually achieved by continuous administration of combination antiretroviral therapy (ART). An exception to this scenario is represented by HIV-1 infected nonprogressors (NP) which maintain relatively high circulating CD4+ T cells without clinical symptoms for several years in the absence of ART. Several lines of evidence indicate an important role of the T-cell response in the modulation of HIV-1 infection during the acute and chronic phase of the disease. RESULTS: We analyzed the functional and the differentiation phenotype of Nef- and Tat-specific CD8+ T cells in a cohort of HIV-1 infected NP in comparison to progressors, ART-treated seropositive individuals and individuals undergoing a single cycle of ART interruption. We observed that a distinctive feature of NP is the presence of Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma. This population was present in 7 out of 11 NP. CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells were not detected in HIV-1 infected individuals under ART or withdrawing from ART and experiencing a rebounding viral replication. In addition, we detected Nef-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells in only 1 out of 10 HIV-1 infected individuals with untreated progressive disease. CONCLUSION: The novel antigen-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cell population represents a new candidate marker of long-term natural control of HIV-1 disease progression and a relevant functional T-cell subset in the evaluation of the immune responses induced by candidate HIV-1 vaccines.

The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT.

BACKGROUND: T-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-gamma-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-gamma production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level. RESULTS: The cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-gamma producing T-cells were also producing MIP-1beta whereas T-cells characterized by the sole production of IFN-gamma were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-gamma+ MIP-1beta+ T-cells was equivalent to the measurement of the total IFN-gamma+ T-cells, we adopted the IFN-gamma+ MIP-1beta+ data analysis system to evaluate IFN-gamma-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-gamma+ MIP-1beta+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay. CONCLUSION: The IFN-gamma+ MIP-1beta+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.

Nef alleles from human immunodeficiency virus type 1-infected long-term-nonprogressor hemophiliacs with or without late disease progression are defective in enhancing virus replication and CD4 down-regulation.

Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.