Feasibility, safety, and efficacy of early prophylactic donor lymphocyte infusion after T cell-depleted allogeneic stem cell transplantation in acute leukemia patients.

Prophylactic donor lymphocyte infusion (DLI) starting at 6 months after T cell-depleted allogeneic stem cell transplantation (TCD-alloSCT) can introduce a graft-versus-leukemia (GvL) effects with low risk of severe graft-versus-host-disease (GvHD). We established a policy to apply low-dose early DLI at 3 months after alloSCT to prevent early relapse. This study analyzes this strategy retrospectively. Of 220 consecutive acute leukemia patients undergoing TCD-alloSCT, 83 were prospectively classified to have a high relapse risk and 43 were scheduled for early DLI. 95% of these patients received freshly harvested DLI within 2 weeks of the planned date. In patients transplanted with reduced intensity conditioning and an unrelated donor, we found an increased cumulative incidence of GvHD between 3 and 6 months after TCD-alloSCT for patients receiving DLI at 3 months compared to patients who did not receive this DLI (0.42 (95%Confidence Interval (95% CI): 0.14-0.70) vs 0). Treatment success was defined as being alive without relapse or need for systemic immunosuppressive GvHD treatment. The five-year treatment success in patients with acute lymphatic leukemia was comparable between high- and non-high-risk disease (0.55 (95% CI: 0.42-0.74) and 0.59 (95% CI: 0.42-0.84)). It remained lower in high-risk acute myeloid leukemia (AML) (0.29 (95% CI: 0.18-0.46)) than in non-high-risk AML (0.47 (95% CI: 0.42-0.84)) due to an increased relapse rate despite early DLI.

Treatment of sporadic Burkitt lymphoma in adults, a retrospective comparison of four treatment regimens.

Burkitt lymphoma is an aggressive B cell malignancy accounting for 1-2% of all adult lymphomas. Treatment with dose-intensive, multi-agent chemotherapy is effective but associated with considerable toxicity. In this observational study, we compared real-world efficacy, toxicity, and costs of four frequently employed treatment strategies for Burkitt lymphoma: the Lymphome Malins B (LMB), the Berlin-Frankfurt-Münster (BFM), the HOVON, and the CODOX-M/IVAC regimens. We collected data from 147 adult patients treated in eight referral centers. Following central pathology assessment, 105 of these cases were accepted as Burkitt lymphoma, resulting in the following treatment groups: LMB 36 patients, BFM 19 patients, HOVON 29 patients, and CODOX-M/IVAC 21 patients (median age 39 years, range 14-74; mean duration of follow-up 47 months). There was no significant difference between age, sex ratio, disease stage, or percentage HIV-positive patients between the treatment groups. Five-year progression-free survival (69%, p = 0.966) and 5-year overall survival (69%, p = 0.981) were comparable for all treatment groups. Treatment-related toxicity was also comparable with only hepatotoxicity seen more frequently in the CODOX/M-IVAC group (p = 0.004). Costs were determined by the number of rituximab gifts and the number of inpatients days. Overall, CODOX-M/IVAC had the most beneficial profile with regards to costs, treatment duration, and percentage of patients completing planned treatment. We conclude that the four treatment protocols for Burkitt lymphoma yield nearly identical results with regards to efficacy and safety but differ in treatment duration and costs. These differences may help guide future choice of treatment.

10-year stability of clinical-grade serum-free γ-retroviral vector-containing medium.

More than 10 years ago, we developed an efficient protocol for serum-free retroviral transduction of human hematopoietic stem cells derived from mobilized peripheral blood. After upscaling of the methodology, serum-free retroviral gibbon-ape leukemia virus (GALV) pseudotype PG13/LN vector supernatant produced under strict good manufacturing practice (GMP) conditions was used in the first clinical gene-marking trial in Germany. In this study, we analyzed the titer and transduction efficiency of this serum-free clinical-grade retroviral supernatant 10 years after production to evaluate the long-term stability. Long-term storage and transport on dry ice resulted in modestly decreased titers and levels of transduction efficiency in CD34+ cells ranging from 38.4 to 49.1%. We conclude that the stability of retroviral vectors in serum-free medium allows extended storage and distribution of approved clinical-grade retroviral vector stocks to distant sites in multicenter clinical trials.

Allogeneic hematopoietic stem cell transplantation for advanced mycosis fungoides and Sézary syndrome. An updated experience of the Lymphoma Working Party of the European Society for Blood and Marrow Transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option in advanced-stage mycosis fungoides (MF) and Sézary syndrome (SS). This study presents an updated analysis of the initial experience of the Lymphoma Working Party of the European Society for Blood and Marrow Transplantation (EBMT) describing the outcomes after allo-HSCT for MF and SS, with special emphasis on the impact of the use of unrelated donors (URD). METHODS AND PATIENTS: Eligible for this study were patients with advanced-stage MF or SS who underwent a first allo-HSCT from matched HLA-identical related or URD between January/1997 and December/2011. Sixty patients have been previously reported. RESULTS: 113 patients were included [77 MF (68%)]; 61 (54%) were in complete or partial remission, 86 (76%) received reduced-intensity protocols and 44 (39%) an URD allo-HSCT. With a median follow up for surviving patients of 73 months, allo-HSCT resulted in an estimated overall survival (OS) of 38% at 5 years, and a progression-free survival (PFS) of 26% at 5 years. Multivariate analysis demonstrated that advanced-phase disease (complete remission/partial remission >3, primary refractory or relapse/progression in patients that had received 3 or more lines of systemic treatment prior to transplant or the number of treatment lines was not known), a short interval between diagnosis and transplant (<18 months) were independent adverse prognostic factors for PFS; advanced-phase disease and the use of URDs were independent adverse prognostic factors for OS. CONCLUSIONS: This extended series supports that allo-HSCT is able to effectively rescue over one third of the population of patients with advanced-stage MF/SS. High relapse rate is still the major cause of failure and needs to be improved with better strategies before and after transplant. The negative impact of URD is a matter of concern and needs to be further elucidated in future studies.

HA-1H T-Cell Receptor Gene Transfer to Redirect Virus-Specific T Cells for Treatment of Hematological Malignancies After Allogeneic Stem Cell Transplantation: A Phase 1 Clinical Study.

Graft-vs.-leukemia (GVL) reactivity after HLA-matched allogeneic stem cell transplantation (alloSCT) is mainly mediated by donor T cells recognizing minor histocompatibility antigens (MiHA). If MiHA are targeted that are exclusively expressed on hematopoietic cells of recipient origin, selective GVL reactivity without severe graft-vs.-host-disease (GVHD) may occur. In this phase I study we explored HA-1H TCR gene transfer into T cells harvested from the HA-1H negative stem-cell donor to treat HA-1H positive HLA-A*02:01 positive patients with high-risk leukemia after alloSCT. HA-1H is a hematopoiesis-restricted MiHA presented in HLA-A*02:01. Since we previously demonstrated that donor-derived virus-specific T-cell infusions did not result in GVHD, we used donor-derived EBV and/or CMV-specific T-cells to be redirected by HA-1H TCR. EBV and/or CMV-specific T-cells were purified, retrovirally transduced with HA-1H TCR, and expanded. Validation experiments illustrated dual recognition of viral antigens and HA-1H by HA-1H TCR-engineered virus-specific T-cells. Release criteria included products containing more than 60% antigen-specific T-cells. Patients with high risk leukemia following T-cell depleted alloSCT in complete or partial remission were eligible. HA-1H TCR T-cells were infused 8 and 14 weeks after alloSCT without additional pre-conditioning chemotherapy. For 4/9 included patients no appropriate products could be made. Their donors were all CMV-negative, thereby restricting the production process to EBV-specific T-cells. For 5 patients a total of 10 products could be made meeting the release criteria containing 3-280 × 106 virus and/or HA-1H TCR T-cells. No infusion-related toxicity, delayed toxicity or GVHD occurred. One patient with relapsed AML at time of infusions died due to rapidly progressing disease. Four patients were in remission at time of infusion. Two patients died of infections during follow-up, not likely related to the infusion. Two patients are alive and well without GVHD. In 2 patients persistence of HA-1H TCR T-cells could be illustrated correlating with viral reactivation, but no overt in-vivo expansion of infused T-cells was observed. In conclusion, HA-1H TCR-redirected virus-specific T-cells could be made and safely infused in 5 patients with high-risk AML, but overall feasibility and efficacy was too low to warrant further clinical development using this strategy. New strategies will be explored using patient-derived donor T-cells isolated after transplantation transduced with HA-1H-specific TCR to be infused following immune conditioning.

[Active immunotherapy of prostate cancer with a focus on dendritic cells]. / Inmunoterapia activa en cáncer de próstata: revisión con atención especial a las células dendríticas.

Recurrent or metastatic prostate cancer is generally considered an incurable disease. Given the transient benefit from hormone deprivation therapy and limited successes of systemic chemotherapy, alternative treatment modalities are needed both in the situation of PSA recurrence and in hormone-refractory disease. Prostate cancer cells express several tumor associated antigens which are currently being evaluated as targets for active and specific immunotherapy approaches. Dendritic cells (DC) are the most powerful antigen-presenting cells (APC), able to prime naive T cells and to break peripheral tolerance and thus induce tumor immune responses. Close to 1000 prostate cancer patients have been treated with DC-based or other forms of active immunotherapy to date. Vaccination-induced immune responses have been reported in two thirds of DC trials, and favorable changes in the clinical course of the disease in almost half of the patients treated. Most responses, however, were modest and transient. Therefore, mechanisms of treatment failure and possibilities to improve vaccination efficacy are being discussed.

Germline mutations predisposing to diffuse large B-cell lymphoma.

Genetic studies of diffuse large B-cell lymphomas (DLBCLs) in humans have revealed numerous targets of somatic mutations and an increasing number of potentially relevant germline alterations. The latter often affect genes involved in DNA repair and/or immune function. In general, defects in these genes also predispose to other conditions. Knowledge of these mutations can lead to disease-preventing measures in the patient and relatives thereof. Conceivably, these germline mutations will be taken into account in future therapy of the lymphoma. In other hematological malignancies, mutations originally found as somatic aberrations have also been shown to confer predisposition to these diseases, when occurring in the germline. Further interrogations of the genome in DLBCL patients are therefore expected to reveal additional hereditary predisposition genes. Our review shows that germline mutations have already been described in over one-third of the genes that are somatically mutated in DLBCL. Whether such germline mutations predispose carriers to DLBCL is an open question. Symptoms of the inherited syndromes associated with these genes range from anatomical malformations to intellectual disability, immunodeficiencies and malignancies other than DLBCL. Inherited or de novo alterations in protein-coding and non-coding genes are envisioned to underlie this lymphoma.

Donor T-cell responses and disease progression patterns of multiple myeloma.

Donor T-cells transferred after allogeneic stem cell transplantation (alloSCT) can result in long-term disease control in myeloma by the graft-versus-myeloma (GvM) effect. However, T-cell therapy may show differential effectiveness against bone marrow (BM) infiltration and focal myeloma lesions resulting in different control and progression patterns. Outcomes of 43 myeloma patients who underwent T-cell-depleted alloSCT with scheduled donor lymphocyte infusion (DLI) were analyzed with respect to diffuse BM infiltration and focal progression. For comparison, 12 patients for whom a donor search was started but no alloSCT was performed, were analyzed. After DLI, complete disappearance of myeloma cells in BM occurred in 86% of evaluable patients. The probabilities of BM progression-free survival (PFS) at 2 years after start of donor search, alloSCT and DLI, were 17% (95% confidence interval 0-38%), 51% (36-66%), and 62% (44-80%) respectively. In contrast, the probabilities of focal PFS at 2 years after start of donor search, alloSCT and DLI, were 17% (0-38%), 30% (17-44%) and 28% (11-44%), respectively. Donor-derived T-cell responses effectively reduce BM infiltration, but not focal progression in myeloma, illustrating potent immunological responses in BM with only limited effect of T-cells on focal lesions.

Effectivity of a strategy in elderly AML patients to reach allogeneic stem cell transplantation using intensive chemotherapy: Long-term survival is dependent on complete remission after first induction therapy.

Intensive chemotherapy followed by allogeneic stem cell transplantation (alloSCT) can cure AML. Most studies on alloSCT in elderly AML report results of highly selected patient cohorts. Hardly any data exist on the effectiveness of prospective strategies intended to bring as many patients as possible to transplant. Between 2006 and 2011 we implemented a treatment algorithm for all newly diagnosed AML patients aged 61-75 years, consisting of intensive chemotherapy cycles to induce complete remission, followed by alloSCT. 44 of 60 (73%) newly diagnosed elderly AML patients started with chemotherapy. By meticulously following our algorithm in almost all patients, we could induce complete remission (CR) in 66% of patients starting with chemotherapy, and transplant 32% of these patients in continuous CR. Main reasons for failure were early relapse (16%), early death (14%), primary refractory disease (9%), and patient or physician decision to stop treatment (16%). Patients in continuous CR after first induction benefit most with 36% long-term survival. Patients not in CR after first induction benefit less; although additional chemotherapy induces CR in 45% of these patients, only 23% are transplanted and no long-term survival is observed, mainly due to relapse. Long-term survival in the group of 44 patients is 9% (median 4.5 years after alloSCT). Considering that 27% of patients do not start with chemotherapy and 64% of patients starting with chemotherapy do not reach alloSCT, the reasons for failure presented here should be used as a guide to develop new treatment algorithms to improve long-term survival in elderly AML patients.

Influence of pre-existing invasive aspergillosis on allo-HSCT outcome: a retrospective EBMT analysis by the Infectious Diseases and Acute Leukemia Working Parties.

Historically, invasive aspergillosis (IA) has been a major barrier for allogeneic hematopoietic stem cell transplantation (allo-HSCT). The influence of invasive IA on long-term survival and on transplant-related complications has not been investigated in a larger patient cohort under current conditions. Our aim was to analyze the long-term outcome of patients undergoing allo-HSCT with a history of prior IA. We used European Society for Blood and Marrow Transplantation database data of first allo-HSCTs performed between 2005 and 2010 in patients with acute leukemia. One thousand one hundred and fifty patients with data on IA before allo-HSCT were included in the analysis. The median follow-up time was 52.1 months. We found no significant impact of IA on major transplant outcome variables such as overall survival, relapse-free survival, non-relapse mortality, cumulative incidence of acute GvHD grade II-IV, chronic GvHD, pulmonary complications and leukemia relapse. However, we found a trend toward lower overall survival (P=0.078, hazard ratio (HR) (95% confidence interval (CI)): 1.16 (0.98, 1.36)) and higher non-relapse mortality (P=0.150, HR (95% CI): 1.19 (0.94, 1.50)) in allo-HSCT recipients with pre-existing IA. Our data suggest that a history of IA should not generally be a contraindication when considering the performance of allo-HSCT in patients with acute leukemia.

HTLV-I-associated adult T cell leukemia/lymphoma in two patients from Bucharest, Romania.

Two middle-aged patients with T cell lymphoma, both natives of Bucharest, Romania, tested positive for HTLV-I antibodies. Malignant cells had the typical phenotype and morphology of adult T cell leukemia/lymphoma (ATL). Both cases presented with extranodal manifestation, hypercalcemia, early recurrence after initial responses to therapy, and subsequent resistance to conventional and intensified chemotherapy. Infection with HTLV-I was confirmed by PCR analyses of serial biopsies. Neither patient reported known risk factors for HTLV-I infection. This report points to the possibility that Romania may represent an endemic area for HTLV-I and should heighten the awareness towards HTLV-I infections in Romanian patients.

NTS influence on transgene expression from mono- and bicistronic plasmid vectors after lipofection in a human fibroblast cell line.

We evaluated the survival, transgene production, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing murine sequence nontranscribed spacer (NTS) to the plasmids increased transfection efficiency, survival, and transgene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design.

Induction of tumor-specific cytotoxic T lymphocytes by immunization with autologous tumor cells and interleukin-2 gene transfected fibroblasts.

In a phase I trial designed to study a vaccine composed of autologous tumor cells and interleukin-2 gene transfected fibroblasts we analyzed lymphocytes infiltrating the vaccination site (VIL) in two melanoma patients. Functional studies demonstrated that numbers of MHC class I restricted cytotoxic T cells directed against the autologous tumor had increased at the immunization site in both cases. Analysis of the variability of T cell receptors (TCR) in the VIL of one patient revealed that the cytotoxic T lymphocytes consisted of a predominant population of TCRBV21S3+ T cells. Enrichment of this subpopulation to more than 99% by specific anti-TCRBV21S3 monoclonal antibody linked immunomagnetic beads and sequencing of the TCR-beta chain disclosed exactly the same V-D-J junctional sequence in all eight TCRBV21 transcripts from these VIL. The identical sequence was also detected in all eight TCRBV21 transcripts from the patient's tumor-infiltrating lymphocytes, indicating that the same CTL clone had infiltrated the tumor, circulated in the peripheral blood, and was amplified at the vaccination site. The TCRBV21S3+ T cells were also found to display an MHC class I restricted cytotoxic activity specifically directed against the autologous tumor cells. At the beginning of treatment these cells were undetectable at the vaccination site and delayed-type hypersensitivity testing was negative, contrasting with the positive results after therapy. Thus it is likely that vaccination with autologous tumor cells plus interleukin-2 gene transfected allogeneic fibroblasts had induced not only local accumulation but also an increase in the frequency of circulating tumor specific CTL.

Chlamydophila psittaci-negative ocular adnexal marginal zone lymphomas express self polyreactive B-cell receptors.

The pathogenesis of Chlamydophila psittaci-negative ocular adnexal extranodal marginal zone lymphomas (OAEMZLs) is poorly understood. OAEMZLs are monoclonal tumors expressing a biased repertoire of mutated surface immunoglobulins. Antigenic activation of the B-cell receptor (BCR) may have a role in the pathogenesis of these lymphomas. We have analyzed the reactivity of recombinant OAEMZL immunoglobulins. OAEMZL antibodies reacted with self-human antigens, as demonstrated by enzyme-linked immunosorbent assays, HEp-2 immunofluorescence and human protein microarrays. All the analyzed recombinant antibodies (rAbs) exhibited polyreactivity by comprehensive protein array antibody reactivity and some rAbs also demonstrated rheumatoid factor activity. The identity of several reactive antigens was confirmed by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry. The tested rAbs frequently reacted with shared intracellular and extracellular self-antigens (for example, galectin-3). Furthermore, these self-antigens induced BCR signaling in B cells expressing cognate surface immunoglobulins derived from OAEMZLs. These findings indicate that interactions between self-antigens and cognate OAEMZL tumor-derived BCRs are functional, inducing intracellular signaling. Overall, our findings suggest that self-antigen-induced BCR stimulation may be implicated in the pathogenesis of C. psittaci-negative OAEMZLs.

Primary fibroblasts from human adults as target cells for ex vivo transfection and gene therapy.

Diploid fibroblast (dFb) cultures were established from a total of 106 skin and serosa biopsies of human adults. Using an optimized enzymatic dissociation procedure, 10(11) dFb/cm2 skin were obtained from patients younger than 60 years after an average time of 89 +/- 8 days, with a mean population doubling time of 3.87 +/- 1.4 days. Enzymatic dissociation of skin biopsies yielded cultures of significantly higher growth capacity of dFb than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. The plating efficiency that may be crucial for clonal selection of transfected cells was negligible when dFb were plated without feeder cells at low density, while it was enhanced to 9-24% by the addition of a feeder layer of irradiated human embryonal fibroblasts. DFb secreted various cytokines with spontaneous release of interleukin-6 (IL-6) in high quantities of up to 20 ng/10(6) cells/24 hr. In addition, one-third of the culture secreted substantial amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF), while low amounts of tumor necrosis factor-alpha (TNF-alpha) were detectable in some cases after irradiation of the cells. Comparison of various transfection methods by a transient luciferase expression assay demonstrated that receptor-mediated gene transfer was approximately 10-fold more efficient than cationic lipofection of dFb, while electroporation resulted in substantially less expression of the reporter gene. We conclude that primary dFb can be obtained reproducibly from human adults and represent a suitable target cell population for receptor-mediated gene transfer and cationic lipofection.(ABSTRACT TRUNCATED AT 250 WORDS)

Systematic evaluation of chimeric marker genes on dicistronic transcription units for regulated expression of transgenes in vitro and in vivo.

Plasmid expression vectors combining human cytokine cDNAs and selectable marker genes on dicistronic transcription units were functionally characterized in vitro and in vivo. The internal ribosome entry sequence (IRES) of encephalomyocarditis virus mediated cap-independent translation of the downstream cistron. After cationic lipofection of cells with a dicistronic construct containing the Neor gene downstream of a human interleukin-2 (IL-2) cDNA, all G418-resistant clones secreted high amounts of IL-2. Reversal of the order of the cDNAs was associated with less efficient transgene expression and represented no advantage in comparison to separate expression cassettes. To combine direct in vitro selection of expression with in vivo elimination of cytokine-secreting cells, an improved chimeric cDNA of the Neor and herpes simplex virus (HSV) thymidine kinase (TK) genes was constructed and shown to confer sensitivity to ganciclovir concentrations that can be achieved in human patients. This chimeric marker was coupled on dicistronic constructs with a granulocyte colony-stimulating factor (G-CSF) cDNA as a molecule with easily detectable bioactivity in vivo. Subcutaneous implantation of pCMV.GCSF.ires TK/NEO-transfected CMS-5 cells into syngeneic BALB/c mice resulted in excessive leukocytosis and progressively growing tumors. Treatment with ganciclovir led to normalization of leukocyte counts in all animals, whereas complete regression of tumors was observed in only 3/5 mice. Hypermethylation of the transfected promoter was demonstrated in both ganciclovir-resistant tumors. Thus, transcription units combining selectable markers and genes of interest allow selection of high producer cells in vitro and efficient elimination of transgene-expressing cells in vivo. However, cells that hypermethylate transfected genes to terminate gene expression in vivo may escape conditional ablation.

Systemic hematological effects of granulocyte colony-stimulating factor produced by irradiated gene-transfected fibroblasts.

Although long-term expression of therapeutic molecules is necessary for the treatment of permanent deficiencies, short-term expression of therapeutic molecules inducing local or systemic effects is preferable in clinical situations where temporary substitution is the goal. One such clinical setting is the administration of hematopoietic growth factors in cancer chemotherapy-induced myelosuppression. Several plasmid vectors containing the human granulocyte colony-stimulating factor (G-CSF) gene under transcriptional control of different regulatory elements were constructed. In vitro production of G-CSF by nonvirally transfected murine fibroblast clones initially increased after lethal irradiation and was detectable for at least 12 days. We also demonstrate that a single injection of irradiated G-CSF-secreting fibroblasts leads to accelerated hematopoietic recovery and mobilization of committed peripheral blood progenitor cells equivalent to that achieved by twice daily s.c. administration of high doses of recombinant human G-CSF. Using dicistronic vectors, high levels of G-CSF secretion were also obtained in human fibroblasts.

Detection of low-level tumor cells in allergic contact dermatitis induced by mechlorethamine in patients with mycosis fungoides.

Two patients with histologically proven mycosis fungoides, a malignancy of phenotypically mature T cells, received a topical challenge with mechlorethamine to areas of clinically uninvolved skin to exclude possible hypersensitivity reactions to this chemotherapeutic agent. In both patients, allergic contact dermatitis (ACD) developed at the sites of the application and resolved completely after withdrawal of mechlorethamine. The lesions were biopsied and analyzed for the presence of clonal T-cell receptor (TCR)-gamma gene rearrangements using two polymerase chain reaction (PCR)-based assays involving denaturing gradient gel electrophoresis (PCR/DGGE) and ribonuclease protection analysis (PCR/RPA). The former method has a clonal detection threshold of 10(-3)-10(-2), while the latter has a sensitivity of 10(-5). In both cases, the ACD lesions were polyclonal by PCR/DGGE. In contrast, PCR/RPA detected tumor-specific TCR-gamma gene rearrangements in these same lesions. This indicates that the ACD lesions contained tumor cells at a density within the 10(-5)-10(-2) range. Analysis of peripheral blood mononuclear cells from both patients failed to detect the malignant clone and showed the same result as blood from four normal individuals. The normal skin from one skin patient also lacked detectable TCR-gamma gene rearrangements. These results indicate that mycosis fungoides tumor are present within ACD lesions induced in mycosis fungoides patients and that this phenomenon does not appear to be due to the ubiquitous presence of detectable levels of these tumor cells in the blood or skin. These findings might be explained by nonspecific recruitment of malignant T cells to sites of local inflammation mediated by non-neoplastic antigen-specific T cells. Alternatively, they might be due to the local proliferation of very rare tumor cells in apparently normal skin in response to cytokines generated during the ACD reaction. In either case, the present study offers evidence that the malignant cells in myosis fungoides retain at least some capability of responding in vivo to physiologic stimuli.

Molecular staging of cutaneous T-cell lymphoma: evidence for systemic involvement in early disease.

Biopsies of various tissues from eight patients with confirmed cutaneous T-cell lymphoma were analyzed for lymphomatous involvement using V-J junctional sequences in rearranged T-cell receptor-gamma genes as specific molecular markers for the malignant clone. The patients included one stage IA, one stage IB, and six stage IVA. Twenty-five specimens were analyzed including 14 skin, five lymph node, four blood, and two bone-marrow samples. Ten skin samples and four lymph node samples were histologically positive for lymphoma. The other specimens were morphologically uninvolved. An assay involving polymerase chain reaction (PCR) amplification of T-cell receptor-gamma gene rearrangements and denaturing gradient gel electrophoresis was used to identify the tissue specimen containing the greatest tumor clone density in each case. This specimen was then used to generate a tumor-specific RNA probe that was used to molecularly stage each patient by means of an assay involving PCR gene amplification and RNase protection analysis (PCR/RPA). This assay detected malignant cells in all available biopsies, including morphologically uninvolved extracutaneous tissue samples (two blood, one lymph node, and one bone marrow) obtained from the two patients in pathologic stage I. Microscopic examination and the less sensitive PCR/denaturing gradient gel electrophoresis technique failed to detect lymphomatous involvement in 11 (44%) and eight (32%) of these 25 specimens, respectively. We conclude that molecular biologic staging using PCR/RPA is able to demonstrate morphologically occult dissemination of cutaneous T-cell lymphoma in early disease. In addition, PCR/RPA may be able to monitor tumor response to therapy and detect early recurrence of malignant lymphomas during clinical remission.

Detection of clonal T-cell receptor gamma gene rearrangements in early mycosis fungoides/Sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE).

We used a gene amplification strategy to analyze T-cell receptor (TCR) gene rearrangements in 185 specimens, including mycosis fungoides/Sezary syndrome (MF/SS), other cutaneous neoplasms, inflammatory dermatoses, reactive lymphoid tissues, and normal skin. Genomic DNA was extracted from lesional tissues and rearrangements of the TCR-gamma chain gene were amplified using the polymerase chain reaction (PCR) with primers specific for rearrangements involving V gamma 1-8 or V gamma 9 gene segments. The resulting PCR products were then separated according to their nucleotide sequence as well as size by denaturing gradient gel electrophoresis (DGGE). Dominant clonal TCR-gamma gene rearrangements were detected in 61 of 68 MF/SS cases by PCR/DGGE. This sensitivity of 90% compared to a sensitivity of only 59% when dominant clonality was sought in 17 of these same cases by Southern blot analysis of TCR-beta gene rearrangements. This difference in sensitivity was greatest in early, minimally infiltrated skin lesions. PCR/DGGE was also more sensitive than Southern blot analysis for detecting peripheral blood involvement in two cases of early MF. Among 12 additional specimens of suspected MF/SS, nine (75%) showed clonal TCR-gamma gene rearrangements by PCR/DGGE including six of eight cases with a previously confirmed diagnosis of MF/SS and three of four cases without prior known MF/SS. Among 105 non-MF/SS specimens, dominant TCR-gamma gene rearrangements were detected in only six cases (6%). Four were diagnosed as chronic dermatitis and two were diagnosed as cutaneous lymphoid hyperplasia. We conclude that the large majority of MF/SS cases, including patch phase disease, possess dominant clonal TCR-gamma gene rearrangements. PCR/DGGE is more sensitive than Southern blot analysis for detecting dominant clonality and staging disease in patients with a confirmed diagnosis of MF/SS. However, because PCR/DGGE is sensitive enough to detect dominant TCR-gamma gene rearrangements in a subset of patients with chronic dermatitis, it cannot be used as the sole criterion for establishing a diagnosis of T-cell lymphoma. As with other molecular biologic clonality assays, clinicopathologic correlation is essential. Nevertheless, the detection of dominant clonality in some cases of histologically nonspecific dermatitis allows the identification of a previously unrecognized subset of patients, i.e., those with "clonal dermatitis." It will be important to determine the long-term risk of MF/SS among these patients because our study indicated that MF/SS can sometimes present with lesions indistinguishable from clonal dermatitis.