Luigia Luciano

Cytotoxic T lymphocytes directed to the preferentially expressed antigen of melanoma (PRAME) target chronic myeloid leukemia

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed in many hematologic malignancies, including chronic myeloid leukemia (CML). The sensitivity of CML to donor lymphocyte infusion after allogeneic stem cell transplantation suggests this tumor can be highly susceptible to cellular immunotherapy targeted to tumor associated antigens. We therefore tested whether functional PRAME-specific cytotoxic T lymphocytes (PRAME CTLs) could be generated and expanded from healthy donors and CML patients, or whether the limited immunogenicity of this CTA coupled with tumor-associated anergy would preclude this approach. Using optimized culture conditions and HLA-A*02-restricted PRAME-peptides, we have consistently generated PRAME CTLs from 8/9 healthy donors and 5/6 CML patients. These CTLs released IFNgamma in response to PRAME peptides (between 113 +/- 8 and 795 +/- 23 spot forming cells/10(5) T cells) and lysed PRAME peptide-loaded cells (45 +/- 19% at an effector:target [E:T] ratio of 20:1) in a MHC-restricted fashion. Importantly, these CTLs recognized and had cytotoxic activity against HLA-A*02(+)/PRAME(+) tumor cell lines, and could recognize and respond to primary CML cells. PRAME CTLs were generated almost exclusively from the naive T-cell compartment, and clonal analysis showed these cells could have high alphabetaTCR-peptide avidity. PRAME CTLs or vaccines may thus be of value for patients with CML.

High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies.

SHP-1 expression accounts for resistance to imatinib treatment in Philadelphia chromosome-positive cells derived from patients with chronic myeloid leukemia

We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL-independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P < .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Selective strong synergism of Ruxolitinib and second generation tyrosine kinase inhibitors to overcome bone marrow stroma related drug resistance in chronic myelogenous leukemia.

The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co-cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.

Trisomy 13 in a patient with leukemic progression of myelodysplasia

Four months after the diagnosis of refractory anemia, a 60-year-old patient developed acute leukemia with blast cells that were poorly differentiated by morphology and clearly myeloid by immunophenotyping. Cytogenetic analysis performed at leukemization showed trisomy 13. An extra copy of chromosome 13 has already been reported in a few cases of acute leukemia and myelodysplastic syndrome.

Rotation of nilotinib and imatinib for first-line treatment of chronic phase chronic myeloid leukemia

The introduction of second‐generation tyrosine‐kinase inhibitors (TKIs) has generated a lively debate on the choice of first‐line TKI in chronic phase, chronic myeloid leukemia (CML). Despite the TKIs have different efficacy and toxicity profiles, the planned use of two TKIs has never been investigated. We report on a phase 2 study that was designed to evaluate efficacy and safety of a treatment alternating nilotinib and imatinib, in newly diagnosed BCR‐ABL1 positive, chronic phase, CML patients. One hundred twenty‐three patients were enrolled. Median age was 56 years. The probabilities of achieving a complete cytogenetic response, a major molecular response, and a deep molecular response (MR 4.0) by 2 years were 93%, 87%, and 61%, respectively. The 5‐year overall survival and progression‐free survival were 89%. Response rates and survival are in the range of those reported with nilotinib alone. Moreover, we observed a relatively low rate of cardiovascular adverse events (5%). These data show that the different efficacy and toxicity profiles of TKIs could be favorably exploited by alternating their use. Am. J. Hematol. 91:617–622, 2016.

Systemic dasatinib fails to prevent development of central nervous system progression in a patient with BCR-ABL unmutated Philadelphia chromosome―positive leukemia

To the editor: Porkka et al demonstrated that dasatinib crosses the blood-brain barrier and exerts antitumor activity in a mouse model of intracranial chronic myelogenous leukemia (CML).[1][1] They reported that systemic dasatinib induced tumor cell clearance from cerebrospinal fluid (CSF) in