Cabanela, Miguel E.

Abstract The objective of this study was to investigate the underlying molecular alteration in multiple myeloma at the protein level in order to identify regulators of pathogenesis, discover novel targets of therapy, and compare genetic and proteomic alterations. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between MM and normal plasma cells. The antibody array is a new technique enabling protein differences to be assayed directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto glass slides spotted with 512 different monoclonal antibodies. CD138+purified plasma cells were obtained from cryopreserved bone marrow samples of 12 newly diagnosed patients with MM. The labeling index was high (1% cutoff) in 7 samples and low in the other 5. Control plasma cells were obtained from 9 pooled CD138+ purified normal donor bone marrow plasma cells. Interphase FISH analysis for 17p deletion, 13q deletion, and t(11:14) were performed. To assess differential expression, the mean of the ratios of Cy5/Cy3 for each sample were analyzed using the Clontech software to calculate an internally normalized ratio. The normalized data were analyzed by the Genespring software. Unsupervised clustering identified 4 groups of MM. Changes of protein expression ≥2 fold in 70% of the samples as compared to control were identified. There were 6 proteins differentially expressed between all MM samples and control cells including proteins in the ras signaling pathway (KSR-1), the ubiquitin pathway (Ubc-H6), cyclin-dependent kinases (CDK4), cytokines (IL-6), DNA toposisomerase II, and the rho-interacting serine-threonine kinase CRIK. Proteins differentially expressed in MM groups 1 and 2 compared to normal control included cell cycle regulators (cul-2, MCM6, PCNA, TGFb1), kinases (p70S6K, PKC), and chromatin regulators (Ran, AKAP450, Rad50). Protiens differentially expressed in MM group 3 included cell cycle regulators (CDK2, CLK1, MENA), apoptosis regulators (XIAP, caspase 4, perforin) kinases (IKKa and RAC1 in the Wnt signaling pathway) and P53 regulators, while proteins identified in MM group 4 included NFkB/ubiquitin proteins (IKKa and Ubch6), cell cycle regulators (c-myc, CDK4), p53 pathway proteins (53bp2), ras-signaling proteins (KSR1), and the kinase CRIK. There were no differences in protein expression between the high and low labeling index groups. 13q was identified in 5 (42%), 17 p in 1(8%) and t(11:14) in 1(8%) patients. 80% of the 13 q deletion cases clustered in MM group 1 and 2 patients. Cyclin D-1 was upregulated in 5 (42%) patients including the patient with (11:14) translocation. This is the first proteomic study of patients with MM. The results are consistent with previously identified genetic alterations in MM indicating that this novel technique could be used in identifying molecular changes in MM. It identifies novel proteins dysregulated in MM that differ between the 4 MM groups. These results may be used in the future to individualize therapy based on the proteins dysregulated in each group. For example, IKK inhibitors may be useful in group 3 MM patients, while mTOR inhibitors (upstream of p70S6K) could be used in groups 1 and 2 patients. Future correlations with gene expression arrays and prognosis in a larger cohort of patients is warranted.

Abstract The objective of this study was to analyze molecular changes that occur at the protein level in WM and IgM-MGUS in order to identify proteins dysregulated at disease progression and design new therapies. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between WM, IgM-MGUS and normal lymphoplasmacytic cells as control. The antibody array is a novel technique that assays protein differences directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto a glass slide spotted with 512 different monoclonal antibodies specific for human proteins. CD 19+ and CD138+ purified lymphoplasmacytic cells were obtained from cryopreserved bone marrow samples of 5 newly diagnosed patients with WM and 5 patients with IgM-MGUS. Control plasma cells were obtained from 3 pooled CD19+ and CD138+ purified normal donor bone marrow cells. To assess differential expression, the mean of the ratios of Cy5/Cy3 for each sample were analyzed using the Clontech software to calculate an internally normalized ratio. The normalized data were analyzed by the Genespring software and changes of protein expression ≥2 fold in 60% of the samples as compared to normal control were identified in both WM and MGUS groups. There were 20 proteins upregulated in WM and 21 proteins in MGUS as compared to normal control cells These included proteins from the small GTPases such as the Ras, Rho and Ran families (Rab 4, MONA, RCC1, and CDC42GAP), Kinases/signaling proteins (ROK alpha, p62 dok, JAK, 14-3-3e), cyclin dependent kinase (CDK2), fatty acid synthase (HDAC3), NFkB pathway (IKKalpha, IKBe), and histone deacetylase. The WM and IgM MGUS differed in only 3 proteins using the 2-fold threshold. The WM samples showed overexpression of the heat shock protein Hsp90, the Ras family protein CDC25C, and the chemotaxis protein p43/EMAPII as compared to the MGUS samples. This is the first proteomic study of WM and MGUS patients. It identifies novel proteins dysregulated in WM that may serve in future clinical trials as targets for therapy such as Hsp90, NFkB, HDAC and fatty acid synthase. In addition, it identifies 3 proteins that identify disease progression from MGUS to WM, which could be useful for monitoring MGUS patients and designing targeted therapies to prevent progression to WM.

We surveyed 343 young women with 420 total hip arthroplasties (THAs) regarding pregnancy and childbirth after THA. The mean age at surgery was 35 years (18 to 45). The mean length of follow-up after the initial arthroplasty was 16 years (6 to 27).Of these 343 women, 47 (13.7%) had a successful pregnancy after their primary THA. The first baby after a THA was delivered vaginally in 30 patients and by Caesarean section in 17. Of the 343 patients, 138 underwent a revision. For the entire series, the risk of revision at five years was 5%, at ten years 24%, and at 20 years 50%. After adjusting for age at surgical intervention, the risk of revision was not significantly associated with childbirth. Of the 47 patients who had a successful pregnancy, 28 (60%) noted an increase in pain in the hip during pregnancy and ten of these patients had persistent pain after their pregnancy. Seven patients complained of pain in the groin in the replaced hip after childbirth. At the time of this survey, five of these patients (70%) had had revision THA.Childbirth is not affected by the presence of a THA. Pregnancy after THA is not associated with decreased survival of the prosthesis. Pain in the hip is common during pregnancy in these patients. Pain in the groin which persists after delivery commonly leads to revision of the THA.

An analysis of the cement mantle obtained with the Exeter impaction allografting system at one centre showed that it was either deficient or absent in almost 47% of Gruen zones. We therefore examined the mantle obtained using this system at another hospital and compared the results with those from the CPT and Harris Precoat Systems at other centres. The surgical indications for the procedure and the patient details were broadly similar in all four hospitals. There was some variation in the frequency of use of cortical strut allografts, cerclage wires and wire mesh to supplement the impaction allograft. Analysis of the cement mantles showed that when uncertain Gruen zones were excluded, the incidence of zones with areas of absence or deficiency of the cement was 47% and 50%, respectively, for the two centres using the Exeter system, 21% for the CPT system and 18% for the Harris Precoat system. We measured the difference in size between the proximal allograft impactors and the definitive prosthesis for each system. The Exeter system impactors are shorter than the definitive prosthesis and taper sharply so that the cavity created is inadequate, especially distally. The CPT proximal impactors are considerably longer than the definitive prosthesis and are designed to give a mantle of approximately 2 mm medially and laterally and 1.5 mm anteriorly and posteriorly. The Harris Precoat proximal impactors allow for a mantle with a circumference of 0.75 mm in the smaller sizes and 1 mm in the larger. Many reports link the longevity of a cemented implant to the adequacy of the cement mantle. For this reason, femoral impaction systems require careful design to achieve a cement mantle which is uninterrupted in its length and adequate in its thickness. Our results suggest that some current systems require modification.

BACKGROUND: When surgeons perform total hip arthroplasty for hips with a high dislocation related to developmental dysplasia of the hip, obtaining long-term stable implant fixation and optimizing patient function remain challenges. The purpose of this paper was to evaluate the results of cementless arthroplasty with a simultaneous subtrochanteric shortening osteotomy in a group of patients with Crowe type-IV developmental dysplasia of the hip. METHODS: In a retrospective study, we evaluated the results and complications of twenty-eight consecutive primary cementless total hip arthroplasties in twenty-four patients (twenty women and four men), all of whom had Crowe type-IV developmental dysplasia of the hip. The arthroplasty was performed in combination with a subtrochanteric shortening osteotomy and with placement of the acetabular component at the level of the anatomic hip center. The patients were evaluated at a mean of 4.8 years postoperatively. RESULTS: The mean Harris hip score increased from 43 points preoperatively to 89 points at the time of final follow-up (p < 0.01). Twelve (43%) of the twenty-eight hips had an early or late complication or a reoperation. Two (7%) of the twenty-eight subtrochanteric osteotomies were followed by nonunion. There was one instance of isolated loosening of the femoral stem. One acetabular component loosened, and one acetabular liner disengaged. Four hips dislocated postoperatively. All remaining components were well-fixed at the time of the last radiographic follow-up. No sciatic neurapraxic injuries were identified. CONCLUSIONS: Cementless total hip arthroplasty combined with a subtrochanteric femoral shortening osteotomy in patients with a high hip dislocation secondary to developmental dysplasia was associated with high rates of successful fixation of the implants and healing of the osteotomy site and a mean postoperative Harris hip score of 89 points. The complication rate, however, was substantially higher than that associated with primary total hip arthroplasty in patients with degenerative arthritis. LEVEL OF EVIDENCE: Therapeutic Level IV . See Instructions to Authors for a complete description of levels of evidence. ORIGINAL ABSTRACT CITATION: “Total Hip Arthroplasty with Shortening Subtrochanteric Osteotomy in Crowe Type-IV Developmental Dysplasia” (2009;91:2213-21).

Abstract To better understand the molecular changes that occur in Waldenstrom macroglobulinemia (WM), we employed antibody-based protein microarrays to compare patterns of protein expression between untreated WM and normal bone marrow controls. Protein expression was defined as a &gt;2-fold or 1.3-fold change in at least 67% of the tumor samples. Proteins up-regulated by &gt;2-fold included Ras family proteins, such as Rab-4 and p62DOK, and Rho family proteins, such as CDC42GAP and ROKα. Other proteins up-regulated by &gt;1.3-fold included cyclin-dependent kinases, apoptosis regulators, and histone deacetylases (HDAC). We then compared the samples of patients with symptomatic and asymptomatic WM and showed similar protein expression signatures, indicating that the dysregulation of signaling pathways occurs early in the disease course. Three proteins were different by &gt;2-fold in symptomatic versus asymptomatic, including the heat shock protein HSP90. Elevated protein expression was confirmed by immunohistochemistry and immunoblotting. Functional significance was validated by the induction of apoptosis and inhibition of proliferation using specific HDAC and HSP90 inhibitors. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated in WM, which both enhance our understanding of disease pathogenesis and represent targets of novel therapeutics. [Cancer Res 2007;67(8):3777–8]