Blood taken immediately after fatal resuscitation attempts yields higher quality DNA for genetic studies as compared to autopsy samples.

BACKGROUND: The out-of-hospital cardiac arrest (OHCA) in the young may be associated with a genetic predisposition which is relevant even for genetic counseling of relatives. The identification of genetic variants depends on the availability of intact genomic DNA. DNA from autopsy may be not available due to low autopsy frequencies or not suitable for high-throughput DNA sequencing (NGS). The emergency medical service (EMS) plays an important role to save biomaterial for subsequent molecular autopsy. It is not known whether the DNA integrity of samples collected by the EMS is better suited for NGS than autopsy specimens. MATERIAL AND METHODS: DNA integrity was analyzed by standardized protocols. Fourteen blood samples collected by the EMS and biomaterials from autopsy were compared. We collected 172 autopsy samples from different tissues and blood with postmortem intervals of 14-168 h. For comparison, DNA integrity derived from blood stored under experimental conditions was checked against autopsy blood after different time intervals. RESULTS: DNA integrity and extraction yield were higher in EMS blood compared to any autopsy tissue. DNA stability in autopsy specimens was highly variable and had unpredictable quality. In contrast, collecting blood samples by the EMS is feasible and delivered comparably the highest DNA integrity. CONCLUSIONS: Isolation yield and DNA integrity from blood samples collected by the EMS is superior in comparison to autopsy specimens. DNA from blood samples collected by the EMS on scene is stable at room temperature or even for days at 4 °C. We conclude that the EMS personnel should always save a blood sample of young fatal OHCA cases died on scene to enable subsequent genetic analysis.

A new technological approach in diagnostic pathology: mass spectrometry imaging-based metabolomics for biomarker detection in urachal cancer.

Urachal adenocarcinomas (UrC) are rare but aggressive. Despite being of profound therapeutic relevance, UrC cannot be differentiated by histomorphology alone from other adenocarcinomas of differential diagnostic importance. As no reliable tissue-based diagnostic biomarkers are available, we aimed to detect such by integrating mass-spectrometry imaging-based metabolomics and digital pathology, thus allowing for a multimodal approach on the basis of spatial information. To achieve this, a cohort of UrC (n = 19) and colorectal adenocarcinomas (CRC, n = 27) as the differential diagnosis of highest therapeutic relevance was created, tissue micro-arrays (TMAs) were constructed, and pathological data was recorded. Hematoxylin and eosin (H&E) stained tissue sections were scanned and annotated, enabling an automized discrimination of tumor and non-tumor areas after training of an adequate algorithm. Spectral information within tumor regions, obtained via matrix-assisted laser desorption/ionization (MALDI)-Orbitrap-mass spectrometry imaging (MSI), were subsequently extracted in an automated workflow. On this basis, metabolic differences between UrC and CRC were revealed using machine learning algorithms. As a result, the study demonstrated the feasibility of MALDI-MSI for the evaluation of FFPE tissue in UrC and CRC with the potential to combine spatial metabolomics data with annotated histopathological data from digitalized H&E slides. The detected Area under the curve (AUC) of 0.94 in general and 0.77 for the analyte taurine alone (diagnostic accuracy for taurine: 74%) makes the technology a promising tool in this differential diagnostic dilemma situation. Although the data has to be considered as a proof-of-concept study, it presents a new adoption of this technology that has not been used in this scenario in which reliable diagnostic biomarkers (such as immunohistochemical markers) are currently not available.

Double adenomas of the pituitary reveal distinct lineage markers, copy number alterations, and epigenetic profiles.

PURPOSE: Pituitary adenoma (PA) constitutes the third most common intracranial neoplasm. The mostly benign endocrine lesions express no hormone (null cell PA) or the pituitary hormone(s) of the cell lineage of origin. In 0.5-1.5% of surgical specimens and in up to 10% of autopsy cases, two or three seemingly separate PA may coincide. These multiple adenomas may express different hormones, but whether or not expression of lineage-restricted transcription factors and molecular features are distinct within multiple lesions remains unknown. METHODS: Searching the data bank of the German Pituitary Tumor Registry 12 double pituitary adenomas with diverse lineage were identified among 3654 adenomas and 6 hypophyseal carcinomas diagnosed between 2012 and 2020. The double adenomas were investigated immunohistochemically for expression of hormones and lineage markers. In addition, chromosomal gains and losses as well as global DNA methylation profiles were assessed, whenever sufficient material was available (n = 8 PA). RESULTS: In accordance with the literature, combinations of GH/prolactin/TSH-FSH/LH adenoma (4/12), GH/prolactin/TSH-ACTH adenoma (3/12), and ACTH-FSH/LH adenoma (3/12) were observed. Further, two out of 12 cases showed a combination of a GH/prolactin/TSH adenoma with a null-cell adenoma. Different expression pattern of hormones were confirmed by different expression of transcription factors in 11/12 patients. Finally, multiple lesions that were molecularly analysed in 4 patients displayed distinct copy number changes and global methylation pattern. CONCLUSION: Our data confirm and extend the knowledge on multiple PA and suggest that such lesions may origin from distinct cell types.

Glioblastoma multiforme: Metabolic differences to peritumoral tissue and IDH-mutated gliomas revealed by mass spectrometry imaging.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. High infiltration rates and poor therapy responses make it the deadliest glioma. The tumor metabolism is known to differ from normal one and is influenced through various factors which can lead to longer survival. Metabolites are small molecules (< 1500 Da) that display the metabolic pathways in the tissue. To determine the metabolic alterations between tumor and peritumoral tissue in human GBMs, mass spectrometry imaging (MSI) was performed on thin sections from 25 resected tumors. In addition, the GBMs were compared with six gliomas harboring a mutation in the isocitrate dehydrogenase (IDH1) gene (IDH1). With this technique, a manifold of analytes can be easily visualized on a single tissue section. Metabolites were annotated based on their accurate mass using high resolution MSI. Differences in their mean intensities in the tumor and peritumoral areas were statistically evaluated and abundances were visualized on the tissue. Enhanced levels of the antioxidants ascorbic acid, taurine, and glutathione in tumor areas suggest protective effects on the tumor. Increased levels of purine and pyrimidine metabolism compounds in GBM areas indicate the high energy demand. In accordance with these results, enhanced abundances of lactate and glutamine were detected. Moreover, decreased abundance of N-acetylaspartate, a marker for neuronal health, was measured in tumor areas. Obtained metabolic information could potentially support and personalize therapeutic approaches, hence emphasizing the suitability of MSI for GBM research.

RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance.

BACKGROUND: Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10-20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma. METHODS: We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series. RESULTS: Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript. CONCLUSIONS: The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.

Unusual clinico-pathological features in primary Hodgkin's lymphomas of the central nervous system.

Primary Hodgkin's lymphomas of the central nervous system (CNS) as well as cerebral involvement as the first manifestation of a systemic Hodgkin's disease are very rare. CNS involvement usually occurs in patients with advanced or relapsing systemic disease. Because primary CNS Hodgkin's lymphoma may present unexpected and sometimes misleading clinical and neuroradiological features, the description of unusual cases is important for expanding the awareness of this rare disease of the central nervous system. We describe three cases of primary Hodgkin's lymphoma of the CNS with peculiar features. None of the three patients had a previous clinical history of systemic Hodgkin disease. Case 1 and case 2 presented an unusual localization in the cerebellar hemisphere and in the brainstem, respectively. The third case occurred as a temporal lesion in the settings of a Richter transformation of a chronic lymphocytic leukemia.

Subtyping non-small cell lung cancer by histology-guided spatial metabolomics.

PURPOSE: Most cancer-related deaths worldwide are associated with lung cancer. Subtyping of non-small cell lung cancer (NSCLC) into adenocarcinoma (AC) and squamous cell carcinoma (SqCC) is of importance, as therapy regimes differ. However, conventional staining and immunohistochemistry have their limitations. Therefore, a spatial metabolomics approach was aimed to detect differences between subtypes and to discriminate tumor and stroma regions in tissues. METHODS: Fresh-frozen NSCLC tissues (n = 35) were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of small molecules (< m/z 1000). Measured samples were subsequently stained and histopathologically examined. A differentiation of subtypes and a discrimination of tumor and stroma regions was performed by receiver operating characteristic analysis and machine learning algorithms. RESULTS: Histology-guided spatial metabolomics revealed differences between AC and SqCC and between NSCLC tumor and tumor microenvironment. A diagnostic ability of 0.95 was achieved for the discrimination of AC and SqCC. Metabolomic contrast to the tumor microenvironment was revealed with an area under the curve of 0.96 due to differences in phospholipid profile. Furthermore, the detection of NSCLC with rarely arising mutations of the isocitrate dehydrogenase (IDH) gene was demonstrated through 45 times enhanced oncometabolite levels. CONCLUSION: MALDI-MSI of small molecules can contribute to NSCLC subtyping. Measurements can be performed intraoperatively on a single tissue section to support currently available approaches. Moreover, the technique can be beneficial in screening of IDH-mutants for the characterization of these seldom cases promoting the development of treatment strategies.

Impact of left atrial appendage fibrosis on atrial fibrillation in patients following coronary bypass surgery.

OBJECTIVES: We aimed to assess the relationship of left atrial appendage (LAA) fibrosis with atrial fibrillation (AF) and postoperative events in patients receiving coronary artery bypass graft surgery (CABG). BACKGROUND: Increased atrial fibrosis has been associated with AF and worse outcome following catheter ablation. Only limited data exists focusing on the impact of LAA fibrosis on AF after CABG. METHODS: LAA tissue from 164 CABG-patients was stained with Masson-Goldner trichrome. The histological landscape was scanned and segmented into superpixels for software analysis (QuPath). A classification algorithm was extensively trained to detect fibrotic superpixels for quantification. In 43 propensity score matched pairs with AF or sinus rhythm (SR), LAA fibrosis was compared. Moreover, subgroups of mitral valve regurgitation (MR) were analyzed as follows: SR, SR + MR, AF and AF + MR. The predictive value of LAA fibrosis postoperative stroke, postoperative AF and mortality was assessed. RESULTS: Fibrotic remodeling (%) showed no significant difference for the total cohort between the SR and AF group (SR: 30.8 ± 11.4% and AF: 33.8 ± 16.0%, respectively, p = .32). However, significant fibrotic remodeling was observed for SR and AF subgroups (SR: 27.2 ± 12.2% vs. AF: 35.3 ± 13.7%; respectively, p = .049) and between SR and SR + MR subgroups (SR: 27.2 ± 12.2% vs. SR + MR: 34.9 ± 9.1%, respectively, p = .027). LAA fibrosis was not significantly associated with postoperative stroke, postoperative AF or overall mortality (all p > .05). CONCLUSION: LAA fibrosis may contribute to an individual arrhythmia substrate for AF in patients with AF but also in those with SR and coincidence of MR. LAA fibrosis was not found to be predictive for clinical events in patients after CABG.

The emergency medical service has a crucial role to unravel the genetics of sudden cardiac arrest in young, out of hospital resuscitated patients: Interim data from the MAP-IT study.

BACKGROUND: Genetics of sudden cardiac deaths (SCD) remains frequently undetected. Genetic analysis is recommended in undefined selected cases in the 2021 ERC-guideline. The emergency medical service and physicians (EMS) may play a pivotal role for unraveling SCD by saving biomaterial for later molecular autopsy. Since for high-throughput DNA-sequencing (NGS) high quality genomic DNA is needed. We investigated in a prospective proof-of-concept study the role of the EMS for the identification of genetic forms of SCDs in the young. METHODS: We included patients aged 1-50 years with need for cardiopulmonary resuscitation attempts (CPR). Cases with non-natural deaths were excluded. In two German counties with 562,904 residents 39,506 services were analysed. Paired end panel-sequencing was performed, and variants were classified according to guidelines of the American College of Medical Genetics (ACMG). RESULTS: 769 CPR-attempts were recorded (1.95% of all EMS-services; CPR-incidence 68/100,000). In 103 cases CPR were performed in patients < 50y. 58% died on scene, 26% were discharged from hospital. 24 subjects were included for genotyping. Of these 33% died on scene, 37.5% were discharged from hospital. 25% of the genotyped patients were carriers of (likely) pathogenic (ACMG-4/-5) variants. 67% carried variants with unknown significance (ACMG-3). 2 of them had familial history for arrhythmogenic cardiomyopathy or had to be re-classified as ACMG-4 carriers due to whole exome sequencing. CONCLUSION: The EMS contributes especially in fatal OHCA-cases to increase the yield of identified genetic conditions by collecting a blood sample on scene. Thus, the EMS can contribute significantly to primary and secondary prophylaxis in affected families.

Multicenter Evaluation of a Novel Automated Rapid Detection System of BRAF Status in Formalin-Fixed, Paraffin-Embedded Tissues.

The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology.

Interactions between human colon carcinoma cells, fibroblasts and monocytic cells in coculture--regulation of cathepsin B expression and invasiveness.

To elucidate the importance of the tumor/host interaction in malignant tumors, we investigated the colon carcinoma cell line HT-29 in coculture with monocytic cells (THP-1) and fibroblasts (175BR) for cathepsin B expression and activity. The tumor cells were grown in monolayer cultures or as multicellular tumor spheroids. After coculture, the three cell types were separated by labeled magnetic beads for cathepsin B mRNA and protein analysis. The invasive potential was studied in in vitro invasion assays. The expression level of cathepsin B was found to be 10-fold increased in three dimensional spheroids of HT-29 compared to HT-29 monolayers. The coculture of HT-29 with THP-1 cells and/or human fibroblasts led to a considerable increase in cathepsin B mRNA expression in both tumor and tumor-associated cells. The invasive potential of the tumor cells was 5 times increased by adding monocytic cells to the assay system. This is dependent on the functional activity of cathepsin B as shown by specific siRNA's and seems to be regulated by activation of ERK1/2 and p38 signal transduction pathways.

Parallel screening for ALK, MET and ROS1 alterations in non-small cell lung cancer with implications for daily routine testing.

OBJECTIVES: ALK, MET and ROS1 are prognostic and predictive markers in NSCLC, which need to be implemented in daily routine. To evaluate different detection approaches and scoring systems for optimal stratification of patients eligible for mutation testing in the future, we screened a large and unselected cohort of NSCLCs for all three alterations. MATERIAL AND METHODS: Using tissue microarrays, 473 surgically resected NSCLCs were tested for ALK and MET expression by IHC and genomic alterations in the ALK, MET and ROS1 gene by FISH. For MET IHC, two different criteria (MetMAb and H-score), for MET FISH, three different scoring systems (UCCC, Cappuzzo, PathVysion) were investigated. RESULTS: ALK and ROS1 positivity was seen in 2.6% and 1.3% of all ADCs, respectively, but not in pure SCCs. One ROS1 translocated tumor showed additional ROS1 amplification. MET IHC+/FISH+ cases were found in both histological subtypes (8.6% in all NSCLCs; 10.6% in ADCs; 5.0% in SCCs) and were associated with pleural invasion, lymphatic vessel invasion and lymph node metastasis. MET altered ADCs more frequently showed a papillary growth pattern. Whereas ALK testing revealed homogenous results in IHC and FISH, we saw discordant results for MET in about 10% of cases. Both METIHC scoring systems revealed almost identical results. We did not encounter any combined FISH positivity for ALK, MET or ROS1. However, three ALK positive cases harbored MET overexpression. CONCLUSION: In daily routine, IHC could support FISH in the identification of ALK altered NSCLCs. Further research is needed to assess the role of discordant MET results by means of IHC and FISH as well as the relevance of tumors with an increased ROS1 gene copy number.

ALK-FISH borderline cases in non-small cell lung cancer: Implications for diagnostics and clinical decision making.

BACKGROUND: Fluorescence in-situ hybridization (FISH) for the detection of ALK-rearrangements in non-small cell lung cancer (NSCLC) is based on at first sight clear cut-off criteria (≥15% of tumor cells) for split signals (SS) and single red signals (SRS). However, NSCLC with SS-counts around the cut-off may cause interpretation problems. MATERIAL AND METHODS: Tissue microarrays containing 753 surgically resected NSCLCs were independently tested for ALK-alterations by FISH and immunohistochemistry (IHC). Our analysis focused on samples with SS/SRS in the range between 10% and 20% (ALK-FISH borderline group). To better understand the role of these samples in routine diagnostics, we performed statistical analyses to systematically estimate the probability of ALK-FISH-misclassification (false negative or positive) for different numbers of evaluated tumor cell nuclei (30, 50, 100, and 200). RESULTS: 94.3% (710/753) of the cases were classified as unequivocally (<10% or ≥20%) ALK-FISH-negative (93%; 700/753) or positive (1.3%; 10/753) and showed concordant IHC results. 5.7% (43/753) of the samples showed SS/SRS between 10% and 20% of the tumor cells. Out of these, 7% (3/43; ALK-FISH: 14%, 18% and 20%) were positive by ALK-IHC, while 93% (40/43) had no detectable expression of the ALK-protein. Statistical analysis showed that ALK-FISH misclassifications occur frequently for samples with rearrangements between 10% and 20% if ALK-characterization is based on a sharp cut-off point (15%). If results in this interval are defined as equivocal (borderline), statistical sampling-related ALK-FISH misclassifications will occur in less than 1% of the cases if 100 tumor cells are evaluated. CONCLUSION: While ALK status can be determined robustly for the majority of NSCLC by FISH our analysis showed that ∼6% of the cases belong to a borderline group for which ALK-FISH evaluation has only limited reliability due to statistical sampling effects. These cases should be considered equivocal and therapy decisions should include additional tests and clinical considerations.

Comparative genomic hybridization. Synchronous occurrence of focal nodular hyperplasia and hepatocellular carcinoma in the same liver is not based on common chromosomal aberrations.

Occasionally hepatocellular carcinomas (HCCs) occur synchronously with or within focal nodular hyperplasias (FNHs), raising the question of a putative causal relationship. In the present study, we used comparative genomic hybridization to investigate the occurrence of genomic aberrations in FNHs, which might lead to hepatocarcinogenesis. Tissue samples from FNHs and nonlesional liver tissue were obtained from 7 women. None of the patients had a chronic diffuse liver disease. A synchronous HCC not spatially related to FNH was present in 1 patient. Two patients had received oral contraceptives. Genomic aberrations were found in only 1 FNH. No aberration was found in the FNH occurring synchronously with HCC, but the HCC included gains at chromosomes 1q, 5, 12, and 19q and losses at 4p, 7q22-q35, 9p, 17p, 21q, and 22q. No aberrations were found in nonneoplastic liver tissues. Our findings support the notion that FNH is not a preneoplastic lesion for the occurrence of HCC in humans and that the synchronous occurrence of FNH and HCC is coincidental in our case.

Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection.

BACKGROUND: Constitutive expression and localization of antimicrobial human beta-defensin-1 (HBD-1) in human kidneys as a potential mechanism of antimicrobial defense has been previously reported. Inducible expression of human beta-defensin-2 (HBD-2) has been described in various epithelial organs but not for the urogenital tract. METHODS: We investigated the gene- and protein expression of HBD-1 and HBD-2 by reverse transcriptase-polymerase chain reaction, and immunohistochemistry in 15 normal human kidney samples and 15 renal tissues with chronic bacterial infection. Additionally, cell culture experiments were performed to study HBD gene expression by real-time RT-PCR in response to inflammatory cytokines TNFalpha and IL-1beta as well as lipopolysaccharide from Gram-negative bacteria. RESULTS: Constitutive HBD-1 gene- and protein expression was detected in normal renal tissue and kidneys with chronic infection. As a novel finding, inducible HBD-2 gene- and protein expression was demonstrated in tubulus epithelia with chronic infection but not in normal renal tissue. In pyelonephritic kidneys HBD-1 and HBD-2 expression showed a similar pattern of localization in distal tubules, loops of Henle and in collecting ducts of the kidney. Furthermore, real-time RT-PCR of kidney derived cell lines stimulated with inflammatory agents TNF-alpha, IL-1beta and LPS revealed a strong increase in relative HBD-2 transcription level and also a slight increase in relative HBD-1 transcription level. CONCLUSIONS: Upregulated HBD-2 expression in renal tubulus epithelium indicates a role of a wider range of human defensins for antimicrobial host defense in the urogenital tract than previously recognized.

Epithelial cell preparation for proteomic and transcriptomic analysis in human pancreatic tissue.

Standardized sample preparation procedures constitute a prerequisite for obtaining reliable and reproducible results in gene expression research in humans. In particular, in diseases such as pancreatic cancer and pancreatitis, isolating epithelial cells is an important step preceding such research. In pancreatic tissue, the high amount of RNAases is a further problem when it comes to obtaining high-quality RNA, and the presence of secreted proteases accelerates protein degradation. We developed a successful method that addresses these different problems. This method, which uses epithelial cell surface antibody Ber-Ep4, proteases, and RNAases inhibitors, leads to a significant enrichment (> 95% purity) of epithelial cells from fresh human tissue samples and allows for both proteomics (Western Blot, 2D PAGE) and transcriptomics studies (rtPCR, cDNA microarray). Compared with other cell purification procedures, this method is characterized by several advantages: a large quantity of cells available for downstream analysis, combined transcriptomics and proteomics studies using the same samples, better reproducibility of proteomics studies, and an acceptable yield (63%) for gene expression arrays studies. Moreover, a quality control protocol addressing the needs of the industry and the requirements of regulatory agencies is proposed.

[Detection and analysis of bcl-1/IgH rearrangement in mantle cell lymphoma]

OBJECTIVE: To investigate the specificity and sensitivity of the PCR technique in the identification of bcl-1/IgH gene rearrangement in mantle cell lymphoma (MCL) and to characterize the bcl-1/IgH junction DNA sequences. METHODS: A semi-nested PCR method to amplify bcl-1/IgH gene rearrangement in DNA from fresh frozen lymphonode specimen was established. Twenty-eight cases of mantle cell lymphoma were analyzed for the presence of bcl-1/IgH gene rearrangement. The rearrangement products was cloned and sequenced to recognize the junction sequences, the breakpoints in the bcl-1 region, and J(H) gene involved in the rearrangement. RESULTS: A bcl-1/IgH gene rearrangement was detected in 17 out of 28 cases of MCL, while only 9 cases was detected with single step PCR method (X(2)=4.59, P<0.05). The rearrangement product varied in size between 74 to 162 base pairs, and the length of the junction sequences ranged from 6 to 24 base pairs. Ten different bcl-1 breakpoints were clustered within 65 base-pair spans, among which, 5 breakpoints (located at 430, 440, 451, 486 and 492) were never reported by other authors. The most common J(H) gene segments utilized in the translocation were J(H) 4 (8/18), then J(H)5 (3/18), J(H)6 (2/18), J(H)4/5 (2/18). J(H)1 2/18, and J(H)3 (1/18). CONCLUSION: These results indicate that the semi-nested PCR is a specific and sensitive method for the detection of bcl-1/IgH gene rearrangement in mantle cell lymphoma, which has implications for both the diagnosis and clinical management of mantle cell lymphoma. The recognization the potential mechanism of bcl-1/IgH gene rearrangement will help us to know the exact pathogenic machanisms of MCL.

Molecular testing for lymph node metastases as a determinant of colon cancer recurrence: results from a retrospective multicenter study.

PURPOSE: Recurrence risk assessment to make treatment decisions for early-stage colon cancer patients is a major unmet medical need. The aim of this retrospective multicenter study was to evaluate the clinical utility of guanylyl cyclase C (GCC) mRNA levels in lymph nodes on colon cancer recurrence. METHODS: The proportion of lymph nodes with GCC-positive mRNA (LNR) was evaluated in 463 untreated T3N0 patients, blinded to clinical outcomes. One site's (n = 97) tissue grossing method precluded appropriate lymph node assessment resulting in post hoc exclusion. Cox regression models tested the relationship between GCC and the primary endpoint of time to recurrence. Assay methods, primary analyses, and cut points were all prespecified. RESULTS: Final dataset contained 366 patients, 38 (10%) of whom had recurrence. Presence of four or more GCC-positive lymph nodes was significantly associated with risk of recurrence [hazard ratio (HR) = 2.46, 95% confidence interval (CI), 1.07-5.69, P = 0.035], whereas binary GCC LNR risk class (HR = 1.87, 95% CI, 0.99-3.54, P = 0.054) and mismatch repair (MMR) status (HR = 0.77, 95% CI, 0.36-1.62, P = 0.49) were not. In a secondary analysis using a 3-level GCC LNR risk group classification of high (LNR > 0.20), intermediate (0.10 < LNR ≤ 0.20), and low (LNR ≤ 0.10), high-risk patients had a 2.5 times higher recurrence risk compared with low-risk patients (HR = 2.53, 95% CI, 1.24-5.17, P = 0.011). CONCLUSIONS: GCC status is a promising prognostic factor independent of traditional histopathology risk factors in a contemporary population of patients with stage IIa colon cancer not treated with adjuvant therapy, but GCC determination must be performed with methodology adapted to the tissue procurement and fixation technique.

TGF-ß: an emerging player in drug resistance.

The transforming growth factor ß (TGF-ß) pathway acts as a double-edged sword in tumorigenesis. By constraining epithelial cell growth, TGF-ß is a potent tumor suppressor. However, TGF-ß also acts as a key player in the induction of epithelial-to-mesenchymal transition (EMT), thereby enhancing invasiveness and metastasis. Furthermore, TGF-ß signaling has recently been correlated with resistance against both targeted and conventional anticancer agents. Here, we present data demonstrating a role for TGF-ß in chemotherapy resistance in colorectal cancer (CRC). We discuss these results in the context of recent findings indicating TGF-ß signaling as an emerging player in cancer drug resistance.