Flow Cytometry for Detection and Quantification of Micrometastases in Sentinel Lymph Nodes from Patients with Primary Melanoma.

BACKGROUND: Detection of micrometastases in the regional lymph nodes is one of the most important prognostic factors for melanoma patients. Our aim was to evaluate the suitability of flow cytometry for rapid quantification of disseminated melanoma cells in sentinel lymph nodes (SLN). METHODS: 132 SLNs from 104 patients diagnosed with melanoma were analyzed by flow cytometry, utilizing the extracellular marker melanoma-associated chondroitin sulfate proteoglycan, in addition to quantitative immunocytology and conventional histopathology, including immunohistochemistry. For quantification, the number of melanoma-positive cells per million lymph node cells (disseminated cancer cell density, DCCD) detected by flow cytometry was compared to the DCCD obtained by immunocytology. RESULTS: Compared to histopathology and immunocytology, flow cytometry exhibited a sensitivity of 50% and a specificity of 85%. DCCDs of immunocytology and flow cytometry of the 37 immunocytologically positive SLNs showed a positive correlation (Spearman's ρ = 0.7, P < 0.0001). In 10 SLNs from 9 patients with high tumor load, the flow cytometric DCCD was 8-fold higher on average than the immunocytologic DCCD. CONCLUSIONS: Although flow cytometry is not yet suitable for early detection of metastatic melanoma, it promises to become a valuable tool for rapidly quantifying tumor load in high-risk patients.

Reduction of Minimal Residual Disease in Pediatric B-lineage Acute Lymphoblastic Leukemia by an Fc-optimized CD19 Antibody.

Prognosis of primary refractory and relapsed pediatric B-lineage acute lymphoblastic leukemia (ALL) is very poor. Relapse rates significantly correlate with persistent minimal residual disease (MRD). In MRD, favorable effector-target ratios prevail and thus this situation might be optimally suited for immunotherapy with antibodies recruiting immunological effector cells. We here report on the generation, preclinical characterization and first clinical application in B-lineage ALL of an Fc-optimized CD19 antibody. This third-generation antibody (4G7SDIE) mediated enhanced antibody-dependent cellular cytotoxicity (ADCC) against leukemic blasts with effector cells from healthy volunteers and B-lineage ALL patients. The antibody was produced in a university-owned production unit and was applied on a compassionate use basis to 14 pediatric patients with refractory and relapsed B-lineage ALL at the stage of MRD. In 10/14 patients, MRD was reduced by ≥ 1 log or below the patient-individual detection limit, and 5/14 patients have achieved ongoing complete molecular remission with a median leukemia-free survival of 428 days. Two additional patients died in complete molecular remission due to complications not related to antibody therapy. Besides profound in vivo B-cell depletion, side effects were negligible. A clinical phase 1/2 study to further assess the therapeutic activity of 4G7SDIE is in preparation.

Hoechst 33342 Staining Identifies the Progenitor Side Population in NOD.Cg-PrkdcscidIL2rgtmWjl/Sz Mice Harboring Pediatric Leukemias.

BACKGROUND: A sub-population of stem cells termed side population (SP) has a unique ability for the efflux of Hoechst 33342 dye. Recently, it was hypothesized that efflux properties might facilitate the efflux of accumulated chemotherapeutic drugs and as such constitute a tumor escape mechanism. MATERIALS AND METHODS: As SP characterization in leukemia is incomplete, we characterized SP frequencies in 19 children diagnosed with acute B cell precursor leukemia (BCP-ALL), AML and T-ALL and described engraftment properties in immune-compromised NOD.Cg-Prkdc(scid)IL2rg(tmWjl)/Sz (NSG) mice. RESULTS: SP cells are detectable in children and mice irrespective of the origin of the leukemia and flow-cytometric analysis reveals that the SP population is a distinct sub-population. Functionally, the SP size remains stable over serial transplantations indicating that the "stemness" potential of our SP sample cohort was overall low. CONCLUSION: SP cells exist in pediatric leukemia and are maintainable in NSG mice. Thus, our observations may facilitate down-stream characterization of LSCs in future studies.

Rapid generation of NY-ESO-1-specific CD4+ THELPER1 cells for adoptive T-cell therapy.

Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4+, IFNγ-producing THelper type 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complex in vitro protocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4+ TH1 cells in vitro for cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4+ T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8+ T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4+ T cells showed strong specific TH1-responses with IFNγ+, TNFα+, IL-2+ and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4+ TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients.

Both mature KIR+ and immature KIR- NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice.

Therapeutic natural killer (NK)-cell-mediated alloreactivity toward acute myeloid leukemia has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B-cell precursor leukemia (BCP-ALL) appears to be resistant to NK-cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. In this study, we demonstrate in a donor-patient-specific NOD.Cg-Prkdc(scid) IL2rg(tmWjl)/Sz (NSG) xenotransplantation model that NK cells mediate considerable alloreactivity toward pediatric BCP-ALL in vivo. Notably, both adoptively transferred mature KIR(+) NK cells and immature KIR(-) NK cells arising early posttransplantation in humanized NSG mice exerted substantial antileukemic activity. Low-dose and long-term treatment of humanized NSG mice with the DNA-demethylating agent 5-aza-cytidine distinctly enhanced the antitumor response, interestingly without inducing common inhibitory KIR expression but rather by promoting the differentiation of various NK-cell precursor subsets. Collectively, these data indicate that the future design of innovative therapy protocols should consider further exploitation of NK-cell-mediated immune responses for poor prognosis pediatric BCP-ALL patients.

Pediatric posttransplant relapsed/refractory B-precursor acute lymphoblastic leukemia shows durable remission by therapy with the T-cell engaging bispecific antibody blinatumomab.

We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 µg/m(2)/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival.

Therapy response correlates with ALDH activity in ALDH low-positive childhood acute lymphoblastic leukemias.

The malignant cells of childhood acute lymphoblastic leukemia (ALL) do not form a homogenous entity but a collection of differently maturated blasts. The most immature leukemia cells may be more resistant to therapy than the bulk of more differentiated blasts. We studied 42 patients with childhood ALL treated according to the ALL-BFM 2000 protocol. At diagnosis, we determined the immunophenotype and the aldehyde dehydrogenase (ALDH) activity of the leukemic cells. Additionally, we investigated the expression of CD34, CD38 and CD45 to define a population of immunophenotypically immature cells (CD34(+)/CD38(-)/CD45(-/low)). We then studied levels of minimal residual disease (MRD) after induction therapy (day 33) to determine therapy response. Including all cases (n = 42), there was no correlation between ALDH positive cells, CD34(+)/CD38(-)/CD45(-/low) cells and MRD levels. A subset of 18 ALLs displayed a more mature phenotype with low-ALDH positivity (< 1%). Analyzing this cohort, ALDH positive blasts overlapped with the CD34(+)/CD38(-)/CD45(-/low) population. The initial rate of ALDH positivity correlated with MRD levels at day 33 of therapy (r = 0.61, P < .01). We conclude that in pediatric ALL, ALDH positivity as a marker of immaturity and stemness has prognostic significance only in phenotypically mature cases when the ALDH activity is not a property of the majority of the leukemic blasts. In case of an immature ALL phenotype, ALDH activity might be an inherent characteristic of the whole leukemia and is not limited to a more immature subpopulation that could confer to resistance and increased MRD-levels during therapy.

High proportion of leukemic stem cells at diagnosis is correlated with unfavorable prognosis in childhood acute myeloid leukemia.

In acute myeloid leukemia (AML), the leukemia-initiating cell is found within the CD34(+)/CD38(-) cell compartment. Over the last years evidence grew that AML is initiated and propagated by leukemic stem cells (LSCs). Conceivably, these most immature leukemia cells are more resistant to therapy and subsequently initiate relapse. The authors studied 17 patients with childhood AML treated according to the AML-BFM 98/04 protocol. At diagnosis, the authors determined the characteristic immunophenotype of the leukemic cells by flow cytometry and investigated the expression of CD34, CD38, and CD45 to define a population of immunophenotypically immature cells (CD34(+)/CD38(-)/CD45(-/low)) enriched for LSCs in many cases of AML. The authors compared the fraction of this population of all myeloid cells at diagnosis with event-free survival. Kaplan-Meier analysis revealed significant higher event free survival of patients with low CD34(+)/CD38(-)/CD45(-/low) cell proportion (<0.68%) compared to patients with high burden of this population (>0.83%; log-rank P < .04). This correlation was not found for the total number of CD34(+) cells. This is the first study to show that a higher proportion of immature CD34(+)/CD38(-)/CD45(-/low) blasts at diagnosis correlates with unfavorable prognosis in childhood AML. The results suggest that a large CD34(+)/CD38(-)/CD45(-/low) population reflects a higher fraction of LSCs, leading to increased chemotherapy resistance and elevated relapse rate. Thus the initial frequency of CD34(+)/CD38(-)/CD45(-/low) cells may serve as a prognostic marker in pediatric AML. Future treatment in childhood AML should specifically target this immature population as well as the mature blast population.

High frequency of immature cells at diagnosis predicts high minimal residual disease level in childhood acute lymphoblastic leukemia.

Prognosis for children with acute lymphoblastic leukemia (ALL) has considerably improved, yet relapse still occurs in a significant proportion of patients. Conceivably, the most immature leukemia cells may be more resistant to therapy and initiate relapse. We studied 42 patients with childhood ALL treated according to the ALL-BFM 2000 protocol. At diagnosis, we determined the characteristic immunophenotype of the leukemic cells by flow cytometry and also investigated the expression of CD34 and CD38 to define a population of immunophenotypically immature cells (CD34(+)/CD38(-)). We then studied levels of minimal residual disease (MRD) after induction therapy (day 33) and after consolidation therapy (week 12). We found a significant, increasing correlation between the prevalence of CD34(+)/CD38(-) cells at diagnosis and MRD levels at day 33 and week 12. Our results suggest that the initial frequency of CD34(+)/CD38(-) cells may serve as a prognostic marker in pediatric ALL.

Paedocypris, a new genus of Southeast Asian cyprinid fish with a remarkable sexual dimorphism, comprises the world's smallest vertebrate.

Paedocypris is a new genus of paedomorphic cyprinid fish from highly acidic blackwater peat swamps in Southeast Asia. It includes two new species, one of which (Paedocypris progenetica) appears to be the smallest fish and vertebrate known, with the smallest mature female measuring a mere 7.9 mm. Paedocypris has many 'larval' features typically associated with paedomorphic fish (e.g. narrow frontals that leave the brain unprotected dorsally by bone and a precaudal larval-fin-fold), but, uniquely among fishes, males also possess highly modified pelvic fins with hypertrophied muscles and a keratinized pad in front of the pelvic girdle, which, we hypothesize, function together as a clasping or holding device, thereby suggesting an unusual reproductive mode. Unfortunately, habitat destruction jeopardizes the survival of these fishes and thus opportunities for further research.