[Bone marrow fibrosis in primary myelofibrosis in relation to myelodysplasia- and age-related mutations of hematopoietic cells]. / Knochenmarkfibrose bei primärer Myelofibrose in Abhängigkeit von myelodysplasie- und altersassoziierten Mutationen der Hämatopoese.

Besides histopathological findings, there are no indicators of increased risk for fibrotic progression in myeloproliferative neoplasms (MPNs). Age-related clonal hematopoiesis (ARCH) or clonal hematopoiesis of indetermined potential (CHIP) are frequent findings in the elderly and combinations with MPN driver mutations (JAK2, MPL, and CALR) have been described. To determine the impact of ARCH/CHIP-related mutations for the development of fibrosis in primary myelofibrosis (PMF), the mutational status of cases with fibrotic progression from grade 0 to grade 2/3 (n = 77) as evidenced by follow-up bone marrow biopsies (median 6.2 years) was compared to prefibrotic PMF samples without the development of fibrosis (n = 27; median follow-up 7.3 years). Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, DNMT3A) demonstrable at presentation were not connected with fibrotic progression. However, mutations that are rarely found in ARCH/CHIP (SRSF2, U2AF1, SF3B1, IDH1/2, and EZH2) were present in 24.7% of cases with later development of fibrosis and not detectable in cases staying free from fibrosis (P = 0.0028). Determination of tumor mutational burden (TMB) in a subgroup of cases (n = 32) did not show significant differences (7.68 mutations/MB vs. 6.85 mutations/MB). We conclude that mutations rarely found in ARCH/CHIP provide an independent risk factor for rapid fibrotic progression (median 2.0 years) when already manifest at first presentation.

[Liquid biopsy in tumor diagnostics : Applications, perspectives, and limitations of the "cancer liquidome"]. / Liquid Biopsy in der Tumordiagnostik : Anwendungen, Perspektiven und Limitationen des Cancer Liquidome.

The detection of tumor-specific genetic alterations in body fluids as an addition to or even replacement for established tissue-based tumor diagnostics is currently a hot topic in academic research and industry. Progress in methods for nucleic acid analyses together with promising results from clinical studies have raised great expectations for cancer screening, diagnosis, prognosis, and therapy monitoring by means of a minimally invasive blood draw. Individual focused assays have already been introduced into routine diagnostics and represent a valuable option in cases where no tissue samples are available. However, before the use of liquid biopsy outside of clinical studies is enforced and more complex markers (like tumor mutational burden) are analyzed, several practical challenges and principal problems have to be addressed. This review focusses on the detection of free-circulating nucleic acids in blood plasma and critically discusses established and future applications as well as challenges and limitations of this new method.

Rare compound heterozygous variants in PNKP identified by whole exome sequencing in a German patient with ataxia-oculomotor apraxia 4 and pilocytic astrocytoma.

Ataxia-oculomotor apraxia type 4 (AOA4) is a rare autosomal recessive neurologic disorder. The phenotype is characterized by ataxia, oculomotor apraxia, peripheral neuropathy and dystonia. AOA4 is caused by biallelic pathogenic variants in the PNKP gene encoding a polynucleotide kinase 3'-phosphatase with an important function in DNA-damage repair. By whole exome sequencing, we identified 2 variants within the PNKP gene in a 27-year-old German woman with a clinical AOA phenotype combined with a cerebellar pilocytic astrocytoma diagnosed at 23 years of age. One variant, a duplication in exon 14 resulting in the frameshift c.1253_1269dup p.(Thr424fs*49), has previously been described as pathogenic, for example, in cases of AOA4. The second variant, representing a nonsense mutation in exon 17, c.1545C>G p.(Tyr515*), has not yet been described and is predicted to cause a loss of the 7 C-terminal amino acids. This is the first description of AOA4 in a patient with central European descent. Furthermore, the occurrence of a pilocytic astrocytoma has not been described before in an AOA4 patient. Our data demonstrate compound heterozygous PNKP germline variants in a German patient with AOA4 and provide evidence for a possible link with tumor predisposition. Localization of the 2 variants in human PNKP NP_009185.2. NM_007254.3:c.1253_1269dup p.(Thr424fs*49) is predicted to cause a frameshift within the kinase domain, NM_007254.3:c.1545C>G p.(Tyr515*) is predicted to cause loss of 2 C-terminal amino acids of the kinase domain and 5 additional C-terminal amino acids.

[Possibilities and limitations of molecular pathology in dermatohistology]. / Möglichkeiten und Grenzen der Molekularpathologie in der Dermatohistologie.

Malignant tumours, infections caused by microorganisms or genodermatoses are diagnosed with additional help of molecular pathology methods. Polymerase chain reaction (PCR), sequencing and in situ hybridisations play an important role. It remains to be seen if methods such as "liquid biopsies" or "single cell genomics" can be developed as routine diagnostics. High technical efforts, high costs and no possibility for resistency testing is accompanied by fast verification, high sensitivity and high specificity. Overall, molecular pathology results have to be combined with the clinical picture, histology or immunohistochemistry and culturing results to achieve a correct diagnosis for the patient.

TEMHEAD: a single-arm multicentre phase II study of temsirolimus in platin- and cetuximab refractory recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) of the German SCCHN Group (AIO).

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is a common disease, which has a poor prognosis after failure of therapy. Activation of the PI3K-AKT-mTOR axis is commonly detected in recurrent or metastatic SCCHN, and provided the rationale for the clinical phase II trial in pretreated SCCHN. PATIENTS AND METHODS: The primary end point was the progression-free survival rate (PFR) at 12 weeks. Forty eligible patients have been recruited after failure of platinum chemotherapy and cetuximab. A preplanned futility analysis was successfully passed after ≥1 success was detected in 20 patients. Secondary objectives consisted of progression-free survival (PFS), disease control rate (DCR), overall survival (OS), safety and tolerability, and predictive biomarkers for KRAS, BRAF, PIK3CA mutations, and HPV status. Archived tumor tissue was analyzed for DNA sequence. RESULTS: A total of 40 patients were eligible. The PFR at 12 weeks was 40% (95% CI 25.0-54.6). The median PFS and OS were 56 days (95% CI 36-113 days) and 152 days (76-256 days), respectively. In 33 assessable patients, disease stabilization occurred in 57.6%, with tumor shrinkage in 13 patients (39.4%). Overall, the treatment was well tolerated. Fatigue (47.5%), anemia (25.0%), nausea (20.0%), and pneumonia (20.0%) were the most common adverse events. Neither PIK3CA mutations, nor HPV status were predictive for success with temsirolimus treatment. No mutations were found for KRAS or BRAF. CONCLUSION: Tumor shrinkage and efficacy parameter indicate that inhibition of the PI3K-AKT-mTOR axis was a putative novel treatment paradigm for SCCHN. We could not identify parameters predictive for treatment success of temsirolimus, which underscores the need for refinement of the molecular analysis in future studies. CLINICAL TRIALS NUMBER: NCT01172769.

Vitamin D is not linked to folate status and mRNA expression of intestinal proton-coupled folate transporter.

PURPOSE: In vitro studies discovered intestinal proton-coupled folate transporter (PCFT) as a vitamin D hormone-responsive gene. In vivo effects of vitamin D on PCFT and folate status are currently not available. METHODS: Three experiments were conducted. At first, vitamin D receptor knockout (VDR(-/-)) mice and corresponding wild-type (WT) mice were compared for their plasma and hepatic folate concentration and PCFT mRNA expression in intestinal mucosa. In a second experiment with rats, we analyzed the folate status of offspring in response to a maternal vitamin D-adequate (1,000 IU/kg) or vitamin D-deficient (0 IU/kg) diet that was fed for 11 weeks. Finally, the plasma folate concentration of healthy individuals was studied at baseline (in winter) and in response to an oral treatment for 8 weeks with 2,000 IU vitamin D3 per day or a placebo, respectively. RESULTS: Here, we show that folate status and intestinal PCFT mRNA abundance did not differ between the VDR(-/-) and the WT mice. No effect of vitamin D on folate status was also found in rat dams and their offspring, and plasma folate levels of individuals did not change in response to vitamin D. CONCLUSIONS: Current data from studies with model animals and humans provide no indication for a vitamin D effect on intestinal uptake and status of folate.

Role of human papillomaviruses in persistent and glucocorticoid-resistant juvenile phimosis.

BACKGROUND: In adults, human papillomaviruses (HPV), lichen sclerosus et atrophicus (LSA) and phimosis are considered to be major risk factors for penile cancer. In boys, a possible association between phimosis, LSA and HPV has been suggested. OBJECTIVE: To investigate the role of HPV in the persistence of phimosis in children. PATIENTS AND METHODS: Out of a cohort of 420 boys presenting with foreskin problems, we prospectively sampled the preputial tissue of 82 patients during circumcision: 46 with steroid-naïve and 36 with steroid-resistant phimosis. All foreskins were assessed clinically and histopathologically with regard to appearance, inflammation, oedema, epithelial degeneration and fibrosis. The viral status of the foreskins was determined by immunohistochemistry and highly sensitive PCR, with subsequent subtyping by DNA hybridization (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 44, 45, 51-54, 56, 58, 59, 61, 62, 66-68, 70, 72, 73, 81-84, 90, 91). RESULTS: The foreskins appeared normal in 62 boys and suggestive of LSA in one single case. Small cracks or white scars were present in seven steroid-naïve and 12 steroid-resistant foreskins. LSA was diagnosed microscopically in two of the steroid-naïve and six of the steroid-pretreated group. No evidence of HPV was found in any of the juvenile foreskins. CONCLUSIONS: Our prospective study has provided evidence that HPV is not usually present in the foreskin of boys with persistent phimosis after their first year of life and that topical glucocorticoid treatment failure is not associated with HPV or any specific histopathological changes.

[Molecular pathological diagnosis in cytopathology of non-small-cell lung cancer. Standardization of specimen processing]. / Molekularpathologische Diagnostik in der Zytopathologie des nichtkleinzelligen Lungenkarzinoms. Standardisierung der Materialprozessierung.

BACKGROUND: Personalized medicine is becoming standard for the treatment of non-small-cell lung cancer. For example, patients with activating EGFR mutations or EML4-ALK translocations largely benefit from targeted therapies with tyrosine kinase inhibitors with better response rates and progression-free survival compared to standard chemotherapy regimens. However, the application of the respective molecular biomarker analyses requires great expertise in the handling of different cell and tissue specimens. A major challenge for reliable analyses is the usually low amount of tumor material. There are currently relatively few standardized and evidence-based guidelines for the processing and analysis of respective specimens as well as for interpretation of the test results. MATERIALS AND METHODS: To establish a basis for standardized predictive cytopathological analyses, different material processing approaches and molecular pathological tests are discussed, and novel concepts and strategies are lined out in order to improve the quality and reliability of the respective diagnostic procedures. RESULTS AND DISCUSSION: Predictive analyses of cytological specimens can be reliably performed using smears, cytospins or cell blocks; there is no need for histological specimens. The diagnostic work-up of cytological probes should be performed as carefully as possible in order to save further tumor material for subsequent predictive analyses. With standardized and reliable procedures at hand cytopathology is an important contribution to the multidisciplinary, complex care, and treatment of lung cancer patients.

[Diagnostic and predictive analyses of cytological specimens of non-small cell lung cancer: strategies and challenges]. / Diagnose und prädiktive Analysen an zytologischen und bioptischen Tumorproben nicht-kleinzelliger Lungenkarzinome: Aktuelle Strategien und Herausforderungen.

Personalised medicine is becoming the standard care for advanced non-small cell lung cancer. Tumour-specific therapies based on biomarker analyses, e. g., EGFR mutations or translocations of the ALK gene locus, result in a superior patient outcome compared to unselected therapy approaches. However, predictive molecular analyses can be challenging and require significant experience with cell- and tissue-based diagnostic methods. The major challenge relates to the sometimes low amount of available tumour material for both diagnostic and predictive analyses. As yet, there are no standardised or evidence-based recommendations concerning biopsies, specimen processing, and analyses. Respective guidelines require combined interdisciplinary actions to consider both clinical and pathological aspects. In order to establish a basis for high quality procedures, different approaches, methods, and protocols were interdisciplinary discussed with an emphasis on cytological specimens. Detailed evaluation of the parameters and consented recommendations might contribute to optimised strategies in the interdisciplinary, more and more complex care of non-small cell lung cancer patients.

[Myeloproliferative neoplasms: histopathological and molecular pathological diagnosis]. / Myeloproliferative Neoplasien: Histopathologische und molekularpathologische Diagnostik.

Myeloproliferative neoplasms (chronic myeloproliferative disorders according to former nomenclature) comprise chronic myeloid leukemia, polycythemia vera, essential thrombocythemia, primary myelofibrosis, chronic eosinophilic leukemia, chronic neutrophilic leukemia and systemic mastocytosis. All disorders have excessive proliferation of one or more hematopoietic lineages in common and progress with different probability to blast crisis or fibrosis. A further common feature is provided by the activating mutation of tyrosin kinases and associated pathways of signal transduction (BCR-ABL, JAK2(V617F), MPL(W515L/K), KIT(D816V) and FIP1L1-PDGFRA) causative for the abnormal proliferation. With regard to diagnosis and therapy these mutations are of utmost importance because they enable the exclusion of reactive processes, contribute with varying specificity to subtyping of MPN and are at least partly sensitive to targeted therapy. The molecular mechanisms of blastic and fibrotic progression are not yet understood.

Lay health workers and HIV programmes: implications for health systems.

One of the consequences of massive investment in antiretroviral access and other AIDS programmes has been the rapid emergence of large numbers of lay workers in the health systems of developing countries. In South Africa, government estimates are 65,000, mostly HIV/TB care-related lay workers contribute their labour in the public health sector, outnumbering the main front-line primary health care providers and professional nurses. The phenomenon has grown organically and incrementally, playing a wide variety of care-giving, support and advocacy roles. Using South Africa as a case, this paper discusses the different forms, traditions and contradictory orientations taken by lay health work and the system-wide effects of a large lay worker presence. As pressures to regularise and formalize the status of lay health workers grow, important questions are raised as to their place in health systems, and more broadly what they represent as a new intermediary layer between state and citizen. It argues for a research agenda that seeks to better characterise types of lay involvement in the health system, particularly in an era of antiretroviral therapy, and which takes a wider perspective on the meanings of this recent re-emergence of an old concept in health systems heavily affected by HIV/AIDS.

[DNA methylation. From basic research to routine diagnostics]. / DNA-Methylierung. Von der Grundlagenforschung zur Routinediagnostik.

On a molecular level cancer is characterized not only by genetic defects, such as deletions, mutations or translocations, but also by epigenetic lesions. The most important epigenetic mechanisms are DNA methylation, Polycomb/trithorax complexes, histon modifications, non-coding RNAs, and chromosomal territories. These epigenetic mechanisms contribute to a stable modification of gene expression without changes in primary DNA sequence. During the development and progression of human tumours a gene-specific hypermethylation with resulting repression of transcription can occur. At the same time, global hypomethylation can very often be observed which contributes to an increase in chromosomal instability. In tumour pathology, the detection of somatic hMLH1 hypermethylation is important for molecular diagnostics of Lynch syndrome. The detection of MGMT gene methylation is a good prognostic and predictive factor for glioblastoma patients. Performing DNA methylation assays for routine diagnostics requires technical as well as theoretical expertise.

[Beyond staging, typing and grading. New challenges in breast cancer pathology]. / Jenseits von Staging, Typing und Grading. Herausforderungen und Perspektiven für die Tumorpathologie der Mamma.

Tumor biological parameters for the prediction and prognosis of invasive breast cancer are gaining in importance compared to traditional pathological categories. The immunohistochemically determined steroid hormone receptor status is not only the decisive factor influencing whether endocrine therapy (which is indicated in cases of even slight expression) is given or not, but also whether chemotherapy is considered. The Her2 status is of similar importance and needs to be analysed in every case of invasive carcinoma, whereby reproducibility, which requires greater efforts than with steroid hormone receptors, has to be assured. Probably only a small portion of breast cancers will benefit from chemotherapy. Since traditional pathological categories are not sufficient for the identification of this subgroup, there has been an intense search for alternative prognostic parameters, whereby gene expression profiling has emerged as the most promising tool. Despite minimal concordance with regard to single genes, the various profiles available converge in the identification of the proliferative signature as the prognostically most relevant. Whether histopathological grading and immunohistochemical determination of the growth fraction with Ki-67 or genetic profiling will be more reliable in the identification of the aggressive subtype has yet to be clarified in clinical studies which encompass central pathological review.

Autophagy delays apoptotic death in breast cancer cells following DNA damage.

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.

Biomarker analysis in polycythemia vera under interferon-alpha treatment: clonality, EEC, PRV-1, and JAK2 V617F.

Three consecutive polycythemia vera (PV) patients were analyzed before and during pegylated-interferon (rIFNalpha) treatment for the following markers: (1) granulocyte and CD34(+) cell clonality, (2) Jak2V617F expression, (3) PRV-1 mRNA overexpression, and (4) Epo-independent colony (EEC) growth. Before rIFNalpha therapy, all patients displayed clonal hematopoiesis, 100% Jak2V617F expression as well as PRV-1 overexpression, and EEC growth. After rIFNalpha treatment, all three patients demonstrated polyclonal hematopoiesis. Nonetheless, Jak2V617F expression, PRV-1 overexpression, and EEC-growth remained detectable, albeit at lower levels. We conclude that reemergence of polyclonal hematopoiesis after rIFNalpha treatment may be achieved in a substantial proportion of patients. However, this does not constitute elimination of the PV clone. These data demonstrate the usefulness of novel markers in monitoring minimal residual disease and caution against discontinuation of rIFNalpha treatment after hematologic remission. Long-term follow-up of large patient cohorts is required to determine whether rIFNalpha treatment can cause complete molecular remissions in PV.