Introduction to the EQIPD quality system.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

Improving quality of preclinical academic research through auditing: A feasibility study.

How much can we rely on whether what was reported in a study was actually done? Systematic and independent examination of records, documents and processes through audits are a central element of quality management systems. In the context of current concerns about the robustness and reproducibility of experimental biomedical research audits have been suggested as a remedy a number of times. However, audits are resource intense and time consuming, and due to their very nature may be perceived as inquisition. Consequently, there is very little experience or literature on auditing and assessments in the complex preclinical biomedical research environment. To gain some insight into which audit approaches might best suit biomedical research in academia, in this study we have applied a number of them in a typical academic neuroscience environment consisting of twelve research groups with about 100 researchers, students and technicians, utilizing the full gamut of state-of-the-art methodology. Several types of assessments and internal as well as external audits (including the novel format of a peer audit) were systematically explored by a team of quality management specialists. An experimental design template was developed (and is provided here) that takes into account and mitigates difficulties, risks and systematic errors that may occur during the course of a study. All audits were performed according to a pre-defined workflow developed by us. Outcomes were assessed qualitatively. We asked for feedback from participating employees in every final discussion of an audit and documented this in the audit reports. Based on these reports follow-up audits were improved. We conclude that several realistic options for auditing exist which have the potential to improve preclinical biomedical research in academia, and have listed specific recommendations regarding their benefits and provided practical resources for their implementation (e.g. study design and audit templates, audit workflow).

Automated Reports for Monitoring and Improving Data Quality in a Translational Research Network.

Standardised, automated quality reports were generated at three pilot locations of the decentralized translational research network DKTK with separated local data warehouses (LDW), for assessing syntactic conformity against common data element definitions deposited in a central metadata repository (MDR). Deviations in the LDW were categorised, and locally corrected. Comparisons of reports from two time points confirm a major improvement in data quality in terms of syntactic conformity, an essential prerequisite for network-wide data integration.

Loss-of-function uORF mutations in human malignancies.

Ribosome profiling revealed widespread translational activity at upstream open reading frames (uORFs) and validated uORF-mediated translational control as a commonly repressive mechanism of gene expression. Translational activation of proto-oncogenes through loss-of-uORF mutations has been demonstrated, yet a systematic search for cancer-associated genetic alterations in uORFs is lacking. Here, we applied a PCR-based, multiplex identifier-tagged deep sequencing approach to screen 404 uORF translation initiation sites of 83 human tyrosine kinases and 49 other proto-oncogenes in 308 human malignancies. We identified loss-of-function uORF mutations in EPHB1 in two samples derived from breast and colon cancer, and in MAP2K6 in a sample of colon adenocarcinoma. Both mutations were associated with enhanced translation, suggesting that loss-of-uORF-mediated translational induction of the downstream main protein coding sequence may have contributed to carcinogenesis. Computational analysis of whole exome sequencing datasets of 464 colon adenocarcinomas subsequently revealed another 53 non-recurrent somatic mutations functionally deleting 22 uORF initiation and 31 uORF termination codons, respectively. These data provide evidence for somatic mutations affecting uORF initiation and termination codons in human cancer. The insufficient coverage of uORF regions in current whole exome sequencing datasets demands for future genome-wide analyses to ultimately define the contribution of uORF-mediated translational deregulation in oncogenesis.

Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer.

BACKGROUND: As screening methods for colorectal cancer (CRC) are limited by uptake and adherence, further options are sought. A blood test might increase both, but none has yet been tested in a screening setting. OBJECTIVE: We prospectively assessed the accuracy of circulating methylated SEPT9 DNA (mSEPT9) for detecting CRC in a screening population. DESIGN: Asymptomatic individuals ≥50 years old scheduled for screening colonoscopy at 32 US and German clinics voluntarily gave blood plasma samples before colon preparation. Using a commercially available assay, three independent blinded laboratories assayed plasma DNA of all CRC cases and a stratified random sample of other subjects in duplicate real time PCRs. The primary outcomes measures were standardised for overall sensitivity and specificity estimates. RESULTS: 7941 men (45%) and women (55%), mean age 60 years, enrolled. Results from 53 CRC cases and from 1457 subjects without CRC yielded a standardised sensitivity of 48.2% (95% CI 32.4% to 63.6%; crude rate 50.9%); for CRC stages I-IV, values were 35.0%, 63.0%, 46.0% and 77.4%, respectively. Specificity was 91.5% (95% CI 89.7% to 93.1%; crude rate 91.4%). Sensitivity for advanced adenomas was low (11.2%). CONCLUSIONS: Our study using the blood based mSEPT9 test showed that CRC signal in blood can be detected in asymptomatic average risk individuals undergoing screening. However, the utility of the test for population screening for CRC will require improved sensitivity for detection of early cancers and advanced adenomas. CLINICAL TRIAL REGISTRATION NUMBER: NCT00855348.

Multicenter clinical validation of PITX2 methylation as a prostate specific antigen recurrence predictor in patients with post-radical prostatectomy prostate cancer.

PURPOSE: Radical prostatectomy is potentially curative in patients with clinically localized prostate cancer. However, biochemical recurrence affects 15% to 30% of men who undergo radical prostatectomy. We previously reported the prognostic potential of PITX2 gene promoter methylation using conventional assays. In the current study we validated PITX2 methylation status as a biochemical recurrence predictor after radical prostatectomy using a novel microarray based platform in a multi-institutional setting. MATERIALS AND METHODS: PITX2 methylation status was assessed in formalin fixed, paraffin embedded prostatectomy tumor tissue samples from 476 patients from a total of 4 institutions on customized EpiChip PITX2 microarrays. Associations between PITX2 methylation and biochemical recurrence were assessed using the log rank test and Cox regression controlling for prostate cancer features. RESULTS: On multivariate analysis men with high methylation status were at significantly higher risk for biochemical recurrence than those with low methylation status (HR 3.0, 95% CI 2.0-4.5, p <10(-5)). The biochemical recurrence-free survival rate 5 years after surgery was 85% and 61% in the low and high methylation groups, respectively. In men with pathological Gleason 7 tumors the relative risk of biochemical recurrence was twice as high for high than for low PITX2 methylation (HR 2.0, 95% CI 1.2-3.3, p = 0.005). CONCLUSIONS: PITX2 methylation status assessed by EpiChip PITX2 identifies patients with prostate cancer who are most likely to have biochemical recurrence. This test independently adds to the prognostic information provided by standard clinicopathological analysis, improving prostatectomy case stratification into those at high and low risk for biochemical recurrence. This new clinical tool would be of particular benefit to assess intermediate risk cases (Gleason 7) in which risk stratification remains a challenge.

An expression module of WIPF1-coexpressed genes identifies patients with favorable prognosis in three tumor types.

Wiskott-Aldrich syndrome (WAS) predisposes patients to leukemia and lymphoma. WAS is caused by mutations in the protein WASP which impair its interaction with the WIPF1 protein. Here, we aim to identify a module of WIPF1-coexpressed genes and to assess its use as a prognostic signature for colorectal cancer, glioma, and breast cancer patients. Two public colorectal cancer microarray data sets were used for discovery and validation of the WIPF1 co-expression module. Based on expression of the WIPF1 signature, we classified more than 400 additional tumors with microarray data from our own experiments or from publicly available data sets according to their WIPF1 signature expression. This allowed us to separate patient populations for colorectal cancers, breast cancers, and gliomas for which clinical characteristics like survival times and times to relapse were analyzed. Groups of colorectal cancer, breast cancer, and glioma patients with low expression of the WIPF1 co-expression module generally had a favorable prognosis. In addition, the majority of WIPF1 signature genes are individually correlated with disease outcome in different studies. Literature gene network analysis revealed that among WIPF1 co-expressed genes known direct transcriptional targets of c-myc, ESR1 and p53 are enriched. The mean expression profile of WIPF1 signature genes is correlated with the profile of a proliferation signature. The WIPF1 signature is the first microarray-based prognostic expression signature primarily developed for colorectal cancer that is instrumental in other tumor types: low expression of the WIPF1 module is associated with better prognosis.

Genome-wide expression patterns of invasion front, inner tumor mass and surrounding normal epithelium of colorectal tumors.

Colorectal tumors have characteristic genome-wide expression patterns that allow their distinction from normal colon epithelia and facilitate clinical prognosis. The expression heterogeneity within a primary colorectal tumor has not been studied on a genome scale yet. Here we investigated three compartments of colorectal tumors, the invasion front, the inner tumor mass, and surrounding normal epithelial tissue by microdissection and microarray-based expression profiling. In both tumor compartments many genes were differentially expressed when compared to normal epithelium. The sets of significantly deregulated genes in both compartments overlapped to a large extent and revealed various interesting known and novel pathways that could have contributed to tumorigenesis. Cells from the invasion front and inner tumor mass, however, did not show significant differences in their expression profile, neither on the single gene level nor on the pathway level. Instead, gene expression differences between individuals are more pronounced as all patient-matched tumor samples clustered in close proximity to each other. With respect to invasion front and inner tumor mass we conclude that the specific tumor cell micro-environment does not have a strong influence on expression patterns: largely similar genome-wide expression programs operate in the invasion front and interior compartment of a colorectal tumor.

A genome-wide map of aberrantly expressed chromosomal islands in colorectal cancer.

BACKGROUND: Cancer development is accompanied by genetic phenomena like deletion and amplification of chromosome parts or alterations of chromatin structure. It is expected that these mechanisms have a strong effect on regional gene expression. RESULTS: We investigated genome-wide gene expression in colorectal carcinoma (CRC) and normal epithelial tissues from 25 patients using oligonucleotide arrays. This allowed us to identify 81 distinct chromosomal islands with aberrant gene expression. Of these, 38 islands show a gain in expression and 43 a loss of expression. In total, 7.892 genes (25.3% of all human genes) are located in aberrantly expressed islands. Many chromosomal regions that are linked to hereditary colorectal cancer show deregulated expression. Also, many known tumor genes localize to chromosomal islands of misregulated expression in CRC. CONCLUSION: An extensive comparison with published CGH data suggests that chromosomal regions known for frequent deletions in colon cancer tend to show reduced expression. In contrast, regions that are often amplified in colorectal tumors exhibit heterogeneous expression patterns: even show a decrease of mRNA expression. Because for several islands of deregulated expression chromosomal aberrations have never been observed, we speculate that additional mechanisms (like abnormal states of regional chromatin) also have a substantial impact on the formation of co-expression islands in colorectal carcinoma.

CGH of microdissected Kaposi's sarcoma lesions reveals recurrent loss of chromosome Y in early and additional chromosomal changes in late tumour stages.

BACKGROUND: It is still unclear if Kaposi's sarcoma (KS) is a monoclonal cell proliferation or a polyclonal, hyperplastic, reactive process. Reports on KS cytogenetics are few and restricted to late stage disease and cell lines. METHOD: We analysed 27 KS, early and late, AIDS related (AKS) and endemic (EKS) by laser microdissection, global DNA amplification and comparative genomic hybridization (CGH). RESULT: Loss of Y chromosome was detected in 20/23 male KS, which was the only recurrent chromosomal aberration in all nine male early (patch) KS. Only one patch EKS showed in addition to the Y loss a loss of Xq. Late (nodular) AKS and EKS showed recurrent copy number changes in chromosomes 16, 17, 21, X and Y, as well as other random changes. The loss of chromosome 16, 17 and Y was confirmed by interphase fluorescence in situ hybridization (FISH) on paraffin sections. EKS showed a higher number of chromosomal abnormalities than AKS, indicating that rapid growth of AKS is less dependent on genetic changes than is EKS, possibly because of the immunosuppressed host environment in AKS. CONCLUSION: Clonal loss of chromosome Y was detected in all early male KS, while additional chromosomal aberrations appeared during development to late KS. This increase in chromosomal abnormalities during tumour growth indicates genetic instability and the selection of survival cell clones establishing late, aggressive sarcoma growth. Our data support the view that KS (in males) develops into a clonal tumour yet initially is a hyperplastic reactive cell proliferation.

AIDS and non AIDS-related malignant lymphoma in Tanzania.

BACKGROUND: Malignant lymphoma (ML) in HIV patients, are second in frequency to Kaposi's sarcoma (AKS) as AIDS-defining tumors. In Africa the frequency of AIDS-related lymphoma (ARL) is rare and the findings are controversial. Kaposi's sarcoma (KS) lesions are now causally associated with KSHV/HHV-8 but whether African ARL shows this association is not clear. METHOD: Cancer registry data was reviewed for retrospective cases. Both retrospective and prospective lymphoma cases were classified according to the revised European-American (REAL) classification. Immunephenotyping was performed on both frozen and fixed paraffin sections. Viral DNA was assessed by polymerase chain reaction (PCR) of formalin fixed or frozen biopsies. In situ hybridization (ISH) was used to determine the presence of EBV encoded RNA (EBER). OBJECTIVES: To determine the frequency and type of AIDS and non-AIDS related malignant lymphoma in Tanzania and a possible co-association with KSHV/HHV-8 and EBV. RESULTS: An overall increasing tendency for ML in Tanzania was observed during 1991-94 and a clear increase from 1993. The tumors were classified as Burkitt's (6), diffuse large cell (10), precursor-B lymphoblastic (1) and Hodgkin's disease (5) from HIV positive and negative patients. Ten (40%) high grade ML and three Hodgkin's lymphoma from HIV patients had HHV-8 DNA. These findings were not related to age, sex or type of lymphoma. There was no association of HHV-8 with the lymphoma cells. Epstein-Barr virus (EBV) was demonstrable in most (13/18; 72%) of the tested tumors and seven (31.8%) had both HHV-8 and EBV. CONCLUSIONS: This study suggests an overall increased frequency of ML patients infected with HHV-8 in Tanzania particularly in HIV patients which may result from the well established high HHV-8 prevalence in the general population, but HHV-8 was not associated with ARL pathogenesis as reflected by lack of tumor cell infection. As opposed to EBV, measures targeting HHV-8 for control of ML may therefore not be appropriate.

Expression levels of the putative zinc transporter LIV-1 are associated with a better outcome of breast cancer patients.

We investigated the expression pattern of the breast cancer associated gene LIV-1 on mRNA and protein level in 111 human breast cancer patients by in situ hybridization as well as immunohistochemistry and focused on the unknown potential of LIV-1 expression levels as a prognostic marker. To our knowledge, this is the first study on endogenous LIV-1 protein expression. Results of our study indicate that LIV-1 mRNA and protein expression levels are only weakly correlated, suggesting posttranscriptional regulatory mechanisms. Furthermore, LIV-1 mRNA quantity in combination with a positive ER status seem to represent a better marker than the progesterone receptor status according to the prognostic significance for relapse free survival (RFS). A negative correlation of LIV-1 protein levels with tumor size, grade and stage reflects an association of LIV-1 protein expression with less aggressive tumors. High LIV-1 protein expression seems to be associated with a longer relapse free and overall survival in breast cancer patients with invasive ductal carcinoma. This association, however, seems to be dependent from other prognostic markers. Our data suggest that LIV-1 is a promising candidate for a novel marker for breast cancer patients with better outcome. Furthermore, our study presents a revised cDNA sequence of LIV-1 and demonstrates the localization of endogenous LIV-1 in the endoplasmic reticulum.

The human LAPTM4b transcript is upregulated in various types of solid tumours and seems to play a dual functional role during tumour progression.

LAPTM4b (lysosome associated protein transmembrane 4 beta) was recently identified as a gene overexpressed in human hepatocellular carcinoma and belongs to the mammalian LAPTM family. By analysing genome-wide expression profiles of microdissected solid tumour samples by the means of Affymetrix GenChip hybridisation, we found LAPTM4b to be upregulated in 88% (23/26) of lung and in 67% (18/27) of colon carcinoma patients. Northern blots revealed additionally an overexpression of LAPTM4b in the majority of carcinomas of the uterus (30/44), breast (27/53) and ovary (11/16). Other members of the LAPTM family were not overexpressed in the investigated tumour samples according to GeneChip hybridisation data. Northen blot and quantitative RT-PCR on different normal tissues, detected highest levels of LAPTM4b mRNA in uterus, heart and skeletal muscle. Due to sequence analysis of bilaterian LAPTM proteins we suggests the presence of four transmembrane helices per protein, which are probably packed together by hydrophobic forces that are excerted by several evolutionary conserved aromatic residues within the alpha-helices. We discuss an active role for LAPTM4b during disease progression of malignant cells and conclude that its putative dual functional involvement in tumour cell proliferation as well as in multidrug-resistance may represent LAPTM4b as a target suitable for development of novel therapeutic agents.

Loss of SFRP1 is associated with breast cancer progression and poor prognosis in early stage tumors.

Aberrant activation of the Wnt signaling pathway plays an important role in the development of solid tumors such as breast and colon cancer. Secreted Frizzled-related protein 1 (SFRP1) is a negative regulator of the Wnt pathway. It has been described that SFRP1 mRNA is strongly down-regulated in breast cancer and a putative tumor suppressor function has been postulated. We have generated and characterized an SFRP1 specific antibody to analyze its expression on protein level and to investigate the association of SFRP1 expression with clinicopathological parameters and patient survival. Analysis of >2000 invasive breast tumors and 56 carcinoma in situ revealed similar frequencies of SFRP1 loss in these tumors (46% and 43% respectively). Therefore, we propose that loss of SFRP1 expression is an early event in breast tumorigenesis. SFRP1 expression was inversely correlated with tumor stage (p<0.001) but not with tumor grade (p=0.14) or lymph node status (p=0.84). Performing a multivariate analysis we could confirm the association between tumor stage and SFRP1 expression (p=0.029). In particular, loss of SFRP1 expression in early stage breast tumors (pT1) was associated with poor prognosis (p=0.04). In conclusion, expression of SFRP1 is commonly lost in breast cancer. SFRP1 expression might be useful as a novel prognostic marker in early stage breast cancer.

Human herpesvirus 8/Kaposi sarcoma herpesvirus cell association during evolution of Kaposi sarcoma.

Kaposi sarcoma (KS) is associated with a herpesvirus (HHV-8/KSHV), which expresses a latency-associated nuclear antigen (LANA). The histopathology of KS is characterized by angiogenesis, inflammatory cells, and the development of CD34+ tumor spindle cells (SCs). However, the cellular basis for the recruitment and dissemination of HHV-8 during the development of KS lesions is not clear. Twenty-nine KS biopsies with AIDS (AKS, n=22) and without HIV infection (endemic KS or EKS, n=7) were immunostained by a triple antibody method to characterize HHV-8-infected and noninfected (LANA+/-) CD34+ SCs, infiltrating CD3+, CD68+, CD20+, and CD45+ leukocytes as well as proliferating (Ki67+) cells. The CD34+/LANA+ SCs were more frequent in late (nodular) as compared with early (patch/plaque) KS stages. However, in late AKS 36.0% of SCs (median of 11 cases) were CD34+/LANA- compared with 20.7% in early cases (median of 11 cases). Furthermore, both AKS and EKS showed, at all stages, a small (4.1-6.5%) population of LANA+/CD34- cells. Proliferating Ki67+ cells were seen (4.5-11.5%) at all KS stages, and were usually more frequent in early AKS, but no significant difference was observed between nodular AKS and EKS. Most of the proliferating cells in the KS lesions were LANA+/CD34+ but a small fraction was LANA+/CD34-. Lesional CD68+ and CD3+ cells varied between AKS (7.3 and 5.2%, respectively) and EKS (4.9 and 3.1%, respectively) but were not clearly stage related. No LANA+ cells were CD3+, CD20+, or CD45+ and very few (<0.5%) were CD68+. These results indicate that not all CD34+ KS SCs were LANA+, suggesting recruitment of noninfected SCs to the lesions. Cell proliferation in general was much higher in early as compared with the late AKS stages. LANA+ SCs could have a proliferative advantage as suggested by higher frequency of cycling (Ki67+) LANA+ SCs. Few macrophages but no lymphocytes are LANA+.

Dichotomy between CD1a+ and CD83+ dendritic cells in lymph nodes during SIV infection of macaques.

The prevalence and differentiation of dendritic cells (DC) in lymphoid tissue of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys was studied during disease progression. Lymph node biopsies were consecutively obtained from clinical rapid and slow progressors until the development of disease consistent with simian acquired immunodeficiency syndrome (sAIDS) occurred. Quantitative evaluation of CD1a+ DC and the expression of DC antigens related to maturation (CD83, DC-LAMP and S100b) were performed at the single cell level by in situ image analysis. Despite a persistent prevalence of CD1a+ DC in lymphoid tissue during disease progression, there was a subsequent drop of mature CD83+, DC-LAMP+ and S100b+ DC, correlating with the decline of CD4+ T cells in blood. Thus, disease progression to sAIDS was associated with impaired maturation of DC, and lack of CD83, DC-LAMP and S100b expression.

Immunotherapy with recombinant SFV-replicons expressing the P815A tumor antigen or IL-12 induces tumor regression.

Replicons based on alphaviruses are emerging as candidate vectors for vaccination and gene therapy purposes. We have reported previously that mice vaccinated with a recombinant Semliki Forest virus (a member of the alphavirus genus) carrying the gene encoding the P815A tumor antigen (rSFV/E-P1A) were protected against a lethal challenge with the P815 tumor. In this study we investigated the anti-tumor therapeutic efficacy of rSFV/E-P1A or rSFV expressing the cytokine interleukin 12 (rSFV/IL12) on Day 5-established tumors. The results show that both antigen-specific and cytokine-mediated rSFV treatments exhibited a significant effect on P815 tumor growth, by delaying tumor progression and even inducing complete tumor regressions in several mice. The therapeutic potency of these vectors was dependent on the size of the treated tumor, as treatment of mice bearing larger tumors showed lower efficacy. In addition, rSFV treatment resulted in long-term immunity as observed by the lack of tumor recurrence in the majority of tumor-regressing mice after rechallenge with the tumor. Furthermore, the anti-tumor therapeutic effect was only achieved by local intratumoral injection of rSFV, as treatment by injection in the contralateral site resulted in tumor progression comparable to control-untreated mice. Accordingly, expression of a rSFV-RNA was localized to the tumor and draining lymph node. These results further demonstrate the potential of rSFV replicons as tumor therapeutic agents.

Simian AIDS-related lymphoma growth in severe combined immunodeficiency mice is independent of karyotypic abnormalities or Bcl-6 mutations.

Simian AIDS-related lymphomas (sARL) of cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied in relation to growth in severe combined immunodeficient (SCID) mice, karyotype abnormalities, and DNA sequence of the first noncoding region of the Bcl-6 gene. The tumors were diffuse large B cell lymphomas and expressed a simian homolog to Epstein-Barr virus (HVMF-1) in 12 of 13 primary tumors and corresponding cell lines. A tested cell line was tumorigenic in SCID mice. Tumors in the SCID mice showed cell growth features similar to those in the original lymphoma, suggesting that no subpopulation with growth advantage was selected for in the mice. Spectral karyotype analysis of sARL cell lines showed normal cytogenetic features except for a trisomy of monkey chromosome 2 (corresponding to human chromosomes 7 and 21) in two of five sARL lines, which was not recovered in SCID tumors established from the same cell line. Sequence analysis of a Bcl-6 gene fragment showed sequence variations indicative of population polymorphism(s) in 10 of 13 sARLs, and no evidence of Bcl-6 mutations. Thus Bcl-6 mutations in the first noncoding region are irrelevant for sARL development in cynomolgus monkeys and for tumorigenicity of sARL cell lines. We also demonstrate that no cytogenetic alterations are needed for the development of highly aggressive lymphomas in the SIV-immunosuppressed host.