The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

No abstract

We examined the sera of six patients before and after i.v. infusions of autologous chronic lymphocytic leukemia (CLL) cells transduced ex vivo with an adenovirus encoding CD154 (Ad-CD154). Five patients made high-titer antibodies against adenovirus and three made IgG reactive with a leukemia-associated surface antigen, which we identified as ROR1. Anti-ROR1 antibodies were not detected in the sera of untreated patients. We generated anti-ROR1 mAbs and found they reacted specifically with the CLL cells of all patients, but not with nonleukemic leukocytes, a wide variety of normal adult tissues, or blood mononuclear cells, including CD5 ؉ B cells of healthy adults. ROR1 could bind Wnt5a, which induced activation of NF-B when coexpressed with ROR1 in HEK293 cells and enhanced the survival of CLL cells in vitro, an effect that could be neutralized by posttreatment anti-ROR1 antisera. We conclude that patients with CLL can break immune tolerance to ROR1, which is an oncofetal surface antigen and survival-signaling receptor in this neoplastic disease.chronic lymphocytic leukemia ͉ neoplasia P atients with chronic lymphocytic leukemia (CLL) typically develop hypogammmaglobulinemia and progressive immune deficiency, which impairs their immune response to vaccines (1-3). Implicated in the abnormal immune function are immunesuppressive factors (4, 5) and an acquired functional deficiency of the CD40 ligand (CD154) (6). Furthermore, CLL cells are particularly inept at antigen presentation, which appears in part secondary to inadequate leukemia cell expression of immune costimulatory and adhesion molecules (7-9).Activation of CLL cells via CD40 ligation can reverse this immune-suppressive phenotype (7,8,10). Although CLL cells express class I and II major histocompatibility complex antigens, CD54 (ICAM-1), CD27, and CD40, these cells have low-to-absent expression of important immune costimulatory molecules, such as CD80, and cannot stimulate even allogeneic T cells in mixed lymphocyte reactions. However, upon interaction with cells that express CD154, CLL cells are induced to express immune costimulatory molecules, allowing them to become effective antigenpresenting cells (7). Similarly, CLL cells transduced with an adenovirus encoding CD154 (Ad-CD154) can induce CD40 activation of both transduced and nontransduced CLL cells, which then can stimulate autologous, leukemia-reactive T cells both in vitro (8) and in vivo (10-13).Conceivably, Ad-CD154-transduced CLL cells also could induce an antibody response against CLL-associated antigens. To test for this, we examined the sera of six CLL patients before and after five biweekly infusions of autologous, Ad-CD154-transduced CLL cells. ResultsBefore the infusions of autologous, Ad-CD154-transduced CLLcells, the six patients had CLL-associated hypogammaglobulinemia with median serum levels of IgM, IgA, or IgG of 28 Ϯ 21 mg/dl (SD), 53 Ϯ 63 mg/dl, or 600 Ϯ 297 mg/dl, respectively. However, 2 weeks after the final infusion, the median serum levels of IgM and IgG had increased to 77...

No abstract

We examined the immunoglobulin (Ig) heavy chain variable region genes (V H genes) used by leukemia cells of 1220 unrelated patients with chronic lymphocytic leukemia (CLL). We found 1188 (97%) expressed Ig encoded by a single Ig V H subgroup, the most common of which was V H 3 (571 or 48.1%), followed by V H 1 (319 or 26.8%) and V H 4 (241 or 20.2%). Using allele-specific primers, we found 13.8% of all samples (n ‫؍‬ 164) used one major V H 1-69 allele, designated 51p1, 163 of which were not somatically mutated. For these cases, there was marked restriction in the structure of the Ig third complementarity determining regions (CDR3s), which were encoded by a small number of unmutated D and J H gene segments. Strik IntroductionThe mutational status of the immunoglobulin (Ig) genes expressed in B-cell chronic lymphocytic leukemia (CLL) can be used to segregate patients into 2 subsets that have significantly different tendencies for disease progression. Those patients with leukemia cells that express unmutated Ig heavy chain variable region genes (V H genes) have a greater tendency for disease progression and shorter survival than those who have leukemia cells that express Ig V H genes with less than 98% nucleic acid sequence homology with their germ-line counterparts. 1-7 Generally, the Ig V H genes expressed by any leukemia cell population do not display significant intraclonal diversity or tendency to accumulate additional somatic mutations over time. 8 As such, the leukemia cells that express mutated Ig genes apparently do not evolve from cases that originally expressed unmutated Ig genes. Because of this, the presence or absence of Ig somatic mutations was thought by some to reflect 2 subtypes of CLL, each with distinctive cytogenesis. 1,2,9 The subtype that expressed unmutated Ig V H genes was considered to be derived from naive or pre-germinal center B cells, whereas the CLL cells expressing mutated receptors were thought likely to be derived from a post-germinal center or "memory-type" B cells. 10 However, molecular analyses of the Ig V H genes expressed by CLL B cells suggest that neither subtype is derived from a naive, nonselected B cell. CLL B cells express an Ig repertoire that appears more restricted than that of adult blood B cells. Several genes, such as the 51p1 allele of V H 1-69, are expressed at high frequency, 11 are rarely mutated, 12 and constitute a large proportion (eg, Ϸ 20%) of the cases that lack Ig somatic mutation. In addition, prior studies found that 51p1-expressing CLL B cells preferentially use certain diversity (D) and J H gene segments with restricted reading frames (RFs), encoding relatively long third complementarity determining regions (CDR3s) with conserved amino acid motifs. 12 The CDR3s of 51p1-expressing CLL B cells contrasted with those of the Ig heavy chains expressed by CLL B cells that used other Ig V H 1 genes or by nonneoplastic tonsillar or blood B cells that used 51p1. [12][13][14] As the CDR3 is the most variable region of the heavy chain and is directly involved ...

Expression of B cell-activating factor (BAFF), a critical B cell survival factor, is elevated in autoimmune and lymphoproliferative disorders. Mice overproducing BAFF develop systemic lupus erythematosus (SLE)-like disease and exhibit B cell activation of classical and alternative NF-kappaB-signaling pathways. We used a genetic approach and found that both NF-kappaB-signaling pathways contributed to disease development but act through distinct mechanisms. Whereas BAFF enhanced long-term B cell survival primarily through the alternative, but not the classical, NF-kappaB pathway, it promoted immunoglobulin class switching and generation of pathogenic antibodies through the classical pathway. Activation of the alternative NF-kappaB pathway resulted in integrin upregulation, thereby retaining autoreactive B cells in the splenic marginal zone, a compartment that contributes to their survival. Thus, both classical and alternative NF-kappaB signaling are important for development of lupus-like disease associated with BAFF overproduction. The same mechanisms may be involved in the pathogenesis of human SLE.

Metastasis is responsible for 90% of cancer-related deaths. Strategies are needed that can inhibit the capacity of cancer cells to migrate across anatomic barriers and colonize distant organs. Here we show an association between metastasis and expression of a type I receptor-tyrosine-kinase-like orphan receptor, ROR1, which is expressed during embryogenesis and by various cancers, but not by normal post-partum tissues. We found that expression of ROR1 associates with the epithelial-mesenchymal transition (EMT), which occurs during embryogenesis and cancer metastasis. Breast adenocarcinomas expressing high-levels ROR1 were more likely to have gene-expression signatures associated with EMT and had higher rates of relapse and metastasis than breast adenocarcinomas expressing low-levels of ROR1. Suppressing expression of ROR1 in metastasis-prone breast-cancer cell-lines, MDA-MB-231, HS-578T, or BT549, attenuated expression of proteins associated with EMT (e.g. vimentin, SNAIL-1/2, and ZEB1), enhanced expression of E-cadherin, epithelial cytokeratins (e.g. CK-19), and tight-junction proteins (e.g. ZO-1), and impaired their migration/invasion capacity in vitro and the metastatic potential of MDA-MB-231 cells in immune-deficient mice. Conversely, transfection of MCF-7 cells to express ROR1 reduced expression of E-cadherin and CK-19, but enhanced expression of SNAIL-1/2 and vimentin. Treatment of MDA-MB-231 with a mAb specific for ROR1 induced down-modulation of vimentin, and inhibited cancer-cell migration and invasion in vitro and tumor metastasis in vivo. Collectively, this study indicates that ROR1 may regulate EMT and metastasis, and that antibodies targeting ROR1 can inhibit cancer progression and metastasis.

Although initially responsive to chemotherapy, many patients with ovarian cancer subsequently develop relapsed and potentially fatal metastatic disease, which is thought to develop from cancer stem cells (CSCs) that are relatively resistant to conventional therapy. Here, we show that CSCs express a type I receptor tyrosine kinase-like orphan receptor (ROR1), which is expressed during embryogenesis and by many different cancers, but not normal postpartum tissues. Ovarian cancers with high levels of ROR1 had stem cell-like gene-expression signatures. Furthermore, patients with ovarian cancers with high levels of ROR1 had higher rates of relapse and a shorter median survival than patients with ovarian cancers that expressed low-to-negligible amounts of ROR1. We found that ROR1-positive (ROR1 + ) cells isolated from primary tumor-derived xenografts (PDXs) also expressed aldehyde dehydrogenase 1 (ALDH1) and had a greater capacity to form spheroids and to engraft immune-deficient mice than did ROR1-negative (ROR1 Neg ) ovarian cancer cells isolated from the same tumor population. Treatment with UC-961, an anti-ROR1 mAb, or shRNA silencing of ROR1 inhibited expression of the polycomb ring-finger oncogene, Bmi-1, and other genes associated with the epithelial-mesenchymal transition. Moreover, shRNA silencing of ROR1, depletion of ROR1 + cells, or treatment with UC-961 impaired the capacity of ovarian cancer cells to form spheroids or tumor xenografts. More importantly, treatment with anti-ROR1 affected the capacity of the xenograft to reseed a virgin mouse, indicating that targeting ROR1 may affect CSC self-renewal. Collectively, these studies indicate that ovarian CSCs express ROR1, which contributes to their capacity to form tumors, making ROR1 a potential target for the therapy of patients with ovarian cancer.ROR1 | ovarian cancer stem cell | monoclonal antibody | PDX mice model