We investigated the relevance of p53 deletions to the clinical outcome of patients with multiple myeloma (MM) treated with highdose chemotherapy and autologous stem cell transplantation. Hemizygous p53 gene deletions were detected by fluorescence in situ hybridization in 10 of 105 (9.5%) patients studied. p53 deletions were associated with higher serum calcium (P ‫؍‬ .0062) and creatinine (P ‫؍‬ .013) levels, but there were no association with patient age, gender, ␤ 2 -microglobulin, C-reactive protein, hemoglobin, albumin or bone lytic lesions, or immunoglobulin isotype. There were no associations of p53 deletions with 13q deletions or translocations t(11;14) or t(4;14). Patients with p53 deletions had significantly shorter progression-free (median, 7.9 versus 25.7 months, P ‫؍‬ .0324) and overall survival (median, 14.7 versus 48.1 months, P ‫؍‬ .0008) than patients without a p53 deletion. A multivariate analysis confirmed p53 deletion was an independent prognostic factor predicting shortened progression-free (P ‫؍‬ .0009) or overall survival (P ‫؍‬ . Introductionp53, a tumor suppressor gene, is implicated in the regulation of cell proliferation, differentiation, and apoptosis. 1 Inactivation of p53 inactivation by mutation or allelic loss has been observed in various human neoplasms and associated with tumor progression. 2,3 In multiple myeloma (MM), p53 mutations are rare and may represent late events in myeloma progression. [4][5][6] The frequency of p53 deletions detected by fluorescence in situ hybridization (FISH) is reported to range from 9% to 34% of MMs. [7][8][9][10] The p53 gene deletions are associated with poor survival in patients with MM treated with conventional chemotherapy regimens, 7,8,10 but there is little information on the relevance of p53 deletions to survival of patients with MM treated with autologous stem cell transplantation (ASCT).We recently reported that t(4;14) but not t(11;14) is an adverse prognostic factor in patients with MM undergoing ASCT. 11 The current study extends our experience of cytoplasmic light-chain immunofluorescence combined with FISH (cIg-FISH) to investigate the frequency and prognostic significance of p53 deletions in a single institutional cohort of patients with MM treated with high-dose chemotherapy followed by autologous stem cell support. Study design PatientsBetween January 1998 and December 2001, 128 consecutive patients were diagnosed and treated for MM with high-dose chemotherapy followed by ASCT at the Princess Margaret Hospital/University Health Network. Informed consent was provided by the patients in this study. The clinical and biologic features were previously reported. 11 Karyotypic analysis was not routinely performed during this study period. All patients received 4 to 5 cycles of the VAD regimen (vincristine, Adriamycin [doxorubicin], and dexamethasone) followed by stem cell mobilization with cyclophosphamide 2.5 g/m 2 and granulocyte colony-stimulating factor (G-CSF; 10 g/kg) and 1 course of melphalan 200 mg/m 2 immediately prior to ASCT. ...

Summary Hemizygous TP53 deletion is an adverse risk factor in multiple myeloma (MM) but its relationship with p53 protein expression is unclear. We investigated 105 newly diagnosed myeloma patients and correlated nuclear p53 protein immunoreactivity with TP53 deletion status, myeloma‐associated genetic risk factors and survival. Fluorescence in situ hybridisation (FISH) detected hemizygous TP53 deletions in 13 (12%) patients while immunohistochemistry detected nuclear p53 protein expression in 12 (11%). Ten (77%) of the 13 del(TP53) cases expressed nuclear p53 protein while 10 (83%) of the 12 nuclear p53 immunoreactive cases had hemizygous TP53 deletions. Hemizygous TP53 deletion and p53 protein expression were strongly correlated (P < 0·001). The overall survival of patients with p53 protein expression was significantly shorter than that of patients without p53 expression (P < 0·001). A multivariate analysis including other myeloma‐associated genetic risk factors confirmed p53 expression as an independent risk factor for survival. Our data indicate that nuclear p53 protein expression, detected by a widely available immunohistochemical method, is strongly associated with TP53 deletion and an adverse clinical outcome for MM.

Mutations in the nucleophosmin (NPM1) exon 12 resulting in delocalization of NPM1 into the cytoplasm occur in 50% to 60% of acute myeloid leukemia cases with a normal karyotype (AML-NK). As recent studies suggest such patients have a favorable prognosis and there are discordant reports of the immunohistochemical detection of cytoplasmic NPM1 (NPMc+) for predicting NPM1 gene mutations, we correlated the immunohistochemical detection of NPMc+, NPM1 gene mutations, and prognosis in 57 cases of AML-NK. All 31 NPMc+ cases (54% of total) had NPM1 mutations, but none of the 26 nucleus-restricted (NPMc-) cases (46% of total) had NPM1 mutations (P < .0001). NPM1 mutations were correlated with FLT3-internal tandem duplication (ITD) (P = .0062), absence of CD34 (P = .0001), and absence of CD7 (P = .041). There was a favorable survival outcome in AML-NK cases that were NPM1 mutated and FLT3-ITD nonmutated. Our data confirm that cytoplasmic NPM1 immunoreactivity predicts NPM1 mutations and warrants inclusion in the routine diagnostic and prognostic workup of AML.

BackgroundCKS1B is a member of the highly conserved cyclin kinase subunit 1 (CKS1) family that interacts with cyclin-dependent kinases and plays an important role in cell cycle progression. We and others have shown that CKS1B amplification located on chromosome 1q21 is an adverse prognostic factor in multiple myeloma, but its relationship with CKS1B nuclear protein expression, is unclear. The aim of this study was to correlate nuclear CKS1B protein immunoreactivity, 1q21 amplification status, p27Kip1 expression and survival in patients with newly-diagnosed multiple myeloma. Design and Methods Nuclear expression of CKS1B and p27Kip1 was evaluated by immunohistochemistry in decalcified, paraffin-embedded bone marrow biopsies from 94 patients with newly diagnosed multiple myeloma. Clonal plasma cells of the bone marrow aspirates from the same cohort were examined for CKS1B gene status by interphase cytoplasmic fluorescence in situ hybridization. ResultsFluorescence in situ hybridization detected the 1q21 amplification in 36 (38%) of the 94 patients and immunohistochemistry showed CKS1B protein expression in 37 (39%). Thirty-two (86%) of the 36 amplified (1q21) cases expressed CKS1B and 31 (84%) of the 37 CKS1B immunoreactive cases had amplified 1q21. 1q21 amplification and CKS1B protein expression were strongly correlated (P<0.0001). CKS1B protein expression was inversely correlated with p27 Kip1 immunostaining (P<0.0001) and was associated with a shorter overall survival (median 44.5 versus 89.3 months, P<0.0001). ConclusionsImmunohistochemistry for CKS1B is a simple, rapid method that appears to predict 1q21 amplification and adverse outcome for risk stratification of patients with multiple myeloma.Key words: multiple myeloma, 1q21, cyclin kinase subunit 1B, immunohistochemistry, fluorescence in situ hybridization. Citation: Chang H, Jiang N, Jiang H, Saha MN, Qi C, Xu W, and Reece D. CKS1B nuclear expression is inversely correlated with p27Kip1 expression and is predictive of an adverse survival in patients with multiple myeloma. Haematologica 2010;95(9):1542-1547. doi:10.3324/haematol.2010 This is an open-access paper. CKS1B nuclear expression is inversely correlated with p27

del(17p13)(TP53) seems to be an independent poor prognostic factor in patients with relapsed/refractory multiple myeloma (MM) receiving lenalidomide. However, whether aberrant p53 nuclear expression detected by immunohistochemical analysis can be used as a surrogate marker for del(17p13)(TP53) in prognostic evaluation of lenalidomide-treated relapsed/refractory MM remains unclear. The p53 expression in myeloma cells from 88 patients was evaluated by immunohistochemical analysis, and 17p13(TP53) gene status was examined by fluorescence in situ hybridization (FISH). FISH detected hemizygous del(17p13)(TP53) in 13 (15%), and immunohistochemical analysis detected p53 nuclear expression in 11 cases (13%). del(17p13) (TP53) and p53 expression were strongly correlated (P < .0001). Furthermore, patients with aberrant p53 nuclear expression had significantly shorter progression-free and overall survival than patients without this abnormality. Our results suggest that p53 nuclear expression is associated with adverse outcome in patients with relapsed/refractory MM receiving lenalidomide-based therapy and that p53 immunohistochemical analysis may serve as a simple, rapid method to predict del(17p13)(TP53) in this patient subgroup.

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Previous literature suggests that cytogenetics may be used for risk-adapted therapy in patients with relapsed/refractory multiple myeloma (MM) treated with lenalidomide and dexamethasone. However, the significance of each abnormality is still unclear, and chromosome 1 abnormalities have yet to be studied in this population. We therefore evaluated genetic risk factors including chromosome 1q gain and 1p loss by cIg-FISH in 143 patients with relapsed/refractory MM treated with lenalidomide and dexamethasone, and correlated the genomic aberrations with patient clinical outcomes. Patients had a median of two (range 1-7) previous therapies in this cohort. A total of 119 out of 143 (83%) patients had an objective response, with median time to progression (TTP) and overall survival (OS) of 11 and 28 months, respectively. Patients with del(1p21) or del(17p) (p53) deletions had a significantly shorter TTP. OS was shorter in patients with 1p21 or 17p deletions, but did not reach statistical significance. Prior bortezomib or thalidomide treatment was associated with shorter TTP and OS. Multivariate analysis identified del(17p), del(1p21), and prior bortezomib or thalidomide therapy as independent risk factors for shorter TTP. Our data suggest that chromosome 17p and 1p21 deletions adversely impact the outcome of lenalidomide and dexamethasone treated patients with relapsed/refractory MM. Improved therapeutic strategies are required for these patients.

Hemizygous TP53 gene deletion is the most important adverse risk factor in chronic lymphocytic leukemia (CLL), but its relationship with p53 protein expression is unclear. We investigated 110 CLL cases and correlated nuclear p53 protein immunoreactivity with TP53 gene deletion status and other CLL-associated genetic risk factors. Fluorescence in situ hybridization detected hemizygous TP53 deletions in 15 cases (13.6%), whereas immunohistochemical analysis detected nuclear p53 protein expression in 14 (12.7%). All cases expressing nuclear p53 protein had hemizygous TP53 deletions. Hemizygous TP53 gene deletion and p53 protein expression were strongly correlated (P < .001). There was no association between p53 expression and del(13q), del(11q) or trisomy 12 in CLL cases. Our data indicate that nuclear p53 protein expression, detected by a widely available immunohistochemical method, is strongly associated with TP53 deletion and that p53 immunohistochemical analysis may be adopted as a rapid, robust diagnostic tool for risk stratification of CLL.