Constanze Jonak

A phase II study of bortezomib in patients with MALT lymphoma

The activity of bortezomib in patients with MALT lymphoma is unclear. This study shows that that bortezomib is active in patients with MALT lymphoma. However, an unexpectedly high rate of toxicities was seen, warranting assessment of combination schedules with bortezomib at a lower dose than given in this study. We have performed a phase II study to evaluate bortezomib in patients with MALT-lymphoma. Sixteen patients entered the trial, 4 had gastric MALT-lymphoma, 7 of the ocular adnexa, one of the colon, and 2 of the parotid, and one patient each the lung and the breast. Bortezomib was given at 1.5 mg/m2 days 1, 4, 8 and 11; repeated every 21 days. The overall response rate was 80% (13/16); 7 patients achieved complete remission (43%), 6 partial response (37%) and 3 stable disease. After a median follow-up of 23 months (range; 8–26), all patients are alive and 4 have relapsed. Fifteen patients required dose reductions due to either neuropathy (7 patients) or diarrhea (8 patients). Bortezomib appears to be active in patients with MALT-lymphoma. However, an unexpectedly high rate of toxicities was seen, warranting assessment of combination schedules with bortezomib at a lower dose than given in our study (ClinicalTrials.govIdentifier: NCT 00373906).

The hsp27 kD heat shock protein and p38-MAPK signaling are required for regular epidermal differentiation

BACKGROUND In human epidermal keratinocytes the expression of hsp27 is closely related to differentiation in vitro and in situ. OBJECTIVE We aimed to gain further insight into the role of hsp27 in epidermal differentiation by specific inhibition through siRNA and inhibition of p38-MAPK, the key enzyme of hsp27 phosphorylation. METHODS Normal human keratinocytes (KC) and organotypic skin cultures (SE-skin equivalents) were used. Expression and phosphorylation of hsp27 was inhibited in these models by siRNA and SB203580, a specific inhibitor of p38-MAPK, respectively. Modification of morphology and expression of hsp27 and other differentiation associated proteins was investigated by immunofluorescence, western blot, and RT-PCR. RESULTS Inhibition of p38-MAPK resulted in a downregulation of hsp27 in KC and SE. Additionally, in the presence of SB203580 Ca(2+) induced expression of pro-filaggrin and loricrin was inhibited at the protein level and expression of filaggrin, keratin 10, and transglutaminase 1 at the mRNA level. Addition of SB203580 to SE, as well as hsp27 knockdown in this model resulted in identical patterns of irregular differentiation, disturbance of epidermal layers, and delayed expression of K10. CONCLUSION These results provide evidence that the expression of hsp27 and its phosphorylation by p38-MAPK are required for keratinocyte differentiation and for the formation of a regularly stratified epidermis.

Heat shock proteins in the skin.

Heat shock proteins (hsp) are expressed in all cells and organisms. Their expression is induced by heat shock (temperatures above 42°C) and other forms of pathophysiological stress. Elevated levels of hsp protect cells from further stress exposure. Hsp are expressed intracellularly. They are highly conserved throughout evolution indicating hsp being necessary for survival under potentially harmful environmental conditions. Hsp are divided into families according to their molecular weight. The majority of hsp function as molecular chaperones. Chaperone function is characterized by binding to other proteins and mediating their folding, transport and interaction with other molecules. In human epidermis hsp are abundantly expressed and have been linked with functions in cell differentiation and photobiology. Recent research has mainly focused on the 27 and 72 kD hsp that are constitutively expressed in human keratinocytes. ultraviolet radiation (UV)‐induced cell death and sunburn cell formation can be inhibited by previous heat shock exposure and UV itself can induce hsp expression. The expression of the 27 kD hsp (hsp27) in epidermal keratinocytes in situ and in culture correlates with differentiation. Expression of hsp27 increases simultaneously with keratinocyte differentiation. For that reason, hsp27 is described as a marker of epidermal differentiation. Changes in the expression and inducibility of hsp have been linked with ageing. In the skin, recent data indicate that hsp72 expression remains remarkably stable with intrinsic ageing. In contrast, levels of hsp27 have been found to be elevated in sun‐protected aged skin indicating a link between hsp27 expression and age‐dependent epidermal alterations. Regulation of hsp can be modified by pharmacological intervention and the development of safe topical and systemic treatments for the prevention of skin damage and disorders of keratinocyte differentiation can be expected for the future.

Proteome Analysis Identified the PPARγ Ligand 15d-PGJ2 as a Novel Drug Inhibiting Melanoma Progression and Interfering with Tumor-Stroma Interaction

Peroxisome proliferator-activated receptors (PPARs) have been originally thought to be restricted to lipid metabolism or glucose homeostasis. Recently, evidence is growing that PPARγ ligands have inhibitory effects on tumor growth. To shed light on the potential therapeutic effects on melanoma we tested a panel of PPAR agonists on their ability to block tumor proliferation in vitro. Whereas ciglitazone, troglitazone and WY14643 showed moderate effects on proliferation, 15d-PGJ2 displayed profound anti-tumor activity on four different melanoma cell lines tested. Additionally, 15d-PGJ2 inhibited proliferation of tumor-associated fibroblasts and tube formation of endothelial cells. 15d-PGJ2 induced the tumor suppressor gene p21, a G2/M arrest and inhibited tumor cell migration. Shot gun proteome analysis in addition to 2D-gel electrophoresis and immunoprecipitation of A375 melanoma cells suggested that 15d-PGJ2 might exert its effects via modification and/or downregulation of Hsp-90 (heat shock protein 90) and several chaperones. Applying the recently established CPL/MUW database with a panel of defined classification signatures, we demonstrated a regulation of proteins involved in metastasis, transport or protein synthesis including paxillin, angio-associated migratory cell protein or matrix metalloproteinase-2 as confirmed by zymography. Our data revealed for the first time a profound effect of the single compound 15d-PGJ2 on melanoma cells in addition to the tumor-associated microenvironment suggesting synergistic therapeutic efficiency.

90Y-ibritumomab tiuxetan (Zevalin) in heavily pretreated patients with mucosa associated lymphoid tissue lymphoma

Radioimmunotherapy using 90Y-ibritumomab tiuxetan has predominantly been used in patients with follicular lymphoma, but little is known about its activity in patients with extranodal marginal zone lymphoma of the mucosa associated lymphoid tissue (MALT). A total of six patients progressing/relapsing following conventional therapy for MALT lymphoma were treated with 90Y-ibritumomab tiuxetan at our institution. Two patients had gastric MALT lymphoma, one suffered from orbital MALT lymphoma, and two had cutaneous MALT lymphoma, while one patient had a widely disseminated lymphoma involving the stomach, lungs, lymph nodes, and salivary glands. All patients were at least in third relapse following various forms of therapy including Helicobacter pylori-eradication, radiation, chemotherapy, and application of rituximab. Following two doses of rituximab at 250 mg/m2 at an interval of 1 week, 90Y-ibritumomab tiuxetan was given immediately at a dose of 0.4 mCi/kg body weight. Treatment was well tolerated apart from one episode of pneumonia requiring hospitalization. Four patients developed a complete remission (ongoing now for 4, 16, 23, and 24 months), one patient had a partial response lasting for 5 months, and one patient had stable disease for 13 months. After a follow-up of 9–29 months, all patients are alive. Application of 90Y-ibritumomab tiuxetan is active and safe in heavily pretreated patients with MALT lymphoma.

Intradermal Indocyanine Green for In Vivo Fluorescence Laser Scanning Microscopy of Human Skin: A Pilot Study

Background In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. Methodology/Principal Findings Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG) is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. Conclusions/Significance Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of ICG in combination with the near infrared laser opens new ways for minimal-invasive diagnosis and monitoring of skin disorders.

Dermal infiltrates of cutaneous T‐cell lymphomas with epidermotropism but not other cutaneous lymphomas are abundant with langerin+ dendritic cells

Background  The Langerhans cell (LC) hypothesis suggests that cutaneous T‐cell lymphomas (CTCL) are diseases of chronic T‐cell stimulation by LC‐mediated antigen presentation.

In vivo fluorescence confocal microscopy: indocyanine green enhances the contrast of epidermal and dermal structures

In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

Rituximab plus dose-reduced cyclophosphamide, mitoxantrone, vincristine, and prednisolone are effective in elderly patients with diffuse large B-cell lymphoma of the thyroid.

BACKGROUND Primary thyroid lymphoma is a rare disease. Although many reports have dealt with surgery followed by chemotherapy or radiation as well as combined chemoradiation, little is known about the value of immunochemotherapy alone. We present the results of systemic treatment using rituximab plus dose reduced mitoxantrone, cyclophosphamide, vincristine, and prednisolone in three elderly patients with primary diffuse large B-cell lymphoma (DLBCL) of the thyroid. PATIENTS AND METHODS Three patients aged between 86 and 93 years were found to have DLBCL of the thyroid. Lymphoma was locally advanced and deemed unresectable in one patient, whereas the remaining two patients were judged unfit for surgery. All patients were given systemic therapy with R 375 mg/m(2) on day 1, mitoxantrone 8 mg intravenously, cyclophosphamide 750 mg intravenously, and vincristine 1 mg (all given on day 2), along with 100 mg oral prednisolone on days 1-5. RESULTS Two patients were given 6 cycles and one patient was given 8 courses of treatment, and all responded with complete remission of the lymphoma. All three patients are alive without evidence of disease recurrence 16, 19, and 25 months after initiation of therapy. Side effects were leukopenia grade III and anemia grade II in one patient each, nausea/emesis grade I in two patients, and lower urinary tract infection and bronchitis in one patient each. CONCLUSION These data suggest that R plus dose-reduced mitoxantrone, cyclophosphamide, vincristine, and prednisolone are feasible and highly effective in elderly patients with DLBCL of the thyroid.

Subcutaneous dissemination pattern in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue lymphoma.

Background Mucosa-associated lymphoid tissue (MALT) lymphoma is among the most common forms of extranodal lymphomas, but little is known about subcutaneous involvement in patients with non-primary cutaneous marginal zone lymphomas. Design and Methods Patients with MALT lymphoma diagnosed and treated at our institution between 1999 and 2010 were analyzed for subcutaneous deposits from MALT lymphoma diagnosed in another organ. Histological, clinical and genetic findings were assessed. Results Among 216 patients with MALT lymphoma, 12 had subcutaneous deposits from MALT lymphoma (5.5%). In two patients, these lesions were present at diagnosis, while they constituted the site of relapse at an interval between 5 to 144 months in the remaining cases. Interestingly, nine of the 12 patients with subcutaneous deposits had originally been diagnosed with MALT lymphoma of the ocular adnexa (total number=51; 20%), and the other three had MALT lymphoma in the breast (total number=5; 60%). None of the patients with gastric (n=86), salivary gland (n=32) or pulmonary (n=19) MALT lymphomas had subcutaneous involvement during a median follow-up time of 87 months (range; 4 to 119 months). Conclusions Our data show that subcutaneous MALT lymphoma involvement is a rare event in patients with prior non-cutaneous extranodal marginal zone lymphoma. However, it seems to be almost exclusively associated with MALT lymphoma of the ocular adnexa and the breast, suggesting as yet undefined interactions between potentially embryonically related organ systems.