The survival impact of CKS1B gains or amplification is dependent on the background karyotype and TP53 deletion status in patients with myeloma.

Gains or amplification (amp) of chromosome 1q21/CKS1B are reported to be a high-risk factor in myeloma. In this retrospective study, we analyzed the impact of CKS1B gain/amp on overall survival in the context of other genetic aberrations, such as TP53 deletion, FGFR3-IGH, IGH-MAF, MYEOV/CCND1-IGH, and RB1, as well as karyotype. The cohort included 132 myeloma patients with CKS1B gain/amp detected by fluorescence in-situ hybridization. There were 72 men and 60 women with a median age of 65 years (range 39-88 years). A normal, simple, or complex karyotype was observed in 39.5%, 5.4%, and 55% of patients, respectively. "Double hit," defined as CKS1B gain/amp coexisting with TP53 deletion, or "triple hit," defined as double hit plus t(4;14)FGFR3-IGH or t(14;16)IGH-MAF, were identified in 25 patients (18.9%) and five patients (3.8%), respectively. Double and triple hit were highly associated with a complex karyotype (p = 0.02). Ninety-nine patients (99/128, 77.3%) received stem cell transplantation. The median follow-up time was 48.2 months (range 2-104 months); 68 patients (51.5%) died, with a median overall survival of 58.8 months. Multivariate analysis (Cox model) showed that double hit with TP53 deletion (p = 0.0031), triple hit (p = 0.01), and complex karyotype (p = 0.0009) were each independently associated with poorer overall survival. Stem cell transplantation was associated with better overall survival, mainly in patients with a double or triple hit and complex karyotype (p = 0.003). These findings indicate that the inferior outcome of myeloma patients with CKS1B gain/amp is attributable to the high number of high-risk patients in this group. The prognostic impact of CKS1B gain/amp depends on the background karyotype and TP53 status.

Clinical implications of cytogenetic heterogeneity in multiple myeloma patients with TP53 deletion.

TP53 deletion (ΔTP53) in myeloma is known to be a high-risk finding associated with poorer prognosis. The prognostic impact of underlying cytogenetic heterogeneity in patients with myeloma associated with ΔTP53 is unknown. We studied 90 patients with myeloma associated with ΔTP53 identified by interphase fluorescence in situ hybridization and assessed the impact of karyotype and coexisting alterations of IGH, RB1, and CKS1B. There were 54 men and 36 women with a median age of 59 years (range 38-84); 14 patients had a normal karyotype (NK/ΔTP53), 73 had a complex karyotype (CK/ΔTP53), and 3 had a non-complex abnormal karyotype. Patients with CK/ΔTP53 showed a significantly poorer overall survival compared with patients with NK/ΔTP53 (P=0.0243). Furthermore, in the CK/ΔTP53 group, patients with IGH rearrangement other than t(11;14)(q13;q32)/CCND1-IGH, designated as adverse-IGH, had an even worse outcome (P=0.0045). In contrast, RB1 deletion, CKS1B gain, ploidy, additional chromosome 17 abnormalities, or ΔTP53 clone size did not impact prognosis. Stem cell transplant did not improve overall survival in either the NK/ΔTP53 or CK/ΔTP53 (P=0.8810 and P=0.1006) groups, but tandem stem cell transplant did improve the overall survival of patients with CK/ΔTP53 (P=0.0067). Multivariate analysis confirmed in this cohort that complex karyotype (hazard ratio 1.976, 95% CI 1.022-3.821, P=0.043), adverse-IGH (hazard ratio 3.126, 95% CI 1.192-8.196, P=0.020), and tandem stem cell transplant independently correlate with overall survival (hazard ratio 0.281, 95% CI 0.091-0.866, P=0.027). We conclude that comprehensive genetic assessment adds to TP53 status in the risk stratification of myeloma patients.

Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRISPR genome editing.

BACKGROUND: Sickle cell disease (SCD) is a disorder of red blood cells (RBCs) expressing abnormal hemoglobin-S (HbS) due to genetic inheritance of homologous HbS gene. However, people with the sickle cell trait (SCT) carry a single allele of HbS and do not usually suffer from SCD symptoms, thus providing a rationale to treat SCD. METHODS: To validate gene therapy potential, hematopoietic stem cells were isolated from the SCD patient blood and treated with CRISPR/Cas9 approach. To precisely dissect genome-editing effects, erythroid progenitor cells were cloned from single colonies of CRISPR-treated cells and then expanded for simultaneous gene, protein, and cellular function studies. RESULTS: Genotyping and sequencing analysis revealed that the genome-edited erythroid progenitor colonies were converted to SCT genotype from SCD genotype. HPLC protein assays confirmed reinstallation of normal hemoglobin at a similar level with HbS in the cloned genome-edited erythroid progenitor cells. For cell function evaluation, in vitro RBC differentiation of the cloned erythroid progenitor cells was induced. As expected, cell sickling assays indicated function reinstitution of the genome-edited offspring SCD RBCs, which became more resistant to sickling under hypoxia condition. CONCLUSIONS: This study is an exploration of genome editing of SCD HSPCs.

Additional-structural-chromosomal aberrations are associated with inferior clinical outcome in patients with hyperdiploid multiple myeloma: a single-institution experience.

Multiple myeloma is cytogenetically heterogeneous and a hyperdiploid karyotype is considered currently to have standard risk. In this study, we investigated the clinical impact of additional-structural-chromosomal aberrations assessed by chromosome analysis in 284 patients with a hyperdiploid karyotype that were subdivided into four groups based on the complexity of additional-structural-chromosomal aberrations: group 1, no additional-structural-chromosomal aberrations (n=35); group 2, one additional-structural-chromosomal aberration (n=46); group 3, two additional-structural-chromosomal aberrations (n=39); group 4, ≥three additional-structural-chromosomal aberrations (n=164). Clinicopathological data among these groups showed no differences, except patients in group 1 had higher hemoglobin (P=0.031) and albumin (P=0.045) levels. The median follow-up was 55 months (range, 3-221). The median overall survival of patients in groups 1-4 was negatively correlated with the number of the additional-structural-chromosomal aberrations: 98, 76, 61, and 48 months, respectively (P<0.0001). In group 4, CKS1B gain, RB1, or TP53 deletions had no additional impact on overall survival; however, trisomy 3 or 15 conferred a much better overall survival, and monosomy 13 and 14 predicted a worse outcome. In addition, the overall survival of patients in groups 3 and 4 was similar to a subset of high-risk multiple myeloma cases (n=21) (P=0.387). About 192 (67.6%) patients who received stem cell transplantation did not show improved overall survival compared with non-stem cell transplantation patients (n=92; P=0.142) overall; however, they did show significantly improved overall survival in patients with refractory disease in group 4 (P=0.0084). Multivariate analysis showed that two or more additional-structural-chromosomal aberrations (P<0.0001), stages (P=0.02 and P=0.002) and relapsed disease (P=0.009) negatively impacted the overall survival. We conclude that hyperdiploid karyotypes in multiple myeloma are associated with additional-structural-chromosomal aberrations and a greater number of additional-structural-chromosomal aberrations predicts poorer clinical outcome. A hyperdiploid karyotype with ≥2 additional-structural-chromosomal aberrations at chromosomal level should be considered an independent high-risk factor.

Whole-arm translocation of der(5;17)(p10;q10) with concurrent TP53 mutations in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS): A unique molecular-cytogenetic subgroup.

Der(5;17)(p10;q10) is a recurrent but rare aberration reported in myeloid neoplasms (MNs). We report 48 such patients including 19 acute myeloid leukemia (AML) and 29 myelodysplastic syndrome (MDS), to characterize their clinicopathological features. There were 29 men and 19 women, with a median age of 61 years (range, 18-80). 62.5% patients had therapy-related diseases (t-MNs), 70.8% had multilineage dysplasia and 83.3% showed complex karyotypes. In 39 patients tested, FLT3, NPM1, CEBPA, KIT were all wild type and NRAS, KRAS, IDH1, APC, TET2 mutations were detected in single case(s) respectively. TP53 mutations were identified in 8 of 10 cases (80%) tested. Median disease-free survival (DFS) and overall survival (OS) were 3 and 10 months, respectively and did not differ between AML or MDS cases, or between de novo versus therapy-related cases, or between the groups with or without complex karyotypes. In 19 patients who achieved complete remission after chemotherapy, and in 9 patients who underwent stem cell transplantation, the OS was better (14 and 17.5 months, P = 0.0128 and P = 0.0086, respectively). The der(5;17)(p10;q10) represents a unique molecular-cytogenetic subgroup in t-MNs and, associated with complex karyotypes. TP53 inactivation, resulting from 17p deletion coupled with TP53 mutation, likely contributes to the poor clinical outcome of these patients.

Detection of immune suppressive neutrophils in peripheral blood samples of cancer patients.

Neutrophils provide first-line defense against infections and are potent effectors in innate and adaptive immunity. Recently neutrophils have been shown to play important roles in multiple antitumor reactions. A subset of mature neutrophils in human systemic inflammation has been identified as a unique circulating population of myeloid cells, which is capable of inhibiting T cell responses. These neutrophils show unique immunophenotype (CD11c bright/CD62L dim/CD11b bright/CD16 bright). This study reports detection of mature neutrophils with similar immunophenotype in the peripheral blood samples of cancer patients using flow cytometry analysis. This population of neutrophils is not detected in peripheral blood samples of normal controls. Thus this finding suggests the involvement of mature neutrophils in antitumor immunity.

Smurf2 regulates the senescence response and suppresses tumorigenesis in mice.

The E3 ubiquitin ligase Smurf2 mediates ubiquitination and degradation of several protein targets involved in tumorigenesis and induces senescence in human cells. However, the functional role of Smurf2 in tumorigenesis has not been fully evaluated. In this study, we generated a mouse model of Smurf2 deficiency to characterize the function of this E3 ligase in tumorigenesis. Smurf2 deficiency attenuated p16 expression and impaired the senescence response of primary mouse embryonic fibroblasts. In support of a functional role in controlling cancer, Smurf2 deficiency increased the susceptibility of mice to spontaneous tumorigenesis, most notably B-cell lymphoma. At a premalignant stage of tumorigenesis, we documented a defective senescence response in the spleens of Smurf2-deficient mice, consistent with a mechanistic link between impaired senescence regulation and increased tumorigenesis. Taken together, our findings offer the genetic evidence of an important tumor suppressor function for Smurf2.

Conditional activation of ß-catenin signaling in mice leads to severe defects in intervertebral disc tissue.

OBJECTIVE: The incidence of low back pain is extremely high and is often linked to intervertebral disc (IVD) degeneration. The mechanism of this disease is currently unknown. This study was undertaken to investigate the role of ß-catenin signaling in IVD tissue function. METHODS: ß-catenin protein levels were measured by immunohistochemical analysis of disc samples obtained from patients with disc degeneration and from normal subjects. To generate ß-catenin conditional activation (cAct) mice, Col2a1-CreER(T2) -transgenic mice were bred with ß-catenin(fx(Ex3)/fx(Ex3)) mice. Changes in disc tissue morphology and function were examined by micro-computed tomography, histologic analysis, and real-time polymerase chain reaction assays. RESULTS: ß-catenin protein was up-regulated in disc tissue samples from patients with disc degeneration. To assess the effects of increased ß-catenin levels on disc tissue, we generated ß-catenin cAct mice. Overexpression of ß-catenin in disc cells led to extensive osteophyte formation in 3- and 6-month-old ß-catenin cAct mice, which were associated with significant changes in the cells and extracellular matrix of disc tissue and growth plate. Gene expression analysis demonstrated that activation of ß-catenin enhanced runt-related transcription factor 2-dependent Mmp13 and Adamts5 expression. Moreover, genetic ablation of Mmp13 or Adamts5 on the ß-catenin cAct background, or treatment of ß-catenin cAct mice with a specific matrix metalloproteinase 13 inhibitor, ameliorated the mutant phenotype. CONCLUSION: Our findings indicate that the ß-catenin signaling pathway plays a critical role in disc tissue function.

The oncofetal protein IMP3: a novel molecular marker to predict aggressive meningioma.

CONTEXT: One of the major clinical challenges is to predict recurrence of meningioma. Recently, we have found that IMP3, an oncofetal RNA-binding protein, is a biomarker to predict aggressive tumors. OBJECTIVE: To investigate whether IMP3 can be used as a new biomarker to predict the recurrence and overall survival of patients with meningiomas. DESIGN: One hundred seven patients with primary meningiomas were investigated for expression of IMP3 by immunohistochemistry and whether expression of this molecule correlated with tumor recurrence and overall survival. RESULTS: Tumor recurrence was found in 13 of 107 patients with primary meningioma. Seven of 107 patients (6.5%) with primary meningiomas expressed IMP3. Kaplan-Meier plots and log-rank tests showed that patients with IMP3-positive tumors had a much higher recurrence rate (P = .004) and a poorer overall survival (P < .001) than did patients with IMP3-negative tumors. The 5-year recurrence-free and overall survival rates were 0% and 36% in IMP3-positive patients versus 89% and 94% in IMP3-negative patients, respectively. Multivariable analysis of IMP3 status (positive versus negative) in primary tumors showed a hazard ratio of 17.89 for recurrence (P = .01), which was much higher than hazard ratios associated with all other known risk factors including higher tumor grades and Ki-67 labeling index. CONCLUSIONS: IMP3 is a potential independent prognostic biomarker that can be used at the time of initial diagnosis of meningioma to identify patients who have a high risk of developing a recurrence.

IMP2 expression distinguishes endometrioid from serous endometrial adenocarcinomas.

Among various endometrial adenocarcinomas, endometrioid carcinoma can be very difficult to separate from serous carcinomas. Various biomarkers have been studied with proven value, including p53, Ki-67, and p16. In this study, we present data on another biomarker, IMP2, which we believe is sensitive and specific. Using 320 endometrial biopsy cases, we demonstrate that IMP2 is normally expressed in all proliferative and inactive endometrial glandular cells. The pattern of such expression is unchanged in serous carcinomas. IMP2 expression is, however, lost in all cases of endometrioid carcinomas by at least 25% to >95% of tumor cell populations. Therefore, loss of IMP2 expression can differentiate endometrioid from serous carcinomas. Such finding of IMP2 expression remained the same in mixed endometrioid and serous carcinomas; IMP2 expression is lost in all endometrioid components by at least 25% of tumor cell population, whereas it remained diffuse and strong in all serous components of carcinomas.

High-level CD34 expression on megakaryocytes independently predicts an adverse outcome in patients with myelodysplastic syndromes.

Expression of CD34 on mature-appearing megakaryocytes can be seen in various intrinsic bone marrow (BM) disorders as well as reactive bone marrows. In this study we investigate the clinical significance of CD34+ megakaryocytes in myelodysplastic syndromes (MDSs). Expression of CD34 on megakaryocytes was assessed on BM biopsies obtained from 202 patients with MDS. High-level (≥20%) CD34 expression on megakaryocytes was found in BM of 29 patients (14%). The expression of CD34 on megakaryocytes is correlated with severe cytopenia, higher numbers of myeloblasts, more frequent and higher risk cytogenetic abnormalities, and a shorter overall survival. Multivariate analysis indicated that the expression of CD34 on megakaryocytes could be a strong and an independent poor prognostic factor in MDS, with a hazard ratio of 2.53.

Chronic basophilic leukemia: a rare form of chronic myeloproliferative neoplasm.

Chronic basophilic leukemia is a rare and poorly characterized entity. Only a limited number of cases have been described. Herein, we report a patient who presented with fatigue, weight loss, leukocytosis, persistent prominent basophilia, and mild eosinophilia. The bone marrow showed features characteristic of a myeloproliferative neoplasm with a marked increase in maturing basophils. The basophils exhibited nuclear hypersegmentation, abnormal granulation, and abnormally low CD38 expression. Conventional karyotyping revealed a t(5;12)(q31;p13). ETV6 but not PDGFRB rearrangement was detected by fluorescence in situ hybridization.

Activation of beta-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult beta-catenin conditional activation mice.

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/beta-catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for beta-catenin in this disease has not been reported. Because tissue-specific activation of the beta-catenin gene (targeted by Col2a1-Cre) is embryonic lethal, we specifically activated the beta-catenin gene in articular chondrocytes in adult mice by generating beta-catenin conditional activation (cAct) mice through breeding of beta-catenin(fx(Ex3)/fx(Ex3)) mice with Col2a1-CreER(T2) transgenic mice. Deletion of exon 3 of the beta-catenin gene results in the production of a stabilized fusion beta-catenin protein that is resistant to phosphorylation by GSK-3beta. In this study, tamoxifen was administered to the 3- and 6-mo-old Col2a1-CreER(T2);beta-catenin(fx(Ex3)/wt) mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of beta-catenin protein was detected by immunostaining in articular cartilage tissues of beta-catenin cAct mice. In 5-mo-old beta-catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8-mo-old beta-catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in beta-catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp-9, Mmp-13, Alp, Oc, and colX, was significantly increased (3- to 6-fold) in articular chondrocytes derived from beta-catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6-fold increase) in these cells. In addition, we also observed overexpression of beta-catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of beta-catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA-like phenotype. This study provides direct and definitive evidence about the role of beta-catenin in the development of OA.

Erythroid-predominant myelodysplastic syndromes: enumeration of blasts from nonerythroid rather than total marrow cells provides superior risk stratification.

In the FAB (French-American-British) and WHO (World Heath Organization) classifications, the blasts in erythroleukemia (M6a) are enumerated from the marrow nonerythroid rather than the total-nucleated cells. However, the method for blast calculation in erythroid-predominant myelodysplastic syndrome (erythroblasts>or=50%) is not specified either in the FAB or WHO classifications. We retrieved the files of 74 erythroid-predominant myelodysplastic syndrome patients (17% of all myelodysplastic syndrome) and 192 myelodysplastic syndrome controls (erythroblasts<50%). In erythroid-predominant myelodysplastic syndrome, by enumerating blasts from marrow nonerythroid cells rather than from total nucleated cells, 41 of 74 (55%) cases would be upgraded, either by disease subcategory or International Prognostic Scoring System. Importantly, the patients with <5% blasts demonstrated a superior survival to patients with >or=5% blasts (P=0.002); this distinction was lost when blasts were calculated from total-nucleated cells. Of cases with >or=5% blasts, cytogenetics rather than blast count correlated with survival. We conclude that in erythroid-predominant myelodysplastic syndrome, blast calculation as a proportion of marrow nonerythroid rather than total nucleated cells can better stratify patients into prognostically relevant groups.

A left knee wound complication by non-Hodgkins lymphoma in bilateral total knee arthroplasties.

A 70-year-old woman with a history of bilateral primary knee osteoarthritis presented with a left knee wound complication, a non-Hodgkins lymphoma, after bilateral total knee arthroplasties. After exploring several etiologies, the evidence in this unusual case suggests a coincidental preexisting lymphoma.

Hypocellularity in myelodysplastic syndrome is an independent factor which predicts a favorable outcome.

Hypocellular myelodysplastic syndrome (MDS) represents only a small portion of MDS, of which, the clinical significance has not been well-defined. By using currently accepted age-adjusted criteria to define hypocellularity as <30% in patients <70 years old, and <20% in >70 years old, we identified 163 (15.5%) hypocelluar MDS from 1049 consecutive adult MDS patients over an 11-year period (1995-2006). Compared to normal/hypercellular MDS, hypocellular MDS patients were younger (p<0.01), less anemic (p=0.02), but more neutropenic (p<0.001) and thrombocytopenic (p=0.05), and had a comparable cytogenetic risk group distribution (p=0.09) and international prognostic scores (IPSS, p=0.13). With a median follow-up of 52 months, hypocellular MDS showed a favorable overall survival (56 months versus 28 months, log-rank p<0.0001) over normal/hypocellular MDS, and this survival preference was also demonstrated in all IPSS groups and cytogenetic risk groups, and was independent of all other risk factors (Cox regression test, p=0.01). In conclusion, our study demonstrated that hypocellular MDS has characteristic clinicopathologic features, and bone marrow hypocellularity in MDS is an independent factor which predicts a favorable outcome.

A distinct cause of necrotizing fasciitis: Aeromonas veronii biovar sobria.

BACKGROUND: Aeromonas veronii biovar sobria (veronii var. sobria) has not been implicated in the pathogenesis of necrotizing fasciitis in North America. METHODS: Case report and literature review. RESULTS: An 80-year-old man with diabetes mellitus and acute myeloblastic leukemia developed a rapidly spreading skin and soft tissue infection of the right lower extremity pathognomonic for necrotizing fasciitis with signs of sepsis. Cultures grew Aeromonas veronii biovar sobria. Despite aggressive management, including an urgent right below-knee amputation, this patient died of multiple organ dysfunction syndrome. CONCLUSIONS: A. veronii var. sobria should be recognized as one of the causative organisms for necrotizing fasciitis in immunocompromised patients. Early administration of proper antibiotics and aggressive surgical treatment may reduce morbidity and mortality rates in such infections.