Background: Germline mutations in BRCA1 and BRCA2 genes confer a high risk for the hereditary breast and/or ovarian cancer (HBOC) syndrome, whereas both germline and somatic mutations are predictive biomarkers for PARP inhibition. MH BRCA® classifies variants based on ACMG guidelines. Clinical interpretation of NGS results by MH GUIDE® provides clinicians with treatment recommendations to cancer based on expert curated biomarker knowledge.

Background: In recent years, parallel sequencing technologies have become integrated into daily clinical practice. Many institutions use amplicon-based approaches for the detection of somatic mutations. However, these assays do not routinely detect chromosomal aberrations or copy number changes (CNVs), which are still widely analysed by FISH and IHC, and struggle with de novo fusions. The development of new technologies to detect all therapeutically relevant genomic alterations in a single assay is an ongoing process. This study aimed to evaluate the TruSight Tumor 170 assay, a hybrid capture-based assay, for the simultaneous detection of somatic gene mutations, gene fusions and CNVs and its implementation into routine diagnostics. Methods: Until now 24 of 48 tumour samples with known genetic aberrations were evaluated including three control samples. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue. After DNA shearing, DNA and RNA libraries were prepared with the TruSight Tumor 170 assay (Illumina) according to the manufacturer's instructions. Sequencing was performed on the NextSeq (Illumina). For data analysis, variant calling was performed using the TruSight Tumor 170 app on BaseSpace Sequence Hub (Illumina), and for variant interpretation BaseSpace Variant Interpreter (Illumina) and the Molecular Health Guide Software were used. Results: DNA and RNA were successfully extracted. 22 of 24 DNA libraries were analysable with DNA concentrations between 9 – 120 ng DNA. All 24 RNA libraries were analysable with RNA concentrations between 1 – 85 ng. All previously known exonic mutations and 1 high-level MET amplification were detected. Intronic variants and low-level MET amplifications were not detected. 9 of 13 fusions and splice variant were confirmed with the TruSight Tumor 170 assay. The remaining 4 aberrations were false positive by the previous methods. Conclusions: The TruSight Tumor 170 assay works well even with very low DNA and RNA concentrations in comparison to other methods and can be used in a routine workflow. The number of samples will be increased to 48 samples to evaluate the assay further. Additionally, automated solutions for DNA/RNA extraction from same slide might be needed. Legal entity responsible for the study: Carina Heydt. Funding: Illumina. Disclosure: K. Stecker: Molecular Health GmbH. J. Neumann: Illumina. All other authors have declared no conflicts of interest.

Background: Pancreatic cancer (PDAC) is highly resistant to cytotoxic chemotherapy and several trials of molecularly targeted drugs have failed. However, some tumors with specific somatic mutations respond to targeted therapy and patients might profit from treatment stratified by genetic biomarker profiles. Methods: We used MH Guide, a proprietary data tool, to annotate somatic mutation in PDAC patients who had received ≥ 1st line palliative therapy. A molecular tumor board (MTB) interpreted suggestions and matched consecutive therapies including off-lable use of any EMA-approved drug to biomarker profiles. Frequency of clinically relevant markers was the primary outcome. Secondary outcomes were choice of therapy, clinical outcomes and AE. Results: We enrolled 39 patients, analyzed 35 samples and had valid results from 31 tumors. The most common mutations were KRAS (n = 24), TP53 (n = 17), SMAD4 (n = 2), BRCA1/2 (n = 4; 3 germline), CDKN2A (n = 9), ATM (n = 6; 3 LOF), APC (n = 4), and MSH3 (n = 15, 1 germline). MH Guide suggested off-label treatment in 28 cases: PARP-inhibitors (n = 26), MEK/RAF inhibitors (n = 20), CDK inhibitors (n = 9), other kinase inhibitors (n = 3), mTOR inhibitors (n = 2). Common toxicity markers were: Platinum (XRCC1 n = 16; 5 homozygous, TPMT n = 1; MUTYH n = 3), paclitaxel (CYP2C8 n = 2), gemcitabine (CDA n = 4; 1homozygous), 5-FU (DPYD n = 1), doxorubicin (G6PD n = 1), docetaxel (GSTP1 n = 2), sunitinib (ABCG2 n = 2), irinotecan (ERCC2, n = 6; 5 homozygous, UGT1A1 n = 1). Three patients deceased before MTB decision and 8 prior to treatment, 5 patients were not eligible for active treatment. Seven patients received targeted treatment: Olaparib (n = 6, 1 still on treatment), palbociclib (n = 1) and remained median 84 days (range 18 – 114) on treatment. Four patients received cytotoxic chemotherapy according to MTB decision (among them one without positive markers) and in four cases cytotoxic therapy was used despite a molecularly targeted suggestion. Conclusions: Unbiased evidence-based molecular stratification of pre-treated patients with PDAC is feasible in a clinical setting. Disease progression and tissue availability are major challenges. Conclusions on clinical outcomes are limited by the study size. Clinical trial identification: NCT02767700. Legal entity responsible for the study: Karolinska University Hospital. Funding: Molecular Health GmbH (Data analysis). Disclosure: M. Kordes: Travel Grant from Molecular Health GmbH. M. Löhr: Consultant or advisory role: Molecular Health GmbH, Pharmcyte. Stock in Centogene AG. Horaria: Abbott, Mylan, Nordmark. Travel Grant: Abbott, Pharmcyte, Molecular Health GmbH. S. Kaduthanam, C. Hülsewig, K. Stecker, S. Brock: Employee of Molecular Health GmbH. J-E. Frödin: Travel grant: Amgen. All other authors have declared no conflicts of interest.

Abstract BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is one of the major dose-limiting side effects in breast cancer patients, with up to 97% of patients receiving an adjuvant taxane experiencing this symptom in the months and years after breast cancer treatment. CIPN often leads to loss of physical function; difficulties in activities of daily living and decreased of quality of life (QOL). Few effective interventions have been developed to alleviate CIPN in this patient population. We conducted a pilot randomized controlled trial to assess the feasibility, safety and preliminary effect of an acupuncture intervention on CIPN in breast cancer survivors. METHODS: Patients with stage I-III breast cancer who were experiencing CIPN after the completion of a taxane-containing adjuvant chemotherapy regimen were enrolled and randomized 1:1 to immediate participation in an acupuncture intervention or to a delayed intervention control group. Participants randomized to the acupuncture arm received 18 sessions of a standardized acupuncture protocol over 8 weeks while the control group received a lower-dose acupuncture protocol consisting of 9 acupuncture sessions over 8 weeks, after the initial 8-week control period. Measures including the Patient Neurotoxicity Questionnaire (PNQ), Functional Assessment of Cancer Therapy Neurotoxicity subscale (FACT-NTX), and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Chemotherapy-induced Peripheral Neuropathy 20 (EORTC QLQ-CIPN20) were collected at baseline and at 8 weeks after enrollment. RESULTS: A total of 40 patients were enrolled; 20 were randomized to the immediate acupuncture group and 20 to control. All enrolled patients were female, median age was 54, median time between enrollment and completion of chemotherapy was 14.3 months, and 72.5% of participants were White. Thirty-two patients (84%) completed at least 80% of the required sessions. No serious acupuncture-related side effects were observed. Participants randomized to the acupuncture arm experienced improvements in the PNQ sensory score (p=0.02), FACT-NTX summary score (p=0.002) and EORTC QLQ-CIPN20 score (p=0.006), respectively equivalent to 40%, 36% and 53% improvement in CIPN symptoms, as compared to controls. MeasurementsTime pointsAcupunctureUsual CareP-valueNMeanSDMeanSDPNQ summary sensory score (0-4)Baseline202.50.82.50.90.97Changes at 8 week15-1.00.9-0.30.60.02FACT-NTX summary score (0-44)Baseline2025.08.422.19.40.40Changes at 8 week159.09.21.25.40.002EORTC QLQ-CIPN20 sensory score (0-100)Baseline2044.919.945.022.30.93Changes at 8 week15-23.818.1-5.16.40.006 CONCLUSIONS: Women with CIPN after adjuvant taxane therapy for early breast cancer experienced a significant and clinically meaningful improvement in neuropathy symptoms as a result of an 8-week acupuncture protocol. Given the prevalence of taxane-induced neuropathy in women treated for early breast cancer, acupuncture could significantly improve QOL and functional status of thousands of women treated for breast cancer every year. Larger studies are needed to confirm these findings and evaluate the impact of acupuncture on functional measures in women with CIPN. Citation Format: Lu W, Giobbie-Hurder A, Freedman R, Yung R, Lin N, Partridge A, Shockro L, Stecker K, O'Connor KA, Rosenthal DS, Ligibel JA. Acupuncture for chemotherapy-induced peripheral neuropathy in breast cancer, preliminary results of a pilot randomized controlled trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD4-01.

To advance preclinical testing of novel targeted drugs in colorectal cancer (CRC) we established a panel of 133 mouse xenograft models from fresh tumor specimens of 239 patients with CRC of all four UICC stages. A subgroup of 67 xenograft models was treated with cetuximab, bevacizumab and oxaliplatin as single agents. Mutation status of KRAS (G12, G13, A146T), BRAF (V600E) and PIK3CA (E542K, E545K, H1047R) was assessed in all xenografts by allelespecific real-time PCR. KRAS codon 61 was assessed by conventional sequencing. AREG and EREG expression levels were analyzed by real-time PCR expression assays. In the treatment experiment we observed response rates of 27% (18/67) for cetuximab, 3% (2/67) for bevacizumab, and 6% (4/67) for oxaliplatin. Classification based on KRAS, BRAF and PIK3CA mutation status identified 15 of the responders (sensitivity 83%, confidence interval at p = 0.05 (CI): 59% - 96%), and 38 nonresponders (specificity 78%, CI: 63% - 88%). If any mutation except in KRAS codon 13 were considered, the classifier reached sensitivity of 94% and specificity of 69%. We improved specificity of the classifiers to 90% and 86% respectively by adding AREG and EREG RNA expression thresholds retrospectively. In patient-derived xenograft models, we found a predictive classifier for response to cetuximab that is more accurate than established biomarkers. We confirmed its potential performance in primary human tumors. For patients, the classifier’s sensitivity promises increased response rates and its specificity limits unnecessary toxicity. Given the scope of our xenograft models across all UICC stages, this applies not only to mCRC but also to the adjuvant setting of earlier stages. The xenograft collection allows to mimic randomized phase II trials and to test novel drugs effectively as single agents or in combinations. It also enables the development of highly accurate companion diagnostics as demonstrated by us for cetuximab.

Context.—Correct diagnosis of the tissue origin of a metastatic cancer is the first step in disease management, but it is frequently difficult using standard pathologic methods. Microarray-based gene expression profiling has shown great promise as a new tool to address this challenge. Objective.—Adoption of microarray technologies in the clinic remains limited. We aimed to bridge this technological gap by developing a real-time quantitative polymerase chain reaction (RT-PCR) assay. Design.—We constructed a microarray database of 466 frozen and 112 formalin-fixed, paraffin-embedded (FFPE) samples of both primary and metastatic tumors, measuring expression of 22 000 genes. From the microarray database, we used a genetic algorithm to search for gene combinations optimal for multitumor classification. A 92-gene RT-PCR assay was then designed and used to generate a database for 481 frozen and 119 FFPE tumor samples. Results.—The microarray-based K-nearest neighbor classifier demonstrated 84% accuracy in classifying 39 tumor types via cross-validation and 82% accuracy in predicting 112 independent FFPE samples. We successfully translated the microarray database to the RT-PCR platform, which allowed an overall success rate of 87% in classifying 32 different tumor classes in the validation set of 119 FFPE tumor samples. Conclusions.—The RT-PCR-based expression assay involving 92 genes represents a powerful tool for accurately and objectively identifying the site of origin for metastatic tumors, especially in the cases of cancer of unknown primary. The assay uses RT-PCR and routine FFPE samples, making it suitable for rapid clinical adoption.

Background. The addition of phosphorothioate (PS) groups to natural phosphodiester (PD) antisense oligodeoxynucleotides (oligo) prevents their in vivo hydrolysis by nucleases allowing an RNase-dependent elimination of targeted mRNA. To further improve oligo function 2′-methoxyethyl (ME) groups were attached to selected nucleotides at the 3′-end because ME groups block RNase activity. Methods/Results. ME modification of PS- or PD/PS-oligo targeting human intracellular adhesion molecule (ICAM)-1 mRNA significantly increased the degree and duration of the in vitro inhibitory effects without compromising selectivity and specificity. A 7-day intravenous or oral therapy with rat ME/PS-modified ICAM-1 antisense oligo extended the survivals of kidney allografts. In addition, ME/PS-modified ICAM-1 antisense oligo reduced ischemic-reperfusion injury in kidneys, as measured by glomerular filtration rate, creatinine levels, and infiltration with leukocytes. Finally, a 14-day treatment with cyclosporine (CsA)-induced nephrotoxicity in syngeneic kidney transplants correlated with both increased ICAM-1 protein expression and infiltration with leukocytes. Graft perfusion and treatment of recipients with ICAM-1 antisense ME/PS-oligo alleviated the nephrotoxic effect and decreased ICAM-1 expression and leukocyte infiltration. Conclusions. ME/PS-modified ICAM-1 antisense oligo is very effective in inhibiting the ICAM–1-dependent mechanism of graft infiltration and tissue damage involved in allograft rejection, ischemic-reperfusion injury, and CsA-induced nephrotoxicity.

Primary extramedullary plasmacytomas are infrequent, typically solitary, plasma cell neoplasms that generally pursue an indolent clinical course but may, rarely, convert to multiple myeloma. Phenotypic differences between these two entities are not well defined. Twenty‐eight cases of primary extramedullary plasmacytoma and 26 cases of both medullary ( n = 17) and extramedullary ( n = 9) multiple myeloma were analysed for the expression of proteins known to play a role in the biology of multiple myeloma. Immunohistochemistry was performed on paraffin wax sections using antibodies against cyclin D1, Bcl‐2, Bcl‐xL, p27, p21, p53, MIB1, CD20, and CD56. Twenty‐three extramedullary plasmacytomas were localized in the upper aerodigestive tract, four in the lymph nodes, and one in the testis. There was a strong male predominance (M : F = 6 : 1). None of the patients died from the disease or progressed to multiple myeloma (mean follow‐up 50 months). Nine patients developed local relapse and one patient's tumour evolved into a B‐cell non‐Hodgkin's lymphoma. In contrast to both intra‐ and extra‐medullary multiple myeloma, extramedullary plasmacytoma showed absence of cyclin D1 ( p < 0.001) and infrequent expression of CD56 ( p < 0.001). Furthermore, extramedullary plasmacytomas were characterized by weaker staining for Bcl‐2 protein and rare overexpression of p21 and p53. In comparison to extramedullary multiple myeloma, extramedullary plasmacytoma showed a more mature morphology and lower proliferation indices ( p = 0.008). There was no association between the phenotypic parameters investigated and clinical outcome in extramedullary plasmacytoma. In summary, extramedullary plasmacytoma and multiple myeloma show significant immunophenotypic differences, some of which may be of both diagnostic utility and biological relevance. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Activation of the cell-surface receptor Fas can lead to apoptosis in parenchymal cells in the liver, and if severe enough, result in fulminant hepatic failure and animal death. In the present study, we have examined the roles played by the Bcl-2 family members Bcl-xL and Bid in regulating this response. To do this, we have developed chemically modified 2'-O-(2-methoxy) ethyl antisense inhibitors of both Bid and Bcl-xL expression. In Balb/c mice, dosing with these antisense oligonucleotides reduced expression of the targeted mRNA by greater than 80% in the liver. This reduction was highly dependent upon oligonucleotide sequence and oligonucleotide dose. Reduction of Bcl-xL expression resulted in a potentiation of Fas-mediated apoptosis in liver and significant increase of the lethality of Fas-mediated fulminant hepatitis (p < 0.0001). In contrast, reduction of Bid expression protected the animals against Fas-mediated fulminant hepatitis and death (p < 0.0001). Simultaneous dosing of mice with Bcl-xL and Bid-targeting antisense oligonucleotides resulted in an inhibition of expression of both targeted proteins and protection of the animals from Fas-mediated apoptosis. These results demonstrate, for the first time, the role of Bcl-xL in regulating responses to proapoptotic Fas signaling in mouse liver. In addition, this is the first reported example demonstrating the ability of antisense inhibitors to reduce expression of multiple proteins in animals by simultaneous dosing.

Purpose. To test the efficacy of a synthetic antisense oligonucleotide inhibitor of an intracellular signal transduction protein, C-raf-1 kinase, as an inhibitor of ocular neovascularization.Methods. A 2'methoxyethyl, 2'deoxy chimeric 20-nucleotide sequence containing a uniform phosphorothioate backbone was synthesized which targets the 3'untranslated region of porcine C-raf-1 mRNA (ISIS 107189). Efficacy of mRNA inhibition was tested in vitro in porcine vascular endothelial cells and treated pigs by Northern blotting. In a pig model of intraocular neovascularization induced by branch retinal vein occlusion, intravitreal injection of ISIS 107189 compound (8 muM calculated intraocular concentration) at baseline and on days 14, 42, and 70, was tested against vehicle as control for inhibition of neovascularization. After enucleation on day 84, ocular tissues were analyzed for ISIS 107189 content by solid-phase extraction and capillary gel electrophoresis. Cryostat sections were immunostained for C-raf-1 kinase protein.Results. The antisense oligonucleotide demonstrated high potency for inhibition of C-raf-1 kinase in the porcine cells lines. Levels of C-raf-1 kinase were also decreased in the retina of pigs following a single 180 mug dose. Pig eyes injected with the multiple doses of 180 mug oligonucleotide demonstrated a marked decrease in neovascularization due to branch retinal vein occlusion 12 weeks after treatment (p = 0.05, Mann-Whitney U-test). Posterior subcapsular cataracts were noted in the treated eyes. Concentrations of oligonucleotide in retina ranged from 2-12 muM in the treated eyes. Qualitative assessment of the expression of C-raf-1 kinase via immunohistostaining of frozen sections demonstrated inhibition of expression in the treated eyes compared to controls.Conclusions. ISIS 107189 successfully inhibited neovascularization in this model, which was correlated with an inhibition of expression of C-raf-1 kinase. While not proven in these studies, these results suggest that C-raf kinase may be important in angiogenesis. Antisense therapy has potential applicability in the therapy of ocular neovascular diseases.

Solid organ transplantation represents a life-saving or prolonging therapy for hundreds of thousands of patients worldwide. Placement of an immunologically foreign graft into the recipient inevitably provokes an immune response, as donor-specific antigens are recognized by the host adaptive immune system. In addition, during the transplant procedure, the organ is rendered temporarily hypoxic, with attendant cellular damage, resulting in activation of the innate immune system by damage-associated signals. Here, we provide an overview of innate and adaptive immune responses to the allograft, including the cells and receptors required to respond to danger signals or graft antigens, and the molecular processes involved in generating alloantigen-specific B and T cells. We will also discuss the effector mechanisms by which cellular and humoral alloimmune responses damage the allograft and consider how transplant tolerance may be achieved. Finally, we describe emerging data implicating factors beyond the graft, including the microbiome, in susceptibility to rejection.

Chen, W.1; Bennett, C. F.2; Condon, T. P.2; Stecker, K.2; Tian, L.1; Kahan, B. D.1; Stepkowski, S. M.1 Author Information