RB1 gene mutations are a distinct predictive factor in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that tends to show local recurrence and metastasis. Typically, MCC is polyomavirus (MCPyV)-associated and cytokeratin 20 (CK20) positive. However, little is known about this tumor and its origins. Here, we aimed to determine the developmental origins of MCC and to identify prognostic clinicopathologic factors. Initial examinations revealed that CK20 and MCPyV expression (CK20+, MCPyV+ (60%); CK20+, MCPyV− (10%); CK20−, and MCPyV− (30%)) did not affect overall survival. With RB1 gene sequencing of FFPE specimens, which covered an entire exon, all RB1 mutation-positive cases showed positive regional lymph node and/or distant metastases (8/8 cases, 100%), whereas the frequency of the metastasis was statistically significantly lower in RB1 mutation-negative cases, (10/16 cases, 62%, P = 0.033). The results were also confirmed with immunohistochemistry, and either RB1 alterations, entire exon sequencing, or immunohistochemistry was associated with the metastasis (P = 0.007). RB1 alterations may be used to access the aggressive clinical course of MCC.     

RB1 gene in Merkel cell carcinoma: hypermethylation in all tumors and concurrent heterozygous deletions in the polyomavirus‐negative subgroup

Sequestration of the tumor suppressor retinoblastoma protein (RB) by the Merkel cell polyomavirus (MCV) is a crucial step in the pathogenesis of Merkel cell carcinoma (MCC). RB expression is frequently lost, particularly in MCV‐negative MCC tumors, through yet unknown mechanisms. We compared the genomic copy number changes of 13 MCV‐positive and 13 ‐negative MCC tumors by array comparative genomic hybridization. The analysis revealed increased genomic instability, amplification of 1p34.3–1p34.2, and losses of 11p in the absence of MCV infection. Deletions of the RB1 locus were also detected at high rates in MCV‐negative tumors. None of the tumors with heterozygous RB1 losses expressed RB in immunohistochemistry. RB1 promoter hypermethylation was studied with a methylation‐specific multiplex ligation‐dependent probe amplification technique. The RB1 promoter was methylated in all tumor specimens at CpG islands located close to the ATG start codon, albeit at low levels. The pattern of hypermethylation was similar in all MCC samples, despite RB expression, survival or MCV status. In conclusion, the frequent heterozygous losses of the RB1 locus could partly explain the decreased RB expression in MCV‐negative MCC tumors, although the effects of RB1 mutations, coinciding promoter hypermethylation and, for example, miRNA regulation, cannot be excluded.     

T cell chronic lymphocytic leukaemia with suppressor phenotype

The peripheral blood cells from a patient with T cell chronic lymphocytic leukaemia were examined for surface marker and functional characteristics. Eighty-91% of the peripheral blood cells formed SRBC rosettes and 22-49% possessed Fc receptors; 73% of the peripheral blood cells were reactive with the OKT8 antiserum and 61% expressed DR antigens. Response to PHA stimulation was markedly reduced, whereas allogeneic responsiveness in mixed leucocyte culture was intact. The ability of Con A-stimulated peripheral blood cells to generate suppressor activity in a mixed leucocyte reaction was deficient, whereas suppression of in vitro immunoglobulin synthesis was greater than normal. The leukaemic peripheral blood cell population expressed a T suppressor phenotype. Functional studies suggest that these cells were derived from the subset of T lymphocytes with regulatory activity for immunoglobulin synthesis as opposed to mitogenic responsiveness.     

Detection of Merkel cell polyomavirus in chronic lymphocytic leukemia T-cells

Chronic lymphocytic leukemia/small cell lymphoma (CLL/SLL) is the most common B-cell leukemia/lymphoma, effecting > 15,000 patients/year. There has been a proposed limited antigenic etiology, at least in some cases, of CLL/SLL based upon immunoglobulin heavy chain stereotypy found across unrelated cases, suggesting viral source may provide such antigenic stimulation. With an established epidemiological link between CLL/SLL and Merkel cell carcinoma (MCC), there has been some interest in investigating a possible leukemogenic role of Merkel cell polyomavirus (MCPyV), which is found in 80% of MCC cases. Recent studies have shown that MCPyV is present in lymphocytes in a small percentage of CLL/SLL cases, but the specific tropism for lymphocytes has not been well-established. In this study, we used quantitative PCR to investigate the presence of MCPyV in fluorescence activated cell sorted purified B- and T-cells from 23 CLL/SLL cases. Three of 23 cases (13%) had detectable MCPyV in T-cells, and none of the cases had detectable MCPyV in B-cells. These findings suggest that MCPyV may have tropism for T-cells in addition to previously reported neoplastic B-cells.     

Phenotypic heterogeneity of B cell chronic lymphocytic leukaemia

The peripheral blood lymphocytes from 39 patients from the Latvian S.S.R.T., U.S.S.R. with chronic lymphocytic leukaemia (CLL) have been phenotyped with various monoclonal antibodies representing the major clusters of differentiation (CD) used for phenotyping B cells. A clear delineation of two groups of patients was evidenced. The major group (33/39) possessed leukaemic cells bearing surface immunoglobulins (SIg) at a low density, Class II HLA, and CD5, CD24 and CD37 molecules but not CD21, CD22 and CD35. CD23 antigen was seen only once under microscope examination, but could be visualized by flow cytometry. CD6 antibody reacted with cells from about 1/3 of this group of patients. In the six patients of the second group the leukaemic phenotype was SIg+, Class II HLA+, CD5+, 24+, 37+, 21+, 22+, 35+, 23+ and 6-. The main finding is the concomitant expression of CD22, CD21 (CR2) and CD35 (CR1) molecules, all involved in B cell activation. It is not yet known whether these observations correlate with different clinical evolutions of the disease.     

Merkel cell carcinosarcoma: Merkel cell carcinoma with embryonal rhabdomyosarcoma-like component

Merkel cell carcinoma is an uncommon primary neuroendocrine neoplasm of the skin that may exhibit divergent differentiation. However, rhabdomyosarcomatous differentiation has only been rarely described and takes the form of isolated rhabdomyoblasts. We describe a case of cutaneous Merkel cell carcinoma with biphasic morphology imparted by discrete patches of embryonal rhabdomyosarcoma-like spindle cells alternating with islands of neuroendocrine small round cells, justifying a designation of "Merkel cell carcinosarcoma." The former component showed positive immunostaining for desmin and myogenin; and the later component, pan-cytokeratin, cytokeratin 20, synaptophysin, and chromogranin. The patient was an elderly man who presented with a temporal skin mass, and the biphasic morphology was evident in the recurrence and metastasis that developed 2 months after incomplete excision of the skin lesion.     

Gaucher's disease associated with chronic lymphocytic leukaemia, gout and carcinoma.

A case report of Gaucher's disease associated with chronic lymphocytic leukaemia is described in a patient with gout and carcinoma of the sigmoid colon. This the first reported case of Gaucher's disease associated with chronic lymphocytic leukaemia.     

Expression of natural killer cell activating receptors in patients with chronic lymphocytic leukaemia

Recent advances in chronic lymphocytic leukaemia (CLL) treatment, more particularly through upfront use of anti-CD20 monoclonal antibodies, have prolonged patient progression-free survival. Nonetheless, apart from allogeneic stem cell transplantation, no curative treatment is available. One possible explanation for the lack of cure in CLL could be a defective immune anti-tumour response. As the result of abnormal HLA class I molecule expression, CLL cells escape from specific T-lymphocyte immunity but should be the target for the innate natural killer (NK) cell-mediated immune response. Defective NK cytotoxicity as the result of decreased expression of the natural cytotoxicity receptors (NCRs) NKp30/NCR3, NKp44/NCR2 and NKp46/NCR1 has been described in haematological malignancies such as acute myeloid leukaemia. This prompted us to focus our attention on NCR expression on NK cells from patients with CLL. Although we failed to detect any difference between CLL patients and healthy age-matched controls, a precise analysis of clinical data showed a correlation between decreased NCR expression and poor prognosis factors such as low haemoglobin level, high (>30×10(9) per litre) lymphocyte count or elevated C-reactive protein. Together, these observations support the rationale for restoration of normal NK cell functions in patients with CLL, putatively through the use of immune therapy protocols that already have demonstrated some benefit in acute myeloid leukaemia such as interleukin-2 plus histamine dihydrochloride.     

Merkel cell carcinoma and chronic lymphocytic leukemia (collision tumor) of the arm: a diagnosis by fine-needle aspiration biopsy

Simultaneous involvement of the same anatomical site by two different primary malignant tumors is rare. Cases of hematopoietic malignancies associated with breast and skin neoplasms have been described. The association of chronic lymphocytic leukemia (CLL) and Merkel cell carcinoma (MCC) has been established, although the cause for this association is still unclear. There are reports of MCC metastatic to lymph nodes involved by CLL. We report the case of a 57-year-old man with history of CLL with concurrent involvement of the arm by CLL and MCC diagnosed on fine-needle aspiration biopsy (FNA). To our knowledge, this is the first reported case of such tumors colliding in a nonlymphoid site, diagnosed by FNA in the English literature.     

Imbalance of T cell subpopulations in patients with chronic lymphocytic leukaemia of the B cell type.

T cell enriched mononuclear cells from the peripheral blood of 20 patients with histologically and immunologically defined chronic lymphocytic leukaemia of the B cell type (B-CLL) and 20 healthy individuals of various ages were investigated with T cell-specific monoclonal antibodies (OKT 4 and OKT 8) with regard to their subpopulation distribution. In B-CLL, a significant increase of lymphocytes reacting with OKT 8 could be demonstrated. Whereas there was a ratio of OKT 4 to OKT 8 of 1 X 72 in the control group, an OKT 4 to OKT 8 ratio of 0 X 67 was found in the B-CLL as a whole. With increasing clinical stage in accordance with the Rai scheme (Rai et al., 1975), a further displacement of this ratio in favour of OKT 8 positive cells was found. These results clearly show that, in peripheral blood of patients with B-CLL, an abnormal distribution pattern of circulating T cell subpopulations is present and that this also has prognostic relevance.     

Monoclonal cryoimmunoglobulin with anti-cytomegalovirus activity associated with T cell chronic lymphocytic leukaemia.

A patient with chronic T cell lymphocytic leukaemia developed a monoclonal immunoglobulin (IgG3 kappa = 14 g/l) which was in part cryoprecipitable. At the same time, a subclinical CMV infection occurred which was associated with a neutropenia and thrombocytopenia, and which led to a rise in anti-CMV antibodies. The F(ab')2 fragment of IgG3 kappa, obtained by enzymatic cleavage, was examined for several antiviral activities and it was found to have a strong anti-CMV activity using the immunofluorescence test with anti-kappa conjugate. This is one of the few examples of a cryoglobulin with specific antiviral activity. The leukaemia, possibly together with immunosuppressive therapy, may have been responsible for the uncontrolled proliferation of the clone producing the cryoimmunoglobulin.     

Association of Merkel cell polyomavirus infection with morphologic differences in Merkel cell carcinoma

Recently, it has been shown that approximately 80% of Merkel cell carcinomas harbor a novel polyomavirus named Merkel cell polyomavirus, thought to be a carcinogenic agent. However, it is not fully elucidated whether Merkel cell carcinomas differ with regard to the presence or absence of Merkel cell polyomavirus. To address this, we investigated morphologic differences between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas by morphometry. Using polymerase chain reaction and real-time quantitative polymerase chain reaction, Merkel cell polyomavirus was detected in 20 (77%) of 26 Merkel cell carcinoma cases, including 4 Merkel cell carcinomas combined with squamous cell carcinomas. Interestingly, Merkel cell polyomavirus was detected only in ordinary (pure) Merkel cell carcinomas; none of the 4 combined Merkel cell carcinomas + squamous cell carcinomas was positive for Merkel cell polyomavirus (P = .001). Morphometric analyses revealed that Merkel cell polyomavirus-negative Merkel cell carcinomas had more irregular nuclei (P < .001) and more abundant cytoplasm (P = .001) than Merkel cell polyomavirus-positive Merkel cell carcinomas, which had uniform round nuclei and scant cytoplasm. Reliability of the morphometry was confirmed using intraobserver and interobserver reliability tests. These results demonstrated statistically significant differences in tumor cell morphology between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas and reconfirmed the absence of Merkel cell polyomavirus in combined tumors. Furthermore, the results strongly suggest fundamental biological differences between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas, supporting that Merkel cell polyomavirus plays an important role in the pathogenesis of Merkel cell polyomavirus-positive Merkel cell carcinoma.     

Angiogenesis in the bone marrow of patients with chronic lymphocytic leukaemia

Angiogenesis is a process of formation of new vessels from the preexisting ones. It is involved in many physiological processes, at the same time, however, it is involved also in the progress of tumoral growth. Although a lot is known about angiogenesis in solid tumors where it plays a role in tumoral invasion and its metastatic potential, in hematological malignancies it has been appreciated only recently. However, the results of studies on abnormal angiogenesis in hematological malignancies are inconsistent. Angiogenesis can be studied at different levels; histologically, it is studied in the infiltrated tissues (lymph nodes, bone marrow) and quantified as microvessel density (MVD). The aims of our study were to introduce the method of MVD quantification in the bonemarrow using immunohistochemical detection of endothelial markers (fVIII) and then evaluate MVD in bone marrow samples in a group of patients with chronic lymphocytic leukaemia (CLL) and compare the results with a control group of patients (CON). CLL is a typical malignancy of the hematopoietic tissue but the course and the prognosis of patients with this disease vary considerably. For this reason there is urgent need for novel prognostic markers in order to assess individual patient prognosis and tailor treatment. Angiogenesis is one of the possible markers which may add more informations about the course of this disease. So far only few studies have been published about angiogenesis measured as MVD in CLL patients andthe results are inconsistent. In our study, both the number and the area of microvessels were increased in bone marrow of patients with CLL, but the number and area of sinuses were not. It can be concluded that there are signs of abnormal angiogenesis in bone marrow of patients with CLL but larger study with longer follow-up is needed to give more specific information about prognostic value of these findings.     

Bone marrow aplasia in B cell chronic lymphocytic leukaemia: successful treatment with antithymocyte globulin.

Pure red cell aplasia is a rare but well known association of chronic lymphocytic leukaemia (CLL). Pancytopenia due to bone marrow aplasia has not been previously described in CLL. A 42 year old man with B cell CLL became severely pancytopenic with bone marrow aplasia. Bone marrow culture resulted in a greatly reduced colony formation. High dose corticosteroids and intravenous immunoglobulin treatment were unsuccessful. Prompt and complete marrow recovery ensued after administration of antithymocyte globulin.     

Blood dendritic cells in patients with chronic lymphocytic leukaemia

Myeloid and plasmacytoid dendritic cells (MDC, PDC) play a key role in the initiation of immune responses. We found a reduction of both DC subsets in 42 patients with chronic lymphocytic leukaemia (CLL) at diagnosis (P<0.0001 and 0.0001 vs. controls, respectively), likely related to the high secretion of CCL22 and CXCL12 (P=0.04 and 0.008 vs. controls, respectively) by leukaemic cells. However, CD14+ monocytes from CLL patients could give rise to functional IL-12p70-secreting monocyte-derived DCs, capable of inducing a type 1 polarization immunostimulatory profile. These monocyte-derived DCs from CLL patients efficiently migrate in response to CCL19/MIP-3beta chemokine, suggesting that functional autologous DCs can be generated for immunotherapeutic purposes to circumvent DC defects in CLL.     

Advances in therapy of chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is a lymphoproliferative disorder characterized by the progressive accumulation of clonal small mature-looking lymphocytes, usually of B cell origin. In addition to a better understanding of many biological features, during the last 10-15 years a great progress has been made in the prognostic characterization of the disease. The therapeutic achievements, however, have been less impressive. The possibility to identify patients with different prognosis has renewed interest in the treatment of this disorder. A considerable number of controlled trials have been performed or are in course, and new perspectives for CLL treatment are emerging. Basically four different kinds of measures are used: 1) chemotherapy, 2) radiotherapy, 3) adjuvant measures and 4) new modalities.     

Impaired production of mononuclear cell procoagulant activity in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is associated with a low incidence of thrombotic complications, or disseminated intravascular coagulation (DIC), or both, despite the frequent occurrence of severe infections. We have investigated the capacity of blood mononuclear cells to produce procoagulant activity when stimulated with bacterial endotoxin in 16 patients with untreated chronic lymphocytic leukaemia (CLL). Procoagulant activity generated by patients' cells after prolonged incubation with endotoxin was significantly lower than that produced by cells from a matched control group (p less than 0.001). The defect could not be attributed to an inhibitory effect of leukaemic lymphocytes. It is suggested that in CLL the monocyte has an intrinsic functional abnormality of the procoagulant response to endotoxin and possibly to other stimuli. These findings help explain why CLL patients do not develop thrombotic complications despite the high incidence of severe infectious diseases.     

Immunoglobulin VH gene expression in human B cell lines and tumors: biased VH gene expression in chronic lymphocytic leukemia

We have studied frequencies of VH gene utilization in a panel of monoclonal Epstein-Barr virus (EBV)-transformed B cell lines derived from human adult and fetal tissues as well as in monoclonal B cells obtained from fresh chronic lymphocytic leukemia (CLL) samples. The results show that IgM-secreting EBV cell lines from both fetal and adult tissues utilize VH genes from particular families roughly in proportion to estimated family size, suggesting that the repertoire of sigM-positive B cells in both fetal and adult organs is 'normalized' with respect to the V(H) gene family. In contrast, we find a highly biased pattern of VH gene expression in CLLs. The significance of these findings is discussed in the context of mechanisms that could be involved in normal B cell repertoire development and in the process of malignant transformation of precursors of CLL.