Leonhard Müllauer

ALK-positive anaplastic large cell lymphoma limited to the skin: clinical, histopathological and molecular analysis of 6 pediatric cases. A report from the ALCL99 study

Anaplastic large cell lymphomas are peripheral T-cell lymphomas that are characterized by a proliferation of large anaplastic blasts expressing CD30. In children, systemic anaplastic large cell lymphomas often present at advanced clinical stage and harbor translocations involving the anaplastic lymphoma kinase (ALK) gene leading to the expression of chimeric anaplastic lymphoma kinase (ALK)-fusion proteins. Primary cutaneous anaplastic large cell lymphoma is regarded as an ALK-negative variant confined to the skin and is part of the spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders. Thirty-three of 487 pediatric patients registered within the Anaplastic Large Cell Lymphoma-99 trial (1999 to 2006) presented with a skin limited CD30-positive lympho-proliferative disorder. In 23 of the 33 patients, material for international histopathological review was available, and the cases were studied for histopathological, immunophenotypical and clinical features as well as for breaks within the ALK gene. Five of 23 cases and one additional case (identified after closure of the trial) expressed ALK-protein. Complete staging excluded any other organ involvement in all children. Expression of ALK proteins was demonstrated by immunohistochemistry in all cases and the presence of breaks of the ALK gene was genetically confirmed in 5 evaluable cases. The histopathological and clinical picture of these skin-restricted ALK-positive lymphomas was indistinguishable from that of cutaneous anaplastic large cell lymphoma. Five children presented with a single skin lesion that was completely resected in 4 and incompletely resected in one. Three of these patients received no further therapy, 2 additional local radiotherapy, and one chemotherapy. All children remain in complete remission with a median follow up of seven years (range 1–8 years). We present 6 pediatric cases of ALK-positive primary cutaneous anaplastic large cell lymphomas. After thorough exclusion of systemic involvement, therapy confined to local measures seems to be sufficient to induce cure.

A phase II study of lenalidomide in patients with extranodal marginal zone B-cell lymphoma of the mucosa associated lymphoid tissue (MALT lymphoma)

Mucosa associated lymphoid tissue lymphoma shares certain features with multiple myeloma. In view of this and the activity of lenalidomide in various B-cell lymphomas, we have initiated a phase II study of lenalidomide in patients with mucosa associated lymphoid tissue lymphoma. Patients with histologically verified advanced stages of this lymphoma were included in the study. Treatment consisted of oral lenalidomide 25 mg Days 1–21, with a 7-day break after each cycle. A total of 18 patients were included in the trial: 5 had gastric and 13 had extragastric mucosa associated lymphoid tissue lymphoma, but 2 discontinued therapy during the first course of therapy. In the intent to treat analysis, an overall response rate of 61% was seen (11 of 18; 6 complete and 5 partial remissions). Three patients had stable disease while 2 progressed. Side effects were manageable and included neutropenia (grade III in 3 patients) as the leading hematotoxicity. After a median follow up of 20.3 months, one patient has died from lymphoma while the remaining patients are alive and relapse-free. These data suggest activity of lenalidomide monotherapy in mucosa associated lymphoid tissue lymphoma. The study protocol had been approved by the Ethical Board of the Medical University Vienna (EK-No.: 146/09), and before opening the trial, it had been registered at www.clinicaltrials.gov. (identifier: NCT00923663).

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).

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.

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.

Identification of genes that exhibit increased expression after flat reversion of NIH3T3 cells transformed by human activated Ha-ras oncogene

By differential hybridization, we have isolated 14 cDNA clones corresponding to genes that are more highly expressed in the flat revertant cell line R1 than in the parental human Ha-ras oncogene-transformed NIH/3T3 cell line (EJ-NIH/3T3). From cross-hybridization experiments, we determined that 5 sequence families accounted for the 14 clones. DNA sequencing revealed that four out of five selected cDNA clones represented mitochondrial genes (cytochrome b, cytochrome c oxidase subunit II, NADH dehydrogenase subunits 1 and 4, respectively), whereas one cDNA clone was homologous to the alpha 2 (type I collagen gene. Although a Southern blot analysis of the studied cell lines showed similar copy numbers of mitochondrial genomes, the transcript levels of the mitochondrial genes were high in R1, intermediate in NIH/3T3 and low in EJ-NIH/3T3 and partially revertant R2 cell lines. alpha 2 (type I) collagen mRNA levels were high in R1 and NIH/3T3, intermediate in R2 and low in EJ-NIH/3T3 cells. These results suggest that a complex alteration of the expression of mitochondrial and extracellular matrix components may be closely associated with the flat reversion of the transformed cells.

Primary mucosa-associated lymphoid tissue (MALT) lymphoma of the liver: clinical, molecular, and microbiological aspects

Dear Editor, Primary hepatic mucosa-associated lymphoid tissue (MALT) lymphomas are rarely reported in the literature. To our knowledge, the largest case series so far consists of four patients [1]. These data were the first to suggest that the liver was in fact an organ with the propensity for the development of MALT lymphoma, even if this is apparently a rare phenomenon. A literature review [2] found a total of 45 primary hepaticMALT lymphomas, again reflecting the rarity of the disease. In keeping with this, we have retrospectively analyzed all patients with primary hepatic MALT lymphoma at our institution (1999–2012) to further characterize their clinicopathological features. We have identified a total of five patients, who were all female. All patients presented with circumscript lesions. Information evaluated included age, initial stage, genetic characteristics of the lymphoma, Helicobacter pylori, hepatitis status, and treatment (see Table 1). All patients presented with normal liver function tests, and circumscript lesions within non-enlarged livers were found on CT scan. In the first attempt to assess a potential infectious cause, we have performed broad-range real-time PCR for the detection of bacterial DNA in paraffin-embedded lymphoma slides from our patients. This was done according to already published methods [3], with the limit of detection of the assay being ≤10 bacterial cells/g tissue. Our proceeding was based on the assumption that bacteria and in particular Helicobacter species might colonize the biliary tract, as seen in rodents with Helicobacter hepaticus and Helicobacter bilis [4]. However, no bacterial DNA could be detected in liver tissue/lymphoma samples in our patients. As opposed to other B cell malignancies, various different genetic aberrations have been identified in MALT lymphoma, including t(11;18)(q21;q21), t(14;18)(q32;21) involving IGH/ MALT, and t(1;14)(q22;q32). The frequency of each translocation may vary markedly according to the involved site, suggesting MALT lymphoma to be a genetically heterogeneous disease. In keeping with previous data obtained in an international cooperative effort in 2004 [5], which had screened 252 MALT lymphomas of different primary sites, we could again confirm that t(14;18)(q32;q21) is a genetic hallmark of MALT lymphoma of the liver with all of our patients testing positive for this translocation. Concerning viral infections, no evidence of hepatitis infection was seen in our patients. This is in contrast to findings from Japan, as three case reports on hepatic MALT lymphoma found infection with each of the three common hepatitis subtypes [2, 6, 7]. However, these findings might not be mutually exclusive, as wide geographic variations in terms of infectious agents in MALT lymphoma have been reported [8, 9]. Due to the small number of patients and the retrospective nature of our analysis, we cannot extrapolate treatment B. Kiesewetter :H. Agis :M. Raderer (*) Department of Internal Medicine I, Division of Oncology, Comprehensive Cancer Center of the Medical University Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria e-mail: markus.raderer@meduniwien.ac.at

Analysis of 10 Adrenocortical Carcinoma Patients in the Cohort of the Precision Medicine Platform MONDTI

Objective: Adrenocortical carcinoma (ACC) is a rare disease with a dismal prognosis. We aimed to evaluate if a personalized medicine approach may be useful for matching patients with ACC to targeted therapies. Methods: This is an analysis of 10 molecularly profiled ACCs that were progressing under standard of care treatment. The profile consisted of a 50-gene next-generation sequencing panel, immunohistochemistry (IHC), and fluorescence in situ hybridization for several proteins or chromosomal aberrations. Results: In 6 (60%) tumor samples, no somatic mutation was detected, while in 3 (30%) tumors 1 mutation was detected and in 1 (10%) tumor 2 mutations were detected. These mutations were CTNNB1 (2 samples), TP53 (1 sample), RB1 (1 sample) and APC (1 sample). Expression of phospho-mTOR and of EGFR was commonly detected by IHC (87.5 and 62.5%). In 4 (50%) samples, IHC revealed a weak expression of progesterone receptor. Less frequent alterations were expression of PDGFR-α, c-KIT, and estrogen receptor, each in 1 case. Conclusions: Based on the molecular profile, no recommendation for targeted therapy was made by the multi-disciplinary team. Currently, ACC might not be suitable for a precision medicine approach according to our tests.