Christoph Thorns

Immunohistochemical Prognostic Markers in Diffuse Large B-Cell Lymphoma: Validation of Tissue Microarray As a Prerequisite for Broad Clinical Applications—A Study From the Lunenburg Lymphoma Biomarker Consortium

PURPOSE The results of immunohistochemical class prediction and prognostic stratification of diffuse large B-cell lymphoma (DLBCL) have been remarkably various thus far. Apart from biologic variations, this may be caused by differences in laboratory techniques, scoring definitions, and inter- and intraobserver variations. In this study, an international collaboration of clinical lymphoma research groups from Europe, United States, and Canada concentrated on validation and standardization of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL. PATIENTS AND METHODS Sections of a tissue microarray from 36 patients with DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM1, and MIB-1 according to local methods. The study was performed in two rounds firstly focused on the evaluation of laboratory staining variation and secondly on the scoring variation. RESULTS Different laboratory staining techniques resulted in unexpectedly highly variable results and very poor reproducibility in scoring for almost all markers. No single laboratory stood out as uniformly poor or excellent. With elimination of variation due to staining, high agreement was found for CD20, HLA-DR, and CD10. Poor agreement was found for bcl-6 and Ki-67. Optimization of techniques and uniformly agreed on scoring criteria improved reproducibility. CONCLUSION This study shows that semiquantitative immunohistochemistry for subclassification of DLBCL is feasible and reproducible, but exhibits varying rates of concordance for different markers. These findings may explain the wide variation of biomarker prognostic impact reported in the literature. Harmonization of techniques and centralized consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.

MicroRNA signatures characterize diffuse large B-cell lymphomas and follicular lymphomas

MicroRNAs (miRNA, miR) are negative regulators of gene expression that play an important role in diverse biological processes such as development, cell growth, apoptosis and haematopoiesis, suggesting their association with cancer. Here we analysed the expression signatures of 157 miRNAs in 58 diffuse large B‐cell lymphoma (DLBCL), 46 follicular lymphoma (FL) and seven non‐neoplastic lymph nodes (LN). Comparison of the possible combinations of DLBCL‐, FL‐ and LN resulted in specific DLBCL‐ and FL‐signatures, which include miRNAs with previously published function in haematopoiesis (MIRN150 and MIRN155) or tumour development (MIRN210, MIRN10A, MIRN17‐5P and MIRN145). As compared to LN, some miRNAs are differentially regulated in both lymphoma types (MIRN155, MIRN210, MIRN106A, MIRN149 and MIRN139). Conversely, some miRNAs show lymphoma‐specific aberrant expression, such as MIRN9/9*, MIRN301, MIRN338 and MIRN213 in FL and MIRN150, MIRN17‐5P, MIRN145, MIRN328 and others in DLBCL. A classification tree was computed using four miRNAs (MIRN330, MIRN17‐5P, MIRN106a and MIRN210) to correctly identify 98% of all 111 cases that were analysed in this study. Finally, eight miRNAs were found to correlate with event‐free and overall survival in DLBCL including known tumour suppressors (MIRN21, MIRN127 and MIRN34a) and oncogenes (MIRN195 and MIRNLET7G).

Prognostic significance of immunohistochemical biomarkers in diffuse large B-cell lymphoma: a study from the Lunenburg Lymphoma Biomarker Consortium

The Lunenburg Lymphoma Biomarker Consortium (LLBC) evaluated the prognostic value of IHC biomarkers in a large series of patients with diffuse large B-cell lymphoma (DLBCL). Clinical data and tumor samples were retrieved from 12 studies from Europe and North America, with patients treated before or after the rituximab era. Using tissue microarrays from 1514 patients, IHC for BCL2, BCL6, CD5, CD10, MUM1, Ki67, and HLA-DR was performed and scored according to previously validated protocols. Optimal cut points predicting overall survival of patients treated in the rituximab era could only be determined for CD5 (P = .003) and Ki67 (P = .02), whereas such cut points for BCL2, BCL6, HLA-DR, and MUM1 could only be defined in patients not receiving rituximab. A prognostic model for patients treated in the rituximab era identified 4 risk groups using BCL2, Ki67, and International Prognostic Index (IPI) with improved discrimination of low-risk patients. Newly recognized correlations between specific biomarkers and IPI highlight the importance of carefully controlling for clinical and biologic factors in prognostic models. These data demonstrate that the IPI remains the best available index in patients with DLBCL treated with rituximab and chemotherapy.

Immunoblastic morphology but not the immunohistochemical GCB/nonGCB classifier predicts outcome in diffuse large B-cell lymphoma in the RICOVER-60 trial of the DSHNHL

The survival of diffuse large B-cell lymphoma patients varies considerably, reflecting the molecular diversity of tumors. In view of the controversy whether cytologic features, immunohistochemical markers or gene expression signatures may capture this molecular diversity, we investigated which features provide prognostic information in a prospective trial in the R-CHOP treatment era. Within the cohort of DLBCLs patients treated in the RICOVER-60 trial of the German High-Grade Lymphoma Study Group (DSHNHL), we tested the prognostic impact of IB morphology in 949 patients. The expression of immunohistochemical markers CD5, CD10, BCL2, BCL6, human leukocyte antigen (HLA)-DR, interferon regulatory factor-4/multiple myeloma-1 (IRF4/MUM1), and Ki-67 was assessed in 506 patients. Expression of the immunohistochemical markers tested was of modest, if any, prognostic relevance. Moreover, the Hans algorithm using the expression patterns of CD10, BCL6, and interferon regulatory factor-4/multiple myeloma-1 failed to show prognostic significance in the entire cohort as well as in patient subgroups. IB morphology, however, emerged as a robust, significantly adverse prognostic factor in multivariate analysis, and its diagnosis showed a good reproducibility among expert hematopathologists. We conclude, therefore, that IB morphology in DLBCL is likely to capture some of the adverse molecular alterations that are currently not detectable in a routine diagnostic setting, and that its recognition has significant prognostic power.

Chromosomal aberrations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma unspecified: A matrix-based CGH approach

Angioimmunoblastic T‐cell lymphoma (AILT) is a histopathologically well‐defined entity. However, despite a number of cytogenetic studies, the genetic basis of this lymphoma entity is not clear. Moreover, there is an overlap to some cases of peripheral T‐cell lymphoma unspecified (PTCL‐u) in respect to morphological and genetic features. We used array‐based comparative genomic hybridization (CGH) to study genetic imbalances in 39 AILT and 20 PTCL‐u. Array‐based CGH revealed complex genetic imbalances in both AILT and PTCL‐u. Chromosomal imbalances were more frequent in PTCL‐u than in AILT and gains exceeded the losses. The most recurrent changes in AILT were gains of 22q, 19, and 11p11–q14 (11q13) and losses of 13q. The most frequent changes in PTCL‐u were gains of 17 (17q11–q25), 8 (involving the MYC locus at 8q24), and 22q and losses of 13q and 9 (9p21–q33). Interestingly, gains of 4q (4q28–q31 and 4q34–qtel), 8q24, and 17 were significantly more frequent in PTCL‐u than in AILT. The regions 6q (6q16–q22) and 11p11 were predominantly lost in PTCL‐u. Moreover, we could identify a recurrent gain of 11q13 in both AILT and PTCL‐u, which has previously not been described in AILT. Trisomies 3 and 5, which have been described as typical aberrations in AILT, were identified only in a small number of cases. In conclusion, CGH revealed common genetic events in peripheral T‐cell lymphomas as well as peculiar differences between AILT and PTCL‐u.

Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as a prerequisite for broad clinical applications (a study from the Lunenburg Lymphoma Biomarker Consortium).

Background and Aims: The results of class prediction and the determination of prognostic markers in diffuse large B-cell lymphoma (DLBCL) have been variably reported. Apart from biological variations, this may be caused by differences in laboratory techniques, scoring definitions and inter- and intra-observer variation. In this study, an international collaboration of clinical lymphoma research groups has concentrated on validation and standardisation of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL. Methods: Sections of a tissue microarray with 36 cases of DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM-1 and Ki-67 according to local methods. The study was performed in two rounds, firstly focused on the evaluation of laboratory staining variation, and secondly on the scoring variation. Results: Different techniques resulted in highly variable results and poor reproducibility for almost all markers. Reproducibility of the nuclear markers was highly sensitive to technical variations, including immunological enhancement techniques (agreements 34%). With elimination of variation due to staining and uniformly agreed on scoring criteria, significant improvement was seen; however less so for bcl-6 and Ki-67 (agreement 53–58%). Absence of internal controls that preclude scoring, significantly influenced the results for CD10 and bcl-6. Conclusion: Semi-quantitative immunohistochemistry for subclassification of DLBCL is feasible, but with varying rates of concordance for different markers and only using optimised techniques and strict scoring criteria. These findings may explain the wide variation in prognostic impact reported in the literature. Harmonisation of techniques and centralised consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.

miRNA expression profiling in formalin-fixed and paraffin-embedded placental tissue samples from pregnancies with severe preeclampsia

Abstract Aims: Micro RNAs (miRNAs) are small, single-strand RNAs, playing an important role in post-transcriptional gene regulation. The placenta is considered to play a key role in pathogenesis of preeclampsia. The purpose of this study was to demonstrate deregulation of miRNAs in placentas with preeclampsia using formalin-fixed and paraffin-embedded (FFPE) tissues. Methods: Expression levels of 162 miRNAs were measured in FFPE placental tissues (5 with severe preeclampsia, 5 from a control group) using a quantitative qPCR based technique. Results: Six miRNAs were more than 2-fold over-expressed in severe preeclampsia: let-7b, miRNA-302*, miRNA-104, miRNA-128a, miRNA-182* and miRNA-133b. Gene ontology analyses were performed using the algorithms “TargetScanS”, “microRNA”, and “PicTar”. Conclusions: Two of the up-regulated miRNAs (miRNA-182* and miRNA-133b) are putative regulators of the transcript variants 1 and 2 of the BCL2-like gene, which controls apoptosis. miRNA-182* is also a probable angiogenesis regulator via angiogenin and VEGF-B. Apoptosis and angiogenesis are major mechanisms presumed to be involved in the pathogenesis of preeclampsia. Moreover, usability of qPCR technique based miRNA profiling for FFPE tissues was proofed. Hence FFPE tissue is the most widely used material for retrospective clinical studies, this method has a great property for future investigations in placenta research.

Prognostic factors (including HPV status) for irradiation of locally advanced squamous cell carcinoma of the head and neck (SCCHN)

Background and PurposeThe prognosis of patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN) is generally poor. However, prognostic factors can help optimize the care for the individual patient. This study investigated potential prognostic factors, including HPV status, for locoregional control (LRC), metastases-free survival (MFS), and survival (OS).Patients and MethodsTwelve potential prognostic factors were investigated in 170 patients irradiated for stage III or IV SCCHN, including age (≤ 60 vs > 60 years), gender, ECOG performance score (0–1 vs 2), preradiotherapy hemoglobin level (< 12 vs ≥ 12 g/dl), tumor site (oropharynx, oral cavity, hypopharynx, or larynx), histological grade (G1–2 vs G3), T category (T1–T2 vs T3–T4), N category (N0–N1 vs N2–N3), AJCC stage (III vs IV), surgery (no vs yes), and chemotherapy (no vs yes).ResultsOn multivariate analysis, positive HPV status (RR 2.34; p = 0.014), ECOG performance score 0–1 (RR 1.94; p = 0.017), preRT hemoglobin ≥ 12 g/dl (RR 1.88; p = 0.018), T category T1–T2 (RR 2.72; p < 0.001), and surgery (RR 2.29; p = 0.007) were significantly associated with improved LRC. PreRT hemoglobin ≥ 12 g/dl (RR 1.98; p = 0.040) and T category T1–T2 (RR 3.33; p < 0.001) were significantly associated with improved MFS. Positive HPV status (RR 2.19; p = 0.019), pre-RT hemoglobin ≥ 12 g/dl (RR 2.15; p = 0.002), T category T1–T2 (RR 2.31; p = 0.002), and AJCC stage III (RR 1.91; p = 0.034) were significantly associated with improved OS.Conclusion Improved treatment outcomes were significantly associated with positive HPV status, better performance status, lower tumor stage, and pretreatment hemoglobin levels ≥ 12 g/dl. These factors should be considered in future trials.HintergrundDie Prognose von Patienten mit lokal fortgeschrittenem Plattenepithelkarzinom im Kopf-Hals-Bereich (SCCHN) ist häufig schlecht. Prognosefaktoren können dabei helfen, die optimale Behandlung für den individuellen Patienten auszuwählen. Diese Studie untersuchte mögliche Prognosefaktoren inklusive HPV-Status für die lokoregionale Kontrolle (LRC), das metastasen-freie Überleben (MFS) und das Gesamtüberleben (OS).Material und MethodeZwölf mögliche Prognosefaktoren wurden in einer Serie von 170 Patienten, die aufgrund eines SCCHN im Stadium III oder IV eine Strahlentherapie erhielten, untersucht. Diese Faktoren waren Alter (≤ 60 vs > 60 Jahre), Geschlecht, Allgemeinzustand (ECOG 0–1 vs 2), Hämoglobinwert vor Strahlentherapie (< 12 vs ≥ 12 g/dl), Tumorlokalisation (Oropharynx, Mundhöhle, Hypopharynx, Larynx), Grading (G1–2 vs G3), T-Kategorie (T1–T2 vs T3–T4), N-Kategorie (N0–N1 vs N2–N3), AJCC-Stadium (III vs IV), Operation (nein vs ja) und Chemotherapie (nein vs ja).ErgebnisseIn der multivariaten Analyse waren ein positiver HPV-Status (RR 2,34; p = 0,014), ein besserer Allgemeinzustand (RR 1,94; p = 0,017), Hämoglobinwerte ≥ 12 g/dl (RR 1,88; p = 0,018), eine niedrigere T-Kategorie (RR 2,72; p < 0,001) und eine Operation (RR 2,29; p = 0,007) signifikant mit einer besseren LRC assoziiert. Hämoglobinwerte ≥ 12 g/dl (RR 1,98; p = 0,040) und eine niedrigere T-Kategorie (RR 3,33; p < 0,001) waren signifikant mit einem besseren MFS assoziiert. Ein positiver HPV-Status (RR 2,19; p = 0,019), Hämoglobinwerte ≥ 12 g/dl (RR 2,15; p = 0,002), eine niedrigere T-Kategorie (RR 2,31; p = 0,002) und AJCC-Stadium III (RR 1,91; p = 0,034) waren signifikant mit einem besseren OS assoziiert.SchlussfolgerungenEine Verbesserung der Behandlungsergebnisse war signifikant mit positivem HPV-Status, besserem Allgemeinzustand, niedrigerem Tumorstadium und Hämoglobinwerten ≥12 g/dl vor Therapie assoziiert. Diese Faktoren sollten in zukünftigen Studien berücksichtigt werden.

Detection of genomic aberrations in molecularly defined Burkitt’s lymphoma by array-based, high resolution, single nucleotide polymorphism analysis

Background Knowledge about the genetic lesions that occur in Burkitt’s lymphoma, besides the pathognomonic IG-MYC translocations, is limited. Design and Methods Thirty-nine molecularly-defined Burkitt’s lymphomas were analyzed with high-resolution single-nucleotide polymorphism chips for genomic imbalances and uniparental disomy. Imbalances were correlated to expression profiles and selected micro-RNA analysis. Translocations affecting the MYC locus were studied by fluoresence in situ hybridization. Results We detected 528 copy number changes, defining 29 recurrently imbalanced regions. Five hundred and eighteen regions of uniparental disomy were found, but these were rarely recurrent. Combined imbalance mapping and expression profiling revealed a strong correlation between copy number and expression. Several recurrent imbalances affected the MYC pathway: the micro-RNA-supercluster 17-92 was frequently gained and the transcription factor E2F2 was recurrently deleted. Molecular Burkitt’s lymphoma lacking MYC translocations showed MYC gains. Amplifications of the polymerase iota gene were associated with increased frequency of positions scored as aberrant. Conclusions The present findings suggest that uniparental disomies do not play a major role in the pathogenesis of Burkitt’s lymphoma, whereas some genes may contribute to the development of this lymphoma through gene dosage effects. Amplifications of the polymerase iota gene may be functionally linked with increased genomic alterations in Burkitt’s lymphoma. The pattern and rarity of chromosomal changes detectable, even at the high resolution employed here, together with aberrations of genes regulating MYC activity, support the hypothesis that deregulation of the MYC pathway is the major force driving the pathogenesis of Burkitt’s lymphoma, but show that this deregulation is more complex than previously known.

Prognostic Value of MCM2 Immunoreactivity in Stage T1 Transitional Cell Carcinoma of the Bladder

OBJECTIVE Due to the heterogeneous biologic behavior of stage T1 bladder carcinomas, there is a need for new markers allowing to assess the prognosis more accurately. To our knowledge, there are no reports on studies investigating minichromosome maintenance protein 2 (MCM2) expression in bladder carcinomas. Thus, we investigated the prognostic value of MCM2 immunoreactivity in stage T1 bladder tumors. METHODS Fifty-four tumors were analyzed using Biochip microarrays. Also p53 and Ki67 antigen expression were examined. Immunohistochemical scores were compared with the clinical outcome. RESULTS During a median follow-up of 43 months, tumor recurrence was registered in 43 and progression to stage T2 in 19 patients. Kaplan-Meier curves demonstrated that high-level MCM2 expression was significantly associated with early tumor recurrence when using a cutoff of 60% (p=0.0035 by log-rank test), and with early tumor progression when using a cutoff of 20% (p=0.0454). There was no relationship (p=0.604) between MCM2 and p53, but a tendentious relationship (p=0.082) between MCM2 and Ki67 antigen expression. MCM2 (p=0.006), Ki67 antigen (p=0.035) and p53 expression (p=0.049) as well as tumor grade (p=0.026) and age (p=0.025) were found significantly associated with recurrence-free survival by univariate Cox regression analysis, among which only Ki67 antigen expression (p=0.015) and age (p=0.019) proved to be of independent predictive value by multivariate analysis. Concerning tumor progression, MCM2 expression was identified as the only predictive parameter by log-rank test, but it was not of independent predictive value by multivariate analysis (p=0.101). CONCLUSION Our data suggest that MCM2 expression may bear some prognostic relevance in stage T1 bladder carcinomas.