Search

Patients with primary cutaneous lymphomas (PCLs) are treated with multiple therapeutic regimens, which may increase the risk of subsequent solid and haematological neoplasms. The aim of our study was to assess the incidence of other malignancies in our series of PCLs. From March 1994 to January 2007, 272 patients with PCLs (179 M, 93 F, median age 65 yr, range 14–88) were referred to our center for staging, treatment and follow-up. The clinical charts were reviewed to detect the incidence of malignancies occurred before or after the diagnosis of PCL or concomitantly. The series was composed by 228 patients (150 M, 78 F, median age 66 yrs) with T-cell lymphomas (202 Mycosis Fungoides/MF, 10 Sézary Syndrome/SS, 9 CD30+ PCL, 7 non MF/non CD30+ T cell PCL); 43 patients (28 M, 15 F, median age 60 yrs) with B-cell lymphomas (25 Follicular/FL, 14 marginal/MZL, 3 Leg-type, 1 Lymphoblastic) and one patient with CD4+/CD56+ hematodermic neoplasm. Chemotherapy was administered to 48 patients. During follow-up 12 patients died for the disease and 24 for other causes. A second tumor was observed in 41 patients (15%): 6 of them experienced more than one neoplasms: overall we observed 48 malignancies, 38 solid and 10 haematological. The other neoplasms appeared similarly before (20) and after (21) the diagnosis of PCL; in 7 cases they were diagnosed simultaneously. Solid tumours (17 preceding, 4 concurrent, 17 subsequent) were diagnosed in: skin (11), colon (5), lung (4), breast (3), CNS (3), bladder (2), liver (2), kidney (2), uterus (2), testis (1), prostate (1), stomach (1), thyroid (1). The haematological malignancies (3 preceding, 3 concurrent, 4 subsequent) were: B-cell lymphomas (4), acute myeloid leukemias (3), plasmocytoma (1), T-cell lymphoma (1), Hodgkin’s lymphoma (1). Among the six patients with more than one adjunctive neoplasms one patient had lung and kidney carcinoma preceding PCL; two patients a preceding carcinoma (skin and bladder, respectively) and subsequently a lung carcinoma; other two patients showed both a preceding and a concurrent neoplasm (skin and colon carcinoma, B-cell lymphoma and skin carcinoma, respectively). Finally a patient had a preceding skin carcinoma, a concurrent nodal Hodgkin’s lymphoma and a subsequent nodal B-cell lymphoma. So we have reported 48 other neoplasms in 41 patients within 272 PCLs (15%). The occurrence of the other malignancy was not related to the B/T phenotype of PCLs, as it was observed in 35/228 (15.4%) T-cell lymphomas (32 MF, 2 SS, 1 non MF/non CD30+ T cell lymphoma) and in 6/43 (14%) B-cell lymphomas (3 FL, 3 MZL; χ2 test: P=0.88). The interval of occurrence was longer for tumors preceding (median 60 mo.s, range 8–180) than for tumors following PCL (median 45, range 6–122). The administration of chemotherapy for PCL was not associated with an increased incidence of second neoplasm(χ2 test, P=0.77). Multicentric studies might help in elucidating the role of genetic and immunitary factors in the pathogenesis of multiple neoplasms in patients with PCLs.

Extranodal marginal zone B-cell lymphomas are considered at the present time as “infection-associated” tumors, because their occurrence has been observed in the background of a chronic antigenic stimulation started by some recognised pathogenic microbial agents, which do not cause the lymphoid transformation by a direct infection of the lymphocytes, but by maintaining the cells with a prolonged antigenic stimulation in a continuous proliferative status and thus increasing the probabilities of a neoplastic transformation. Among them, the Helicobacter pilori is known to be active in the stomach, the Campylobacter jejuni in the ileum, the Chlamydia psittaci in the eye, the Hepatitis C virus in the spleen, whereas in the skin Borrelia burgdorferi, the agent of Lyme’s disease, which can present as erithema migrans as well as a lymphocytoma cutis, has been indicated as a potential agent involved in the pathogenesis of B-cell lymphomas. In some instances, the eradication of the Borrelia has been associated with skin lymphoma regression in cases with documented infection. In Italy there is a great variability of incidence of the disease, which is trasmitted by arthropode-bites and few cases have been observed also in our region, the Marche; this region has approximately one and half million of inhabitants. The aim of our study was to analyse by molecular techniques the presence in tissue of Borrelia burgdorferi DNA in 72 cases of primary cutaneous B-cell lymphomas, diagnosed at the Institute of Anatomic Pathology of Polytechnic Marche Region in Ancona from 1990 and 2004. In all the cases the clinical history was retrieved from clinical charts. On the basis of the pathological findings together with a complete phenotypical and clonality analysis, each case was reclassified according to the recent WHO/EORTC scheme, as follows: 16 cases of marginal-zone lymphomas, 33 cases of follicle-center lymphomas, 23 cases of diffuse large B-cell lymphomas, subdivided in “leg-type” (7), and “other” (16). DNA was extracted by paraffin-embedded tissue and amplified as previously reported, together with DNA extracted from colonies of Borrelia burgdorferi as positive control. None of the 72 cases resulted positive. The efficacy of the PCR method was documented by the expected bands obtained with control samples. Based on our results, the role of Borrelia burgdorferi in the pathogenesis of B-cell lymphomas seems to be unlikely in our geographical area, in agreement with a very low incidence of the Lyme’s disease. Therefore, also the specific antibiotic therapy do not seem to have a rationale in lymphoma treatment. Our negative results should represent a basis for searching a different combination of immunological, infectious and genetic factors to explain the occurrence of primary B-cell lymphomas, particularly the marginal types, in our region.

Chronic myeloproliferative syndromes (MPS) are clonal stem cell disorders resulting in excessive proliferation of one or more cell lineages. Rare families exist with first-degree relatives affected by MPS. Familial or hereditary essential thrombocythemia (HT) is described among children with primary thrombocytosis and it is inherited in an autosomal-dominant manner. In HT germ-line activating mutations of thrombopoietin (TPO) gene or its receptor, c-MPL can be recovered. A 31-year-old woman was first referred to our Clinic for an isolated high platelet count. On routine blood testing performed when she was 14 years old the platelet count was 1020 × 103/μl; at that time she refused other laboratory examinations, instrumental and histological diagnostic procedures and subsequent therapy and she remained asymptomatic. During the first pregnancy the platelet count went down to 500 × 103/μl but after the second it raised over one million per microliter. Bone marrow trephine biopsy showed clusters of megakaryocytes with hyperlobulated forms and platelet clumps; the caryotype was normal and bcr-abl rearrangements were negative. Leukocytic alkaline phosphatase was high, there was reduced platelet aggregation, the thrombophilic study didn’t demonstrate either factor V Leiden G1691A or prothrombin Gene G20210A mutations. During the follow up the platelet count remained under 1000 × 103/μl so that a watch-and-wait strategy seemed appropriate in this asymptomatic case and low-dose aspirin has been started. What is remarkable in this case is that it is a not-frequent example of HT since the patient’s father, sister and niece are all affected by essential thrombocytemia (ET); moreover the patient’s own little daughter is affected by neonatal thrombocytosis. The relatives are being treated only with low-dose aspirin, they are completely asymptomatic without thrombohemorrhagic events. Some authors report that HT appears to be a different disease from sporadic ET and with a more benign course. The somatic activating point mutation (V617F) in the JAK2 gene was not demonstrated in our patient; as for TPO and c-MPL mutations we are now completing the genetic study. Among other familiar chronic MPS we describe the occurrence of ET/PV and idiopathic myelofibrosis (IMF) in two monozygotic twin sisters. They both were diagnosed with MPS at the median age of 65 years old. The first had splenomegaly, thrombocytosis and erythrocytosis and was treated with aspirin, hydroxyurea and phlebotomy. The other, affected by IMF, had important epato-splenomegaly and the bone marrow trephine biopsy showed myelofibrosis with >90% collagen fibres. She received low dose hydroxyurea together with nandrolone decanoate; the organomegaly reduced and the hematic crasis improved. The JAK2 gene mutation was demonstrated only in the IMF patient. This second familial case strengthens the concept of a common pathogenesis of chronic MPS and suggests a genetic and hereditary etiology; moreover the occurrence of multiple disease phenotypes in a family is consistent with the theory of MPS as arising from clonal expansion of a pluripotential haematopoietic precursor cell that retains its pluripotentiality and produces an array of inter-related syndromes.

In the skin of 50–75% patients with early mycosis fungoides (MF) a dominant T-cell receptor (TCR) γ gene rearrangement can be detected. An identical T-cell clone is also detectable in the peripheral blood (PB) in 15–40% of these patients, as well as in 39–61% of skin biopsies showing histological remission after therapy. At the present time, it is still unclear if molecular analyses might be helpful to recognize an unfavourable subset of patients less responsive to skin-directed therapy or with a higher risk of disease recurrence. 89 patients (56 men, 33 women) with a median age of 60 years (range, 17–80), with a histologically confirmed diagnosis of early MF, were treated with a combination protocol of PUVA and low dose IFN-α for 14 months and then closely followed up. Only patients with a dominant clone in the affected skin at diagnosis, and in whom peripheral blood samples at diagnosis and a second tissue biopsy at the end of treatment were available, were selected for the present study. Twenty-four patients (10 men, 14 women; median age 62.5 yrs, range 17–77; 6 in stage IA, 15 IB, 3 IIA) met the inclusion criteria. PCR amplification for TCR γ gene was performed on DNA extracted from formalin-fixed and paraffin-embedded skin tissue samples and from frozen PB lymphocytes, as previously reported. Amplification products were visualised by 10% polyacrylamide gel electrophoresis at the same time, on the same gel, thereby allowing precise comparison of the dominant clonal populations. In one patient the identity of T-cell clones in the skin at diagnosis and at the end of therapy was assessed by sequencing analysis. After a mean time of 4 months (range 1–11), all 24 patients responded to the combination therapy, obtaining a clinical complete remission (CCR), even if three of them showed histological persistence of disease. During the follow-up, 11 patients had a disease recurrence (median time 70 months range 27–108). PCR analysis of TCR γ gene in the PB showed a circulating T-cell clone identical to the one detected in the skin in 9 cases (37.5%) at diagnosis. An identical T-cell clone was observed in the skin at the end of therapy in 17 cases (71%). Disease-free survival curves plotted by Kaplan-Meier method showed that patients with or without a peripheral T cell clone did not behave differently, that is, half of them would have experienced a relapse after a similar period of time in any case. The same behaviour was observed in patients showing different molecular responses after therapy. Only in one third of patients a molecular cure for the disease could be obtained by combination IFN-α and PUVA therapy, since a high rate of persistence of monoclonal PCR signals following CR was observed (71% of CR cases with a dominant clone at diagnosis); surprisingly, such a molecular outcome seems not to protect patients from subsequent disease relapses. On the other hand, our data seems to indicate that the combination of a skin-directed therapy like PUVA in addition to the systemic immunoregulatory effects of IFN-α may abolish the negative influence of a circulating clone.

Sézary syndrome (SS) is a cutaneous T cell lymphoma in which the demonstration of a T-cell clone in the skin and in the peripheral blood (PB) together with cytomorpho-immunophenotypical criteria are needed for a correct diagnosis besides the typical erytrhoderma and lymphadenopathies. In March 2004 a 63-year-old white man developed a rapidly growing and slightly itching squamous macular eruption on the interscapular region: skin biopsy revealed superficial dermal band-like infiltrates, consisting of small CD3+ CD4+ CD8- lymphocytes with epidermotropism and a few Pautrier’s microabscesses, suggesting a diagnosis of mycosis fungoides (MF). A simultaneous blood involvement was detected with lymphocytosis (14000/μl), increased CD4, CD3, CD2, CD5, CD7 positive T cells and T cell receptor (TCR) Vβ5.1 chain monoclonality. As for initial staging, bone marrow (BM) trephine biopsy demonstrated an interstitial infiltrate of small CD3+ and CD5+ lymphocytes and CT scan did not show any visceral involvement or lymphoadenopathies. TCRγ and TCRδ gene rearrangement patterns in PB and BM analyzed by PCR showed, respectively, a monoclonal Jpγ and a oligoclonal Vδ3Jδ1 rearrangement. Circulating Lutzner cells were seen by electronic microscopy. Cytogenetic analysis in PB using fluorescence in situ hybridization (FISH) techniques revealed multiple abnormalities (add7q, i8q, 10p+, 12p-). In September 2004 the patient started chemotherapy with fludarabine 25 mg/m2 (days 1 to 5 every 4 weeks) for 5 courses. In April 2005 he was still in good condition, not showing skin lesions; CT scan was negative, but lymphocytosis had increased (45000/μl). A BM trephine biopsy and the cytogenetic analysis of PB confirmed the above mentioned alterations. Cytofluorimetric analysis of the CD4+ CD5+ CD7+ population showed two different pattern as regarding CD3 positivity; half of the T cells was expressing CD3 antigens on the membrane surface, the other half in the citosol; however both isoforms of the molecule expressed the same Vβ5.1 chain; another cytofluorimetric characteristic was the strong expression of CD5 and CD7 in terms of elevated fluorescence intensity, a characteristic feature of T-cell prolymphocytic leukemia (T-PLL). The study of TCRγ and TCRδ gene rearrangement in PB showed, respectively, a monoclonal Jpγ and a monoclonal Vδ3Jδ1 rearrangement. In May 2005 the patient started a second line therapy with 30 mg alemtuzumab administered subcutaneously 3 times a week. At the end of the 5th week a complete haematological, immunophenotypical and cytogenetic response was obtained, even if the molecular analysis showed a persistent clonal expansion of T cells. This case demonstrates that the currently proposed haematological, flowcytometric and molecular criteria for SS are not forceful enough to differentiate between SS, MF with blood involvement and some T-PLL cases. A more detailed characterization of Sezary cells is therefore needed. Some investigators suggest that a loss of T-cell lineage markers together with the amount of CD4+ CD26- cells in PB in an appropriate clinical and histological contest, could be a more sensitive criterion of SS.

In patients with early Mycosis Fungoides (MF), a dominant T-cell receptor (TCR) gene rearrangement can be detected in the skin in 50–75% of cases, while in 15–40%, an identical T-cell clone is detectable also in the peripheral blood This may indicate an unfavourable subset of patients. We observed on omogeneus group of early MF patients all receiving the some protocol. The purpuose of this study were: - T-cell receptor gene rearrangement analisys of peropheral blood samples; - evaluation of the prognostic impact of T-cell monoclonality on CR and relapse rate. 44 patients diagnosed as having MF at early stage (25 M, 19 F; mean age 58.7 yrs, range 34–77; 11 in stage IA, 28 in stage IB, 5 in stage IIB) and showing a dominant T cell clone in the skin lesions at diagnosis, were included in the present study. Peripheral blood samples were collected for DNA extraction at diagnosis. PCR amplification for TCRγ gene was performed as previously reported by Ashton-Key et al. (1997) in all 44 cases both in peripheral blood and skin and reduplicated: amplification products were visualised by 10% polyacrylamide gel electrophoresis. Peripheral blood samples were considered positive for a circulating T cell clone only if the monoclonal signals in peripheral blood and skin were reproducible and overlapping. All patients received the same treatment, consisting of a combination protocol with low-dose IFNα + PUVA for 14 months, and then followed up. Clinical response to the therapy and further disease recurrences were registered. After a mean time of 6.02 months (range, 1–21), 7 patients failed to respond to combination therapy, while 37 obtained a clinical complete remission (CCR). Among them, 15 experienced a disease recurrence during the follow-up (mean time, 29.8 months, range, 1–77 months). PCR analysis of TCRγ gene showed in the peripheral blood the same T-cell clone detected in the skin in 16 cases (36.4%). Failure to obtain a CCR was found in 3 out of 28 cases without a T cell clone in peripheral blood (10.7%) and in 4 out 16 cases with a circulating T cell clone (25%; c2 test: P=0.41, NS). Disease recurrence was observed in 9 out of 25 cases without a T cell clone in peripheral blood (36%) and in 6 out of 12 cases with a circulating T cell clone (50%; c2 test: P=0.65, NS). Disease-free survival curves plotted by Kaplan-Meier method showed that patients with and without a circulating T cell clone did not behave differently and that half of the patients would have experienced a relapse after a similar period of time (34 and 36 months, respectively, for patients with TCRg+ and − peripheral blood; log rank test, P=0.79). PCR analysis of TCRγ gene rearrangement analysis has allowed us to detect monoclonality in the peripheral blood in 36% of early MF cases with a documented monoclonality in the skin. At this stage of the disease a circulating T cell clone could indicate a subset of patients in which skin-directed therapies are more likely to fail to completely eradicate the malignant cells. Interestingly, our data seems to show that the negative influence of a circulating clone can be bypassed by the combination of a skin-directed therapy like PUVA and the systemic immunoregulatory effects of IFNα.

Dendritic cells (DCs) are immune accessory cells involved in the uptake, processing and presentation of antigens to reactive T cells. In Mycosis fungoides (MF), they are increased when compared to healthy skin, although their role has not yet been elucidated. The activation of the so called TILs (tumor infiltrating lymhocytes) is more efficient when DCs have completed their maturational process. Therefore, blockage of DCs maturation by MF neoplastic cells could represent a mechanism of tumoral escape. The distribution and the density of DCs expressing CD207 (the langerin molecule) and CD1a, (the immature stage of their differentiation), together with the distribution and density of CD8 positive reactive lymphocytes (TILs), were analysed in MF skin lesions. Another purpose was to investigate the prognostic impact of langerin positive cells in terms of patients responsiveness to treatment and disease free survival (DFS). 33 MF patients (20 F, 13 M; mean age, 55.7 yrs, range 28–76) were included in the present study. 11 patients were in stage IA, 17 in stage IB, 4 in stage IIA and 1 in stage IIB. After treatment with PUVA and interferonα, 30 obtained a clinical complete remission, while 3 did not respond. After a mean follow-up of 29.3 months (range, 2–79), 8 patients relapsed, whereas 22 are still in complete remission. Immunostaining for langerin, CD1a, and CD8 was performed on skin formalin-fixed paraffin-embedded tissue sections obtained before the beginning of treatment and langerin, Cd1a and CD8 positive cell density was evaluated at light microscopy. Statistical analysis was performed using a SPPS statistical package. Density of CD1a+ and langerin+epidermal DCs was, respectively, low in 6 and 11 cases, and high in 27 and 22. Density of CD1a+ and langerin+ dermal DCs was, respectively, low in 6 and 16 cases, and high in 27 and 17. Density of CD8+ lymphocytes was low in 8 and high in 25 cases. Density of dermal langerin+ DCs was more frequently high in cases with a band-like/nodular infiltrate (78.6%) than in cases with a not confluent infiltrate (31.6%; Fisher’s exact test: P=0.02). Density of langerin+ epidermal DCs was low in all 3 cases in which therapy with PUVA and interferona did not achieve a complete remission (Fisher’s exact test: P=0.03). A shorter DFS was observed in patients with a low density of CD8+ lymphocytes (log rank test, P=0.03), and with a high density of both langerin+ dermal DCs (log rank test, P=0.005) and CD1a+ dermal DCs (log rank test, P=0.06): at 45 months all patients with a high density of langerin+ dermal DCs and a low density of CD8+ lymphocyte had the probability to relapse of 35% compared to 25% of those with a low density of langerin+ dermal DCs and a high density of CD8+ lymphocytes. The presence of many epidermal langerin positive DCs appears to be an optimal base for a good clinical outcome in early MF with a combination of PUVA plus interferonα. A low number of CD8+ lymphocytes and many immature langerin+ dermal DCs seem to be related to an increased risk of disease relapse, and denote an unfavourable disease behaviour since MF neoplastic cells, by blocking dermal DCs maturation, may reduce TILs recruitment, thus impairing the anti-tumour immune response.