Saverio Minucci, Giancarlo Pruneri, Luca Magnani and colleagues identify acquired CYP19A1 amplification as a mechanism of resistance to aromatase inhibitors in ERα metastatic breast cancer. Mechanistically, they show that CYP19A1 amplification results in increased aromatase activity and estrogen-independent ERα binding to target genes. Tumor evolution is shaped by many variables, potentially involving external selective pressures induced by therapies1. After surgery, patients with estrogen receptor (ERα)-positive breast cancer are treated with adjuvant endocrine therapy2, including selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs)3. However, more than 20% of patients relapse within 10 years and eventually progress to incurable metastatic disease4. Here we demonstrate that the choice of therapy has a fundamental influence on the genetic landscape of relapsed diseases. We found that 21.5% of AI-treated, relapsed patients had acquired CYP19A1 (encoding aromatase) amplification (CYP19A1amp). Relapsed patients also developed numerous mutations targeting key breast cancer–associated genes, including ESR1 and CYP19A1. Notably, CYP19A1amp cells also emerged in vitro, but only in AI-resistant models. CYP19A1 amplification caused increased aromatase activity and estrogen-independent ERα binding to target genes, resulting in CYP19A1amp cells showing decreased sensitivity to AI treatment. These data suggest that AI treatment itself selects for acquired CYP19A1amp and promotes local autocrine estrogen signaling in AI-resistant metastatic patients.

Therapy-related Myeloid Neoplasm (t-MN) represents one of the worst long-term consequences of cytotoxic therapy for primary tumors and autoimmune disease. Poor survival and refractoriness to current treatment strategies characterize affected patients from a clinical point of view. In our aging societies, where newer therapies and ameliorated cancer management protocols are improving the life expectancy of cancer patients, therapy-related Myeloid Neoplasms are an emerging problem. Although several research groups have contributed to characterizing the main risk factors in t-MN development, the multiplicity of primary tumors, in association with the different therapeutic strategies available and the new drugs in development, make interpreting the current data still complex. The main risk factors involved in t-MN pathogenesis can be subgrouped into patient-specific, inherited, and acquired predispositions. Although t-MN can occur at any age, the risk tends to increase with advancing age, and older patients, characterized by a higher number of comorbidities, are more likely to develop the disease. Thanks to the availability of deep sequencing techniques, germline variants have been reported in 15-20% of t-MN patients, highlighting their role in cancer predisposition. It is becoming increasingly evident that t-MN with driver gene mutations may arise in the background of Clonal Hematopoiesis of Indeterminate Potential (CHIP) under the positive selective pressure of chemo and/or radiation therapies. Although CHIP is generally considered benign, it has been associated with an increased risk of t-MN. In this context, the phenomenon of clonal evolution may be described as a dynamic process of expansion of preexisting clones, with or without acquisition of additional genetic alterations, that, by favoring the proliferation of more aggressive and/or resistant clones, may play a crucial role in the progression from preleukemic states to t-MN.

The present study describes a series of primary soft tissue lymphomas, including immunohistochemical characterization by tissue microarray and cytogenetic profiling. Formalin-fixed, paraffin-embedded tissue samples were collected from patients who underwent soft tissue biopsy. Cases were selected according to the definition of primary soft tissue lymphoma as a lymphoid malignancy arising in soft tissues without evidence of other nodal or extranodal localization for a period of at least 6 months. Our series comprised seven patients with a mean age of 72 years. There were three diffuse large B-cell lymphomas (DLBCLs); one B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma; one DLBCL derived from follicular lymphoma; one ALK-negative anaplastic large cell lymphoma; and one follicular lymphoma. Immunohistochemical and molecular profiles were consistent with the histological diagnoses. The present study contributes to our knowledge about uncommon presentation of lymphoid neoplasms and confirms previously published clinical-pathological data. We present, for the first time, the complete immunohistochemical profile and molecular cytogenetic studies of these lymphoid neoplasms. A rare case of a primary soft tissue ALK-negative anaplastic large cell lymphoma is described in detail.

Fludarabine, cyclophosphamide, and rituximab (FCR) has represented a significant treatment advancement in chronic lymphocytic leukemia (CLL). In the new scenario of targeted agents, there is an increasing interest in identifying patients who gain the maximum benefit from FCR. In this observational multicenter retrospective analysis of 404 CLL patients receiving frontline FCR, the combination of three biomarkers that are widely tested before treatment (IGHV mutation status, 11q deletion and 17p deletion; available in 80% of the study cohort) allowed to identify a very low-risk category of patients carrying mutated IGHV genes but neither 11q or 17p deletion that accounted for 28% of all cases. The majority of very low-risk patients (71%) remained free of progression after treatment and their hazard of relapse decreased after 4 years from FCR. The life expectancy of very low-risk patients (91% at 5 years) was superimposable to that observed in the matched normal general population, indicating that neither the disease nor complications of its treatment affected survival in this favorable CLL group. These findings need a prospective validation and may be helpful for the design of clinical trials aimed at comparing FCR to new targeted treatments of CLL, and, possibly, for optimized disease management.

We read with interest the study by Rossi and coworkers, reporting CD49d expression as risk factor of treatment free survival (TFS) in Binet A CLL patients.[1][1] In this paper, a close association between CD49d and CD38, LDH and β2-m is also described. We would like to add further information about

Modulating protein ubiquitination via proteasome inhibition represents a promising target for cancer therapy, because of the higher sensitivity of cancer cells to the cytotoxic effects of proteasome inhibition. Here we show that CEP-18770 is a novel orally-active inhibitor of the chymotrypsin-like activity of the proteasome that down-modulates the nuclear factor-kappaB (NF-kappaB) activity and the expression of several NF-kappaB downstream effectors. CEP-18770 induces apoptotic cell death in multiple myeloma (MM) cell lines and in primary purified CD138-positive explant cultures from untreated and bortezomib-treated MM patients. In vitro, CEP-18770 has a strong antiangiogenic activity and potently represses RANKL-induced osteoclastogenesis. Importantly, CEP-18770 exhibits a favorable cytotoxicity profile toward normal human epithelial cells, bone marrow progenitors, and bone marrow-derived stromal cells. Intravenous and oral administration of CEP-18770 resulted in a more sustained pharmacodynamic inhibition of proteasome activity in tumors relative to normal tissues, complete tumor regression of MM xenografts and improved overall median survival in a systemic model of human MM. Collectively, these findings provide evidence for the utility of CEP-18770 as a novel orally active proteasome inhibitor with a favorable tumor selectivity profile for the treatment of MM and other malignancies responsive to proteasome inhibition.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to aryl hydrocarbon receptor (AhR), a member of the erb-A family, allowing, after DNA binding, gene expression regulation. TCDD has a large number of biological effects, as skin and cardiovascular disease, diabetes and cancer. An increase in myeloid leukemia, Hodgkin's and non-Hodgkin's lymphomas, was observed after 15 years in the population exposed to TCDD after the 1976 accident in Seveso, Italy. In the present study, we analyzed the in vitro effect of TCDD exposure on human CD34+ progenitor cells from G-CSF stimulated leukapheresis of 4 normal donors. Gene expression modulation induced by TCDD was analysed on highly purified (>96%) CD34+ cells, after exposure to 10 nM of TCDD for 12 hrs. Gene expression profiles have been generated by high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed with a supervised approach (DNA-Chip Analyzer, dChip 2006). The differential expression of 257 transcripts (150 up regulated and 106 down-regulated) distinguished the 4 TCDD treated from the 4 untreated control samples. Interestingly, a number of the differentially expressed genes were involved in skin and cardiovascular diseases, diabetes, and different cancers, all of which have been associated with TCDD exposure. Among skin diseases, defects in laminin beta 3, ALOX12B and keratin 2a, all downregulated in TCDD exposed CD34+ cells, are associated with development of epidermolysis bullosa, erythroderma ichthyosiform, and Siemens ichthyosis bullosa, respectively. As far as diabetes and cardiovascular disease pathogenesis are concerned, defects of SLC2A4 gene, dowregulated by TCDD, is associated with the development of non insulin dependent diabetes, while epoxide hydrolase 2 and EGR2 are associated with cardiovascular disease. Among cancer related genes, ARHGAP26 and ABL2 are associated with juvenile myelomonocytic leukemia and acute myeloid leukemia with eosinophilia, respectively. Nevertheless, the expression of numerous other genes potentially involved in hemopoiesis/leukemogenesis, was modulated by TCDD exposure. Among these examples are c-kit ligand, LIF receptor, pre B lymphocyte gene 1, piwi-like 2, FLT3 ligand, chemokine ligands 14 and 15, and cdk2, that were all up regulated, while MLL4, wnt inhibitory factor 1, chemokine ligand 7, and lymphoid blast crisis oncogene, were down regulated. In conclusion, the gene expression pattern induced by TCDD exposure on the CD34+ normal progenitor cells is consistent with the spectrum of TCDD induced toxicities/diseases. In particular, it provides the basis for a possible role of TCDD in the neoplastic transformation of hemopoietic stem cells and support the epidemiologic data of increased hematologic cancer risk in the population exposed accidentally to the substance.

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 ( FGFR3 ) and the putative transcription factor WHSC1/MMSET . Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the effects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at different levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14).

Molecular genetic (genotypic) analysis elucidates gene rearrangements within lymphocytes that are responsible for either immunoglobulin production in B-lymphocytes or the expression of cell-surface antigen recognition receptors in T-lymphocytes. Molecular genetic analysis is far more sensitive than immunophenotypic methods for the detection of small clones of lymphocytes because as few as 2 to 5% of cells in an infiltrate can be discovered to possess the same rearranged DNA sequences with genetic probes. In truly polyclonal proliferations, each lymphocyte reorganizes its immunoglobulin or T-antigen receptor genes in a unique manner, resulting in an almost infinite number of combinations of genetic rearrangement and the absence of any new hybridizing bands upon Southern blotting. In monoclonal proliferations, a new, homogeneous, nongermline band is identified on Southern blotting because a sufficiently large number of lymphocytes exhibit an identical genetic rearrangement. In a group of five orbital lymphoid tumors that appeared to be benign reactive hyperplasias by light microscopy and that were polyclonal by immunophenotypic methods, three were found by molecular genetic analysis to harbor small clones of B-lymphocytes with new rearrangement bands on Southern blotting. No clonal abnormalities of T-lymphocytes were found in these five lesions, despite the fact that they were the preponderant cells in the tumors. These observations suggest that "reactive lymphoid hyperplasia" of the orbit may be an unstable lesion, owing to a T-cell immunoregulatory imbalance, with the potential for developing clonal expansions of B-lymphocytes that nonetheless usually remain localized to the orbit.