Alex Rufle

KIT (CD117)-Positive Breast Cancers Are Infrequent and Lack KIT Gene Mutations

Purpose: KIT (CD117) is a transmembrane tyrosine kinase representing a target for STI571 (Glivec) therapy. Some KIT-overexpressing solid tumors have responded favorably to STI571, potentially because of the presence of KIT-activating mutations. Experimental Design: To investigate the epidemiology of KIT overexpression and mutations, we investigated a series of 1654 breast cancers. All tumors were analyzed by immunohistochemistry in a tissue microarray format. Results: KIT expression was always present in normal breast epithelium. However, cancer analysis revealed the only 43 of 1654 (2.6%) tumors were KIT-positive. KIT expression was more frequent in medullary cancer (9 of 47 positive; 19.1%) than in any other histological tumor subtype (P < 0.001). KIT expression was significantly associated with high tumor grade (P < 0.0001) but unrelated to pT and pN categories or patient survival. Mutation analysis of exons 2, 8, 9, 11, 13, and 17 was negative in 10 KIT-positive tumors. Conclusions: Overall, our data show that a high level of KIT expression occurs infrequently in breast cancer. KIT-positive breast cancers may not reflect “KIT up-regulation” because KIT is also expressed in normal breast epithelium. The lack of KIT mutations also argues against the therapeutic efficacy of STI571 in breast cancer.

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features†‡

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision‐making. We report a comprehensive analysis of clinico‐pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour‐ and host‐related protein markers characterizing CIMP‐high (CIMP‐H), ‐low, and ‐negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP‐H: ≥ 4/5 methylated genes), MSI (MSI‐H: ≥ 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP‐H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI‐H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP‐low or ‐negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra‐epithelial lymphocytes (p < 0.0001) were related to CIMP‐H, independently of MSI status. In addition to the expected clinico‐pathological and molecular associations, CIMP‐H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T‐lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP‐H in both microsatellite‐stable (MSS) and MSI‐H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP‐H. Copyright

Sequence analysis and high-throughput immunhistochemical profiling of KIT (CD 117) expression in uveal melanoma using tissue microarrays

We aimed to immunohistochemically examine the expression of KIT (CD 117) in human posterior uveal melanoma and to analyze KIT-positive tumors for gene mutations. Brought into a tissue microarray (TMA) format were 101 formalin-fixed, paraffin-embedded posterior uveal melanomas. Immunhistochemistry was performed using the polyclonal anti-CD117 antibody from Dako (A4502). In ten selected KIT-positive tumors, exons 2, 8, 9, 11, 13 and 17 were sequenced. Of the 101 cases, 89 (88%) could be evaluated on the TMAs. Immunohistochemistry for CD 117 was weakly positive in 5 cases (6%), moderately positive in 10 cases (12%) and strongly positive in 57 cases (69%). No KIT mutations were detected in the analyzed exons. In conclusion, human posterior uveal melanoma frequently expresses CD117 at high levels. Although KIT mutations could not be found, it appears justified to investigate the utility of imatinib mesylate in the treatment of these patients.

Ultrastructural and molecular analysis in fatal neonatal interstitial pneumonia caused by a novel ABCA3 mutation

Pulmonary surfactant is essential to maintain alveolar patency, and invariably fatal neonatal lung disease has been recognized to involve mutations in the genes encoding surfactant protein-B or ATP-binding cassette transporter family member ABCA3. The lipid transporter ABCA3 targets surfactant phospholipids to lamellar bodies that are lysosomal-derived organelles of alveolar type II cells. ABCA3−/− mice have grossly reduced surfactant phosphatidyl glycerol levels and die of respiratory failure soon after birth. We studied lung biopsy samples of two siblings with a novel homozygous ABCA3 mutation at nucleotide position 578 (c.578C>G), leading to a Pro193Arg amino-acid exchange, who died at 55 and 105 days of age. Light microscopy revealed thickened alveolar septa with abundant myxoid interstitial matrix, marked hyperplasia of type II pneumocytes, desquamation of alveolar macrophages and focal alveolar proteinosis. Surfactant protein-B was detected by immunohistochemistry after antigen retrieval. Transmission electron microscopy showed rare cytoplasmic inclusions with concentric membranes and eccentrically placed electron-dense aggregates. These ‘fried-egg’-appearing lamellar bodies differed both from normal lamellar bodies and the larger, poorly formed composite bodies with multiple vesicular inclusions observed in surfactant protein-B deficiency. In conclusion, our findings underscore that the implications of interstitial lung disease in infant lungs differ from those in adults. In infants with a desquamative interstitial pneumonitis pattern, surfactant or ABCA3 mutations should be evaluated. Importantly, these findings support the notion that electron microscopy is useful in distinguishing between surfactant protein-B and ABCA3 deficiency, and has an important role in evaluating biopsies or autopsies of term infants with unexplained severe respiratory failure and interstitial lung disease.

The impact of CpG island methylator phenotype and microsatellite instability on tumour budding in colorectal cancer.

Zlobec I, Bihl M P, Foerster A, Rufle A & Lugli A 
(2012) Histopathology 61, 777–787

Stratification and Prognostic Relevance of Jass’s Molecular Classification of Colorectal Cancer

Background: The current proposed model of colorectal tumorigenesis is based primarily on CpG island methylator phenotype (CIMP), microsatellite instability (MSI), KRAS, BRAF, and methylation status of 0-6-Methylguanine DNA Methyltransferase (MGMT) and classifies tumors into five subgroups. The aim of this study is to validate this molecular classification and test its prognostic relevance. Methods: Three hundred two patients were included in this study. Molecular analysis was performed for five CIMP-related promoters (CRABP1, MLH1, p16INK4a, CACNA1G, NEUROG1), MGMT, MSI, KRAS, and BRAF. Methylation in at least 4 promoters or in one to three promoters was considered CIMP-high and CIMP-low (CIMP-H/L), respectively. Results: CIMP-H, CIMP-L, and CIMP-negative were found in 7.1, 43, and 49.9% cases, respectively. One hundred twenty-three tumors (41%) could not be classified into any one of the proposed molecular subgroups, including 107 CIMP-L, 14 CIMP-H, and two CIMP-negative cases. The 10 year survival rate for CIMP-high patients [22.6% (95%CI: 7–43)] was significantly lower than for CIMP-L or CIMP-negative (p = 0.0295). Only the combined analysis of BRAF and CIMP (negative versus L/H) led to distinct prognostic subgroups. Conclusion: Although CIMP status has an effect on outcome, our results underline the need for standardized definitions of low- and high-level CIMP, which clearly hinders an effective prognostic and molecular classification of colorectal cancer.

Wnt signaling pathway analysis in renal cell carcinoma in young patients

Renal cell carcinomas in young patients constitute a morphologically and genetically heterogeneous group. Twenty percent belong to the newly recognized Xp11.2 translocation-associated family and rare tumors arise from nephroblastoma. Aberrant Wnt signaling through β-catenin mutation has been implicated in nephroblastoma pathogenesis and has been found to synergize with WT1 mutations. To characterize Wnt signaling activity in renal cell carcinomas in young patients, we gathered 34 tumors (three clear cell, ten Xp11.2 translocation associated, five papillary, two chromophobe, two collecting duct, one neuroblastoma associated, eight unclassified renal cell carcinomas, and three carcinomas combined with nephroblastoma) from patients less than 22 years. Expression of β-catenin, its homologue γ-catenin, and of WT1 was assessed by immunohistochemistry in 30 tumors, and sequence analysis of CTNNB1, CTNNG1, and WT1 genes was performed in 25 tumors. Cytoplasmic β-catenin accumulation was demonstrated in two papillary carcinomas, one neuroblastoma-associated carcinoma, and two carcinomas arising from nephroblastoma. The pattern of γ-catenin expression paralleled that of β-catenin but its signal intensity was lower in 22, equal in 7, and stronger only in 1 tumor, respectively. Four tumors showed nuclear WT1 expression. One Xp11.2 translocation-associated carcinoma presented a rare intronic CTNNB1 single nucleotide polymorphism and cytoplasmic β-catenin accumulation. There were no further CTNNB1 or CTNNG1 sequence alterations. A WT1 mutation was found in the nephroblastoma component of a carcinoma arising from nephroblastoma. These findings suggest Wnt signaling pathway activation only in a minority of renal cell carcinomas in young patients. CTNNB1 mutations are rare events. Cytoplasmic β-catenin accumulation in an Xp11.2-associated carcinoma suggests potential interaction of Wnt signaling components with microphthalmia transcription factor family also in Xp11.2 translocation carcinomas. WT1 mutation in the nephroblastoma component of a mixed-type renal cell carcinoma provides direct evidence for clonal independence of nephroblastoma and carcinoma components in this exceptional tumor.

Unique Composite Hematolymphoid Tumor Consisting of a Pro-T Lymphoblastic Lymphoma and an Indeterminate Dendritic Cell Tumor: Evidence for Divergent Common Progenitor Cell Differentiation

Until recently, hematopoietic neoplasms were considered monoclonal proliferations belonging to one cell lineage. In the last years, evidence for transdifferentiation from one cell lineage to another or divergent common progenitor cell differentiation has accumulated, mainly based on composite hematolymphoid tumors, sharing common genetic abnormalities. We report the case of a 59-year-old woman with a composite pro-T lymphoblastic lymphoma (LBL) and indeterminate dendritic cell tumor infiltrating the lymph nodes, bone marrow and stomach. Genetic analyses revealed that both cell populations bore +21, while a G13D mutation of the NRAS gene and monosomy 18 were detected only in the pro-T LBL. The synchronous appearance of two distinct uncommon hematolymphoid tumors in the same patient, recurrent at three different anatomic locations, with an identifiable common genetic denominator, namely +21, but also with unique genetic anomalies in the pro-T LBL raises the hypothesis of a divergent common progenitor cell differentiation.

KIT, PDGFRα and EGFR analysis in nephroblastoma

Nephroblastoma prognosis has dramatically improved, but an unfavourable prognostic subgroup warrants development of novel therapeutic strategies. Selective KIT, PDGFRα and epidermal growth factor receptor (EGFR) tyrosine kinase inhibition evolved as powerful targeted therapy for gastrointestinal stromal tumours and non-small-cell lung cancer. To investigate a potential role for tyrosine kinase inhibition, we analyzed 209 nephroblastomas for immunohistochemical KIT and EGFR expression, 63 nephroblastomas for mutations in KIT exons 9, 11, 13, EGFR exons 18, 19, 20 and 21, and all 209 nephroblastomas for PDGFRα exons 12, 14 and 18. Twenty-two tumours (10.5%) expressed KIT, 31 (14.8%) EGFR, and 10 (4.8%) both KIT and EGFR, respectively. KIT expression was relatively more common among high-risk tumours (6/27; 22.3%) compared to low-/intermediate-risk tumours (26/181; 14.4%). Nine patients deceased, four of which had high-risk tumours with KIT expression in two of four and EGFR expression in one of four. There were no KIT, PDGFRα or EGFR mutations. Our results suggest no significant contribution of KIT, EGFR or PDGFRα mutations to nephroblastoma pathogenesis. Despite a trend towards association of immunohistochemical KIT and EGFR expression with poor outcome in high-risk nephroblastomas, statistical analysis did not yield significant correlations in this subgroup. Therefore, it remains open if KIT, PDGFRα or EGFR tyrosine kinase inhibition constitute a therapeutic target in nephroblastoma in the absence of KIT, PDGFRα or EGFR mutations.

Novel cell enrichment technique for robust genetic analysis of archival classical Hodgkin lymphoma tissues

Approximately 15% of patients with classical Hodgkin lymphoma (cHL) die after relapse or progressive disease. Comprehensive genetic characterization is required to better understand its molecular pathology and improve management. However, genetic information on cHL is hard to obtain mainly due to rare malignant Hodgkin- and Reed-Sternberg cells (HRSC), whose overall frequencies in the affected tissues ranges from 0.1 to 10%. Therefore, enrichment of neoplastic cells is necessary for the majority of genetic investigations. We have developed a new high-throughput method for marker-based enrichment of archival formalin-fixed and paraffin-embedded (FFPE) tissue-derived HRSC nuclei by fluorescence-assisted flow sorting (FACS) and successfully applied it on ten cHL cases. Genomic DNA extracted from sorted nuclei was used for targeted high-throughput sequencing (HTS) of 68 genes that are frequently affected in lymphomas. Chromosomal copy number aberrations were investigated by the Agilent SurePrint 180k microarray. Our method enabled HRSC nuclei enrichment to 40–90% in sorted populations. This level of enrichment was sufficient for reliable identification of tumor-specific mutations and copy number aberrations. Genetic analysis revealed that components of JAK-STAT signaling pathway were affected in all investigated tumors by frequent mutations of SOCS1 and STAT6 as well as copy number gains of JAK2. Involvement of nuclear factor-κB (NF-κB) pathway compounds was evident from recurrent gains of the locus containing the REL gene and mutations in TNFAIP3 and CARD11. Finally, genetic alterations of PD-L1 and B2M suggested immune evasion as mechanisms of oncogenesis in some patients. In this work, we present a new method for HRSC enrichment from FFPE tissue blocks by FACS and demonstrate the feasibility of a wide-scale genetic analysis by cutting-edge molecular methods. Our work opens the door to a large resource of archived clinical cHL samples and lays foundation to more complex studies aimed to answer important biological and clinical questions that are critical to improve cHL management.This paper describes the enrichment of rare neoplastic Hodgkin and Reed-Sternberg (HRSC) cells of classical Hodgkin lymphoma (cHL) from the archival formalin-fixed and paraffin-embedded tissues. This technique allows enrichment of HRSC from 1-5% in the initial cHL tissue to 40-80% in sorted populations, which enables robust genetic analysis of cHL by targeted high throughput sequencing and array-CGH.