Aberrant gene methylation implicated in the progression of monoclonal gammopathy of undetermined significance to multiple myeloma

Malignant transformation is a multistep process that may involve dysregulation of oncogenes and tumour suppressor genes, and monoclonal gammopathy of undetermined significance (MGUS) is believed to be a precursor of multiple myeloma. To investigate whether aberrant promoter methylation might be involved in the evolution of MGUS to multiple myeloma, we examined the p16, protein tyrosine phosphatase, non-receptor type 6 (SHP1), death-associated protein (DAP) kinase, E-cadherin and oestrogen receptor genes, most being tumour suppressor genes, by methylation-specific polymerase chain reaction. In 32 cases of multiple myeloma and 19 cases of MGUS, significantly more frequent methylation of p16 (p = 0.001), SHP1 (p< or =0.001) and E-cadherin (p< or =0.001) genes was found in multiple myeloma than in MGUS. Methylation of DAP kinase and oestrogen receptor genes was comparable in multiple myeloma and MGUS. In conclusion, methylation of p16, SHP1 and E-cadherin genes might be involved in the progression of MGUS to multiple myeloma.     

Clinical and prognostic significances of nuclear and cytoplasmic KIT expressions in extrahepatic bile duct carcinomas.

After receiving FDA approval as a therapeutic regimen in gastrointestinal stromal tumors, the tyrosine kinase inhibitor imatinib mesylate has been applied to the treatment of other solid malignant neoplasms. To evaluate the usefulness of imatinib mesylate as a possible therapeutic regimen in extrahepatic bile duct carcinomas, an immunohistochemical study for KIT was performed in 289 cases of extrahepatic bile duct carcinomas, and mutational analysis of exon 11 of the c-kit gene was performed in 20 cases that were arbitrarily retrieved from the cases with KIT expression. Cytoplasmic KIT expression was observed in 54 cases (19%) and nuclear KIT in 58 cases (20%) of extrahepatic bile duct carcinoma. Nuclear KIT expression was more frequent in cases with vascular invasion (P<0.001), whereas cytoplasmic KIT expression was more common in tumors of T1-T3 than in those of T4 (P=0.04), and was more frequently observed in cases with a papillary growth pattern (P=0.03). Patients with cytoplasmic KIT-positive tumors had significantly better survival both by univariate (P=0.01) and multivariate analyses (P=0.04). Infrequent cytoplasmic KIT expression without mutation of exon 11 suggests that imatinib mesylate may not be effective for the treatment of extrahepatic bile duct carcinoma. However, immunohistochemical study for KIT may be helpful in routine pathologic examinations for evaluating better prognosis for patients with extrahepatic bile duct carcinoma. In addition, more frequent nuclear expression of KIT in cases with vascular invasion suggests that nuclear KIT expression may contribute to the progression of extrahepatic bile duct carcinoma.     

KIT, PDGFRα and EGFR analysis in nephroblastoma

Nephroblastoma prognosis has dramatically improved, but an unfavourable prognostic subgroup warrants development of novel therapeutic strategies. Selective KIT, PDGFRalpha and epidermal growth factor receptor (EGFR) tyrosine kinase inhibition evolved as powerful targeted therapy for gastrointestinal stromal tumours and non-small-cell lung cancer. To investigate a potential role for tyrosine kinase inhibition, we analyzed 209 nephroblastomas for immunohistochemical KIT and EGFR expression, 63 nephroblastomas for mutations in KIT exons 9, 11, 13, EGFR exons 18, 19, 20 and 21, and all 209 nephroblastomas for PDGFRalpha exons 12, 14 and 18. Twenty-two tumours (10.5%) expressed KIT, 31 (14.8%) EGFR, and 10 (4.8%) both KIT and EGFR, respectively. KIT expression was relatively more common among high-risk tumours (6/27; 22.3%) compared to low-/intermediate-risk tumours (26/181; 14.4%). Nine patients deceased, four of which had high-risk tumours with KIT expression in two of four and EGFR expression in one of four. There were no KIT, PDGFRalpha or EGFR mutations. Our results suggest no significant contribution of KIT, EGFR or PDGFRalpha mutations to nephroblastoma pathogenesis. Despite a trend towards association of immunohistochemical KIT and EGFR expression with poor outcome in high-risk nephroblastomas, statistical analysis did not yield significant correlations in this subgroup. Therefore, it remains open if KIT, PDGFRalpha or EGFR tyrosine kinase inhibition constitute a therapeutic target in nephroblastoma in the absence of KIT, PDGFRalpha or EGFR mutations.     

Expression of KIT, EGFR, HER-2 and tyrosine phosphorylation in undifferentiated thyroid carcinoma: implication for a new therapeutic approach

The KIT, epidermal growth factor receptor (EGFR) and HER-2 oncoproteins have tyrosine kinase activity and are molecular targets in human cancer therapy. To clarify the significance of KIT, EGFR, and HER-2 in undifferentiated thyroid carcinoma (UTC), the expression of these receptors and tyrosine phosphorylation was examined immunohistochemically in resected cases of UTC and papillary thyroid carcinoma (PTC). KIT, EGFR, and HER-2 were also examined at the protein and mRNA levels in five UTC cell lines. KIT expression (1+), EGFR overexpression (2+/3+), HER-2 expression (1+), and tyrosine phosphorylation were detected immunohistochemically in 40%, 70%, 10%, and 50% of the 10 UTC. In 20 PTC, KIT, EGFR, and HER-2 were not detected, but tyrosine phosphorylation was detected in 25% of cases. In the five UTC cell lines, KIT expression (1+), EGFR overexpression (3+), HER-2 expression (1+), and tyrosine phosphorylation were detected immunocytochemically in 60%, 100%, 20%, and 40%, respectively. Western blot analysis did not detect KIT expression, but did detect EGFR and HER-2 expression in all five cell lines. Real-time polymerase chain reaction detected KIT mRNA in two of the cell lines (40%), EGFR in five (100%), and HER-2 in three (60%). The present findings suggest that EGFR overexpression was involved in the proliferation and development of UTC and was frequently accompanied by tyrosine phosphorylation. Expression of KIT and HER-2 appeared to be weak but significant, suggesting a possible role in the development of UTC. Molecular therapies targeting KIT, EGFR, HER-2, and/or tyrosine phosphorylation might be indicated for UTC.     

Positive immunohistochemical staining of KIT in solid-pseudopapillary neoplasms of the pancreas is not associated with KIT/PDGFRA mutations.

Solid-pseudopapillary neoplasms of the pancreas are uncommon neoplasms of low malignant potential and of uncertain histogenesis. A small percentage of patients develop metastatic disease and some succumb to disease. The management of patients with metastatic disease or unresectable tumor, and patients who are just not good surgical candidates is problematic. Novel therapy targets are needed. Successful treatment of metastatic and unresectable gastrointestinal stromal tumors with KIT kinase inhibitor, imatinib mesylate (Gleevec), makes it intriguing to look at the status of KIT in solid-pseudopapillary neoplasms of the pancreas. In this study, we investigated KIT expression in 50 solid-pseudopapillary neoplasms by immunohistochemical staining. Of the 50 (50%) solid-pseudopapillary neoplasms, 25 showed diffuse expression (in >50% neoplastic cells) of KIT and additional five (10%) cases showed focal staining (in 10-50% neoplastic cells). Expression of KIT was not associated with tumor behavior and prognosis. A subset of 11 cases showing diffuse KIT expression detected by immunohistochemical staining were further evaluated for the presence of activating mutations in KIT exons 9, 11, 13 and 17, and PDGFRA exons 12 and 18 using PCR amplification followed by direct sequencing. However, no KIT or PDGFRA mutations were identified in any of these 11 cases tested, suggesting that the overexpression of KIT is probably not due to activating mutations in KIT or PDGFRA. The exact mechanism of KIT overexpression in solid-pseudopapillary neoplasms remains to be elucidated. One possible mechanism is gene dose effect (increased copies of KIT gene). Experience in gastrointestinal stromal tumors and other tumors have shown that mutation-mediated activation of KIT or PDGFRA is a prerequisite for clinical response with imatinib mesylate. Thus, lack of mutations in KIT or PDGFRA in solid-pseudopapillary neoplasms suggests that imatinib mesylate is less likely to be effective in the treatment for patients with metastatic disease or unresectable tumor, and patients who are just not good surgical candidates.     

Tumor angiogenesis in the bone marrow of multiple myeloma patients and its alteration by thalidomide treatment

Angiogenesis in solid tumors is important to tumor growth, invasion and metastasis. Recently, it has been suggested that angiogenesis plays a certain role in the development of hematopoietic malignancies, including leukemia and multiple myeloma. We evaluated tumor angiogenesis in the bone marrow (BM) of multiple myeloma (MM) patients by calculating microvessel density (MVD) in needle-biopsy specimens obtained from 51 cases of untreated MM or monoclonal gammopathy of undetermined significance (MGUS). The MVD in the BM of donors for transplantation and patients with non-hematological diseases was calculated as a control. There was an obvious increase in MVD in the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.     

Tryptase-positive mast cells accompany lymphocytic as well as lymphoplasmacytic lymphoma infiltrates in bone marrow trephine biopsies

AIMS: To investigate the specificity of increased bone marrow mast cell numbers in lymphoplasmacytic lymphoma (LPL) and to evaluate the relationship between mast cell number and the immunoglobulin phenotype of neoplastic lymphoid cells. METHODS AND RESULTS: Retrospective study of bone marrow trephine biopsy specimens from patients with LPL, compared with selected cases representing chronic lymphocytic leukaemia (CLL) and multiple myeloma (MM) of known immunoglobulin light and heavy chain phenotype. Bone marrow mast cells were counted following immunohistochemical staining of sections for mast cell tryptase. We have confirmed previous observations that mast cell numbers are increased in bone marrow infiltrates of LPL. However, we found similarly high mast cell numbers in CLL. High mast cell numbers were associated with neoplastic lymphoid cells expressing an IgM kappa phenotype. Mast cell numbers were low in all cases of MM studied and in controls with no lymphoma present. We observed an apparent bias towards kappa light chain expression in our cases of LPL. CONCLUSIONS: Mast cell number should not be considered a reliable factor in the differential diagnosis of LPL and CLL when assessing bone marrow histology. Possible bias towards kappa light chain expression in LPL requires further study, as do the mechanism and functions of mast cell recruitment by neoplastic lymphoid cells.     

MDS/AML-associated cytogenetic abnormalities in multiple myeloma and monoclonal gammopathy of undetermined significance: evidence for frequent de novo occurrence and multipotent stem cell involvement of del(20q)

Multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are characterized cytogenetically by 14q32 rearrangements, -13/13q-, and various trisomies. Occasionally, karyotypic patterns characteristic of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) occur in MM, often signifying therapy-related (t)-MDS/t-AML. Comparison of cytogenetic features in all published MMs (n = 993) and t-MDS/t-AML post-MM (n = 117) revealed significant differences in complexity and ploidy levels and in most genomic changes. Thus, these features often can be used to distinguish between MM and t-MDS/t-AML. Rarely, myeloid-associated aberrations are detected in MM without any signs of MDS/AML. To characterize such abnormalities in MM/MGUS, we ascertained all 122 MM and 26 MGUS/smoldering MM (SMM) cases analyzed in our department. Sixty-six (54%) MMs and 8 (31%) MGUS/SMMs were karyotypically abnormal, of which 6 (9%) MMs and 3 (38%) MGUS/SMMs displayed myeloid abnormalities, that is, +8 (1 case) and 20q- (8 cases) as the sole anomalies, without any evidence of MDS/AML. One patient developed AML, whereas no MDS/AML occurred in the remaining 8 patients. In one MGUS with del(20q), fluorescence in situ hybridization analyses revealed its presence in CD34+CD38- (hematopoietic stem cells), CD34+CD38+ (progenitors), CD19+ (B cells), and CD15+ (myeloid cells). The present data indicate that 20q- occurs in 10% of karyotypically abnormal MM/MGUS cases and that it might arise at a multipotent progenitor/stem cell level.     

Usefulness and reproducibility of cytomorphologic evaluations to differentiate myeloma from monoclonal gammopathies of unknown significance

We attempted to differentiate monoclonal gammopathies of unknown significance (MGUS) and multiple myeloma (MM) on morphologic grounds and to determine interobserver reproducibility of the differentiation. Cytologists blindly evaluated bone marrow smears from 154 patients with bone marrow plasmacytosis for the proportion of plasma cells with predefined cellular atypias. The single morphologic characteristic that most strongly differentiated MM from MGUS was the presence of nucleoli. The percentage of plasma cells, cytoplasmic contour irregularities, and anisocytosis also predicted a diagnosis of myeloma in multivariate analysis. Six cytologists independently evaluated 68 consecutive cases to determine sensitivity and specificity of these cytomorphologic features. The interobserver coefficient of variation for the plasma cell count was 33%. On consideration of the diagnosis, 36 of 41 MGUS cases and all 24 cases of myeloma were classified correctly. The use of a predesigned score system did not present such a bias, although it did not improve overall efficiency. The plasma cell count is the most predictive characteristic of myeloma from a cytologic viewpoint, but the interobserver variability is high. Interobserver variability is also high in the assessment of morphologic atypia, and atypical traits are not uncommon in plasma cells in MGUS.     

p53 nuclear expression correlates with hemizygous TP53 deletion and predicts an adverse outcome for patients with relapsed/refractory multiple myeloma treated with lenalidomide

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.     

Microvessel density,endothelial-cell proliferation and carbonic anhydrase IX expression in haematological malignancies,bone-marrow metastases and monoclonal gammopathy of undetermined significance

The aim of the study was to compare the angiogenic status, potential qualitative differences in microvessels and carbonic anhydrase IX expression in bone-marrow (BM) metastases and different haematological tumours at time of diagnosis. The microvessel density (MVD), endothelial-cell proliferation (ECP) and carbonic anhydrase IX (CA IX) immunoreactivity were determined on 210 trephine biopsies from 57 patients with multiple myeloma (MM), 13 with acute myeloid leukaemia (AML), 48 with chronic myeloproliferative syndrome (CMPS), 26 with chronic lymphocytic leukaemia (CLL), 25 with epithelial BM metastases, 18 with monoclonal gammopathy of undetermined significance (MGUS) and from a control group composed of 23 patients without haematological neoplasm. There was an increased MVD and ECP in epithelial BM metastases, MM, AML, CMPS and in a part of CLL. While an ECP greater than 0 was detected in 72% of MM, 75% of CMPS and 92% of AML, it was invariably observed (100%) in the BM metastases. The absence of ECP together with a MVD comparable with the control group in our MGUS cases supports the view that MGUS is a pre-angiogenic condition. Qualitative differences in microvessels were associated with growth patterns in MM and CLL and were observed between the different entities of CMPS. In one-third of the epithelial BM metastases, there was a focal CA IX immunoreactivity, which was never observed in the haematological diseases.     

Immunophenotypic characterization of plasma cells from monoclonal gammopathy of undetermined significance patients. Implications for the differential diagnosis between MGUS and multiple myeloma.

Although the immunophenotype of plasma cells (PCs) from multiple myeloma (MM) patients has been extensively explored, information on the phenotypic characteristics of PCs in monoclonal gammopathy of undetermined significance (MGUS) patients is scanty and frequently controversial. Thus, the question of whether or not PCs are phenotypically different in the two disorders and whether this criteria could be useful for the differential diagnosis between MGUS and MM remains to be explored. In the present study, the immunophenotypic profile of bone marrow PCs (BMPCs) from a group of 76 MGUS patients has been analyzed by flow cytometry and compared with that of BMPCs present in both MM patients (n = 65) and control subjects (n = 10). For that purpose, a large panel of monoclonal antibodies against PC-related antigens was used together with a sensitive methodology in which a minimum of 10(3) PCs were studied. In all MGUS cases studied, two clearly defined and distinct PC subpopulations could be identified. One PC subpopulation, population A (33 +/- 31% of total PCs), constantly displayed a high CD38 expression with low forward light scatter (FSC)/side light scatter (SSC) and was positive for CD19 and negative for CD56 (only a small proportion of these PCs were weakly positive for CD56). The other PC subpopulation, population B (67 +/- 31% of total PCs), showed the opposite pattern; the antigen CD56 was strongly positive and CD19 was constantly negative, and it showed a lower CD38 expression and higher FSC/SSC values than population A. Clonality studies (cytoplasmic light chain restriction, DNA content studies, and polymerase chain reaction assessment) confirmed the clonal nature of PCs from population B and the polyclonal origin of PCs from population A. Moreover, the polyclonal PCs from MGUS displayed a phenotypic profile identical to that found in PCs from healthy individuals. By contrast, clonal PCs from all MGUS patients displayed a similar antigenic profile to myelomatous PCs, with clear phenotypic differences with respect to normal PCs: lower intensity of CD38 expression and a variable reactivity for markers that were not expressed in normal PCs, such as CD28, CD117, and sIg. Although the presence of residual polyclonal PCs was a constant finding in MGUS patients, it was a rare event in MM and, when present (only 22% of MM cases), its frequency was significantly lower than that observed in MGUS (0.25% versus 32.9%, respectively; P < 0.0001). Only 1.5% of patients with MM had more than 3% of normal PCs, whereas 98% of patients with MGUS had more than 3%. Moreover, as shown by multivariate analysis, the number of residual polyclonal PCs was the most powerful single parameter for the discrimination between MGUS and MM patients at diagnosis, even when only stage I MM cases were considered.     

Neural cell adhesion molecule expression in plasma cells in bone marrow biopsies and aspirates allows discrimination between multiple myeloma, monoclonal gammopathy of uncertain significance and polyclonal plasmacytosis

AIMS: Differential diagnosis between multiple myeloma (MM), monoclonal gammopathy of uncertain significance (MGUS), and polyclonal plasmacytosis may be difficult in cases with not much bone marrow infiltration. Normal plasma cells express the antigens CD138, CD38, CD19, CD10 and D-related human leucocyte antigen (HLA-DR). Myelomatous plasma cells lack B lymphoid-associated markers and may express cell surface antigens associated with other haematopoietic lineages such as NCAM/CD56 (neural cell adhesion molecule). Recently, a monoclonal antibody, anti-CD56, has become available that can be used in fixed tissues embedded in paraffin, and it has been reported that osteoblastic cells of trabecular bone strongly express NCAM/CD56. METHODS AND RESULTS: We analysed NCAM molecule expression in 35 samples from patients with plasma cell disorders: 14 cases of MM, 16 cases of MGUS, and five cases of polyclonal plasmacytosis using immunohistochemistry in parallel in bone marrow core biopsies processed routinely and in bone marrow smears from the same patients. Of the MM samples 78% were CD56+ in smears and 92% positive in biopsies. We did not find strong CD56 expression in MGUS samples. One of five samples of polyclonal plasmacytosis was CD56+. A case was considered to be positive for CD56 expression if >50% of the CD138+ plasma cells expressed NCAM with an intensity on a par with that of the osteoblasts. CONCLUSION: We conclude that CD56 antibody is a very useful marker in the study of plasma cell proliferation in bone marrow biopsies and in bone marrow aspirates and is a great help in discriminating between MM, MGUS, and polyclonal plasmacytosis, especially in those cases with low infiltration.     

CD19 and immunophenotype of bone marrow plasma cells in monoclonal gammopathy of undetermined significance.

AIMS--To determine whether a particular phenotype or antigen is preferentially related to monoclonal gammopathies of undetermined significance (MGUS). METHODS--Bone marrow specimens from 56 patients with MGUS were stained immunocytochemically (ABC peroxidase) for CD38, CD56, CD9, CD10, CD19, CD20, CD22, and MB2. Specimens from patients recently diagnosed with multiple myeloma and reactive bone marrow samples were studied in parallel. RESULTS--CD38 was expressed on all plasma cells from all MGUS samples tested, while 36% were positive for CD56, CD9 and MB2 were both expressed strongly; CD20 was moderately expressed, and staining for CD10 and CD22 was uncommon. For these five B cell antigens there was no clear difference between their expression in MGUS and in multiple myeloma. A great difference was found for CD19: in MGUS this antigen was expressed on 2-91% of plasma cells (mean 35%) and 77% patients had > 10% positive plasma cells; in multiple myeloma its expression was low and only 12% patients had > 10% positive plasma cells. When these results were converted to numbers of CD19 positive plasma cells per 100 nucleated bone marrow cells, reactive bone marrow and MGUS specimens had a similar number of positive plasma cells. There was no correlation between expression of any of the antigens tested. CONCLUSIONS--Many of the so-called pre-B, B or activation antigens are present on plasma cells from MGUS specimens, and expression of CD9, CD10, CD20, CD22, MB2, and CD38 in MGUS was very similar to that in multiple myeloma. CD56 was frequently expressed in MGUS. In this series CD19 was highly expressed in MGUS but not in multiple myeloma. Plasma cells bearing this antigen could represent the non-neoplastic process and determination of its expression could be useful for the diagnosis of MGUS.     

Secondary Monoclonal Gammopathy of Undetermined Significance Is Frequently Associated with High Response Rate and Superior Survival in Patients with Plasma Cell Dyscrasias

Secondary monoclonal gammopathy of undetermined significance (MGUS) is a special phenomenon that occurs during the treatment of multiple myeloma (MM). The incidence, biological characteristics, and prognostic value of secondary MGUS in patients with MM remain undefined. We proceed with a retrospective systematic review of serum immunofixation electrophoresis studies performed in 438 cases of patients with plasma cell dyscrasias, including 409 cases of newly diagnosed MM and 29 cases of primary plasma cell leukemia. Secondary MGUS was more common in patients with myeloma who had undergone stem cell transplantation than in those who had not (17 [29.8%] of 57 versus 5 [1.4%] of 352, P < .001). The clinical parameters and cytogenetic characteristics in patients with or without secondary MGUS were comparable. The complete response rates in patients with or without secondary MGUS were 81.8% and 21.8% respectively (P < .01). For the cohort as a whole, secondary MGUS was associated with significantly prolonged progression-free survival (median, 52.0 months versus 22.5 months; P = .002) and overall survival (median, not reached versus 35.0 months; P < .001). The presence of secondary MGUS retained independent prognostic value with a moderate impact on overall survival (hazard ratio .128 [95% confidence interval .018 to .922]; P = .041) in the multivariate Cox regression model. However, when analysis was restricted to patients undergoing stem cell transplantation, no statistical differences in progression-free survival and overall survival were found. In conclusion, we observe that secondary MGUS was frequently observed in MM patients after transplantation and conferred a survival prolongation. The favorable survival in patients with secondary MGUS may be explained by beneficial effect from myeloablative therapy.     

Immunophenotypic and DNA content characteristics of plasma cells in multiple myeloma and monoclonal gammopathy of undetermined significance

In the present paper we review the immunophenotypic characteristics of plasma cells (PC) and the PC DNA contents from multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS), and its value for the differential diagnosis between both entities. The strong reactivity for CD38 and the positivity for CD138 are the two best markers for identifying PC. Myelomatous PC display an heterogeneous phenotype consistent with the fact that the neoplastic clone is able to undergo a certain degree of differentiation. In addition, PC from MM patients usually lack surface expression of B-cell associated antigens and frequently display reactivity for markers which are not restricted to the B-cell lineage. In MGUS patients, two clearly defined and distinct PC subpopulations can be identified. One of these PC subpopulations shows phenotypic characteristics identical to those of normal PC, including a very strong reactivity for the CD38 antigen, intermediate/low light scatter characteristics and positivity for CD19, in the absence of CD56, and corresponds to the residual normal bone marrow PC. The second PC subpopulation shows an immunophenotype similar to that of myelomatous PC, characterized by a slightly lower reactivity for CD38 and strong CD56 expression, on the absence of positivity for CD19, these PC corresponding to the clonal counterpart. Using a simultaneous staining for PC and DNA, around 60% of MM and 73% of MGUS patients display DNA aneuploidy, the majority of them being hyperdiploid. However, in contrast to MM patients, in MGUS patients two clearly different PC subsets can be discriminated in most cases (73%): a diploid and an aneuploid (hyperdiploid) subset, corresponding to normal and clonal PC, respectively. Upon comparing hyperdiploid with diploid patients in MM, the former display a better prognosis, in line with the higher incidence of DNA hyperdiploidy in MGUS. A clear correlation between the percentage of S-phase PC and several prognosis features of MM has been found. In spite of these findings, no significant differences in the percentage of pathological S-phase PC are detected between MM and MGUS patients. Regarding the differential diagnosis between MGUS and MM, multivariate analysis shows that the ratio between the number of clonal and normal residual PC is the best single parameter.     

Silent mutations in KIT and PDGFRA and coexpression of receptors with SCF and PDGFA in Merkel cell carcinoma: implications for tyrosine kinase-based tumorigenesis.

Merkel cell carcinoma is a rare and aggressive form of skin cancer of neuroendocrine origin. Its treatment involves wide excision and radiotherapy but no effective therapy exists for advanced disease. Upregulation of the platelet-derived growth factor receptor family of tyrosine kinases, PDGFRA and KIT, has a crucial role in cancer development. Several studies have shown expression of the tyrosine kinase receptor KIT (CD117) in Merkel cell carcinoma. In this study, we examined the expression and mutational status of KIT and PDGFRA in 14 primary and 18 metastatic Merkel cell carcinoma. The expression of KIT and PDGFRA and their respective ligands, stem cell factor (SCF) and PDGFA, was assessed by immunohistochemistry. In addition, we analyzed KIT exons 9, 11, 13 and 17, and PDGFRA exons 10, 12 and 18 for the presence of activating mutations. We found that only 53% of cases of Merkel cell carcinoma expressed KIT, which was mostly seen as diffuse weak staining, and SCF expression was observed only in 31% of cases. In contrast, 87 and 81% of cases expressed PDGFRA and PDGFA, respectively. We observed coexpression of SCF and KIT in only 5 of 32 cases (16%) whereas 25 of 31 cases (81%) showed coexpression of PDGFRA and its ligand PDGFA. While we documented silent mutations in exon 17 of KIT and exons 10, 12 and 18 of PDGFRA, we were not able to identify any known activating mutations. Our results indicate that there is no correlation between positive immunostaining and occurrence of activating mutations in KIT and PDGFRA. Moreover, the presence of KIT/SCF and PDGFRA/PDGFA coexpression in a proportion of cases may indicate an autocrine/paracrine stimulation loop. We think therefore that imatinib mesylate is less likely to be an effective therapy for Merkel cell carcinoma, unless activating mutations exist in other exons of these receptor kinases.     

Cyclin D1 overexpression in multiple myeloma. A morphologic, immunohistochemical, and in situ hybridization study of 71 paraffin-embedded bone marrow biopsy specimens

Cyclin D1 expression was evaluated by immunohistochemical analysis and biotin-labeled in situ hybridization (ISH) in a series of 71 decalcified, paraffin-embedded bone marrow biopsy specimens from patients with multiple myeloma (MM). Cyclin D1 messenger RNA (mRNA) overexpression was detected by ISH in 23 (32%) of 71 cases, whereas cyclin D1 protein was identified by immunohistochemical analysis in 17 (24%) of 71 specimens. All cases that were positive by immunohistochemical analysis also were positive by ISH. Statistically significant associations were found between cyclin D1 overexpression and grade of plasma cell differentiation and between cyclin D1 overexpression and extent of bone marrow infiltration. Our findings demonstrate the following: (1) ISH for cyclin D1 mRNA is a sensitive method for the evaluation of cyclin D1 overexpression in paraffin-embedded bone marrow biopsy specimens with MM. (2) ISH is more sensitive than immunohistochemical analysis in the assessment of cyclin D1 expression. (3) Cyclin D1 overexpression in MM is correlated positively with higher histologic grade and stage.