Metronomic Treatment with Low-Dose Trofosfamide Leads to a Long-Term Remission in a Patient with Docetaxel-Refractory Advanced Metastatic Prostate Cancer

The treatment of metastatic prostate cancer patients refractory to androgen withdrawal and docetaxel therapy is currently discouraging and new therapeutic approaches are vastly needed. Here, we report a long-term remission over one year in a 68-year-old patient with metastatic docetaxel-refractory prostate cancer employing low-dose trofosfamide. The patient suffered from distant failure with several bone lesions and lymph node metastases depicted by a (11) C-Choline positron emission tomography/computerized tomography (PET/CT). After initiation of trofosfamide 100 mg taken orally once a day we observed a steadily decreasing PSA value from initial 46.6 down to 2.1 μg/L. The Choline-PET/CT was repeated after 10 months of continuous therapy and demonstrated a partial remission of the bone lesions and a regression of all involved lymph nodes but one. Taken together we found an astonishing and durable activity of the alkylating agent trofosfamide given in a metronomic fashion. We rate the side effects as low and state an excellent therapeutic ratio of this drug in our patient

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with Tcell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed

Adjunctive Volasertib in Patients With Acute Myeloid Leukemia not Eligible for Standard Induction Therapy: A Randomized, Phase 3 Trial

Terapia de inducción estándar; Volasertib adyuvante; Leucemia mieloide agudaStandard Induction Therapy; Adjunctive Volasertib; Acute Myeloid LeukemiaTeràpia d'inducció estàndard; Volasertib adjuvant; Leucèmia mieloide agudaIn this phase 3 trial, older patients with acute myeloid leukemia ineligible for intensive chemotherapy were randomized 2:1 to receive the polo-like kinase inhibitor, volasertib (V; 350 mg intravenous on days 1 and 15 in 4-wk cycles), combined with low-dose cytarabine (LDAC; 20 mg subcutaneous, twice daily, days 1–10; n = 444), or LDAC plus placebo (P; n = 222). Primary endpoint was objective response rate (ORR); key secondary endpoint was overall survival (OS). Primary ORR analysis at recruitment completion included patients randomized ≥5 months beforehand; ORR was 25.2% for V+LDAC and 16.8% for P+LDAC (n = 371; odds ratio 1.66 [95% confidence interval (CI), 0.95–2.89]; P = 0.071). At final analysis (≥574 OS events), median OS was 5.6 months for V+LDAC and 6.5 months for P+LDAC (n = 666; hazard ratio 0.97 [95% CI, 0.8–1.2]; P = 0.757). The most common adverse events (AEs) were infections/infestations (grouped term; V+LDAC, 81.3%; P+LDAC, 63.5%) and febrile neutropenia (V+LDAC, 60.4%; P+LDAC, 29.3%). Fatal AEs occurred in 31.2% with V+LDAC versus 18.0% with P+LDAC, most commonly infections/infestations (V+LDAC, 17.1%; P+LDAC, 6.3%). Lack of OS benefit with V+LDAC versus P+LDAC may reflect increased early mortality with V+LDAC from myelosuppression and infections.This study was funded by Boehringer Ingelheim

Longitudinal single-cell transcriptomics reveals distinct patterns of recurrence in acute myeloid leukemia

Background: Acute myeloid leukemia (AML) is a heterogeneous and aggressive blood cancer that results from diverse genetic aberrations in the hematopoietic stem or progenitor cells (HSPCs) leading to the expansion of blasts in the hematopoietic system. The heterogeneity and evolution of cancer blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. In this study, we investigated the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs at genetic and transcriptional levels, and unveiled the underlying pathways and genes contributing to recurrence. Methods: Whole-exome sequencing was used to detect somatic mutations and large copy number variations (CNVs). Single cell RNA-seq was performed to investigate the clonal heterogeneity between Dx-Re pairs and amongst patients. Results: scRNA-seq analysis revealed extensive expression differences between patients and Dx-Re pairs, even for those with the same -presumed- initiating events. Transcriptional differences between and within patients are associated with clonal composition and evolution, with the most striking differences in patients that gained large-scale copy number variations at relapse. These differences appear to have significant molecular implications, exemplified by a DNMT3A/FLT3-ITD patient where the leukemia switched from an AP-1 regulated clone at Dx to a mTOR signaling driven clone at Re. The two distinct AML1-ETO pairs share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells evolved from the Dx cells. Conclusions: In summary, the single cell RNA data underpinned the tumor heterogeneity not only amongst patient blasts with similar initiating mutations but also between each Dx-Re pair. Our results suggest alternatively and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence

FLT3 mutations in Early T-Cell Precursor ALL characterize a stem cell like leukemia and imply the clinical use of tyrosine kinase inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup

Identification of Driver and Passenger Mutations of FLT3 by High-Throughput DNA Sequence Analysis and Functional Assessment of Candidate Alleles

SummaryMutations in the juxtamembrane and kinase domains of FLT3 are common in AML, but it is not known whether alterations outside these regions contribute to leukemogenesis. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in AML patients without known FLT3 mutations and experimentally tested the consequences of each candidate leukemogenic allele. This approach identified gain-of-function mutations that activated downstream signaling and conferred sensitivity to FLT3 inhibition and alleles that were not associated with kinase activation, including mutations in the catalytic domain. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation and underscore the importance of functional studies to distinguish “driver” mutations underlying tumorigenesis from biologically neutral “passenger” alterations