Frozen Biopsy Specimens Research Articles

Persistent Aseptic Pyelonephritis After Acute Bacterial Pyelonephritis: Possible Role of Corticosteroids

Persistent Aseptic Pyelonephritis After Acute Bacterial Pyelonephritis: Possible Role of Corticosteroids

Hematopoietic Stem Cell Transplant-Membranous Nephropathy Is Associated with Protocadherin FAT1.

Membranous nephropathy (MN) is a common cause of proteinuria in patients receiving a hematopoietic stem cell transplant (HSCT). The target antigen in HSCT-associated MN is unknown. We performed laser microdissection and tandem mass spectrometry (MS/MS) of glomeruli from 250 patients with PLA2R-negative MN to detect novel antigens in MN. This was followed by immunohistochemical (IHC)/immunofluorescence (IF) microscopy studies to localize the novel antigen. Western blot analyses using serum and IgG eluted from frozen biopsy specimen to detect binding of IgG to new 'antigen'. MS/MS detected a novel protein, protocadherin FAT1 (FAT1), in nine patients with PLA2R-negative MN. In all nine patients, MN developed after allogeneic HSCT (Mayo Clinic discovery cohort). Next, we performed MS/MS in five patients known to have allogeneic HSCT-associated MN (Cedar Sinai validation cohort). FAT1 was detected in all five patients by MS/MS. The total spectral counts for FAT1 ranged from 8 to 39 (mean±SD, 20.9±10.1). All 14 patients were negative for known antigens of MN, including PLA2R, THSD7A, NELL1, PCDH7, NCAM1, SEMA3B, and HTRA1. Kidney biopsy specimens showed IgG (2 to 3+) with mild C3 (0 to 1+) along the GBM; IgG4 was the dominant IgG subclass. IHC after protease digestion and confocal IF confirmed granular FAT1 deposits along the GBM. Lastly, Western blot analyses detected anti-FAT1 IgG and IgG4 in the eluate obtained from pooled frozen kidney biopsy tissue and in the serum of those with FAT1-asssociated MN, but not from those with PLA2R-associated MN. FAT1-associated MN appears to be a unique type of MN associated with HSCT. FAT1-associated MN represents a majority of MN associated with HSCT.

Abstract P5-21-10: Phase 2 study and correlative analyses of ruxolitinib, a selective JAK1/2 inhibitor, in patients with metastatic, triple-negative breast cancer

Abstract Background: Preclinical data supports a role for the IL-6/JAK2/STAT3 signaling pathway in breast cancer (BC). Ruxolitinib is an orally bioavailable receptor tyrosine inhibitor targeting JAK1 and JAK2. We evaluated the safety and efficacy of ruxolitinib in patients with metastatic BC and performed correlative analyses. Methods: This was a non-randomized, phase 2 study of patients with refractory, metastatic, triple-negative BC (TNBC). Patients with inflammatory BC (IBC) of any subtype were also enrolled. The primary endpoint was objective response by RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. The study was designed to enroll only patients whose archival tumor tissue was pSTAT3 moderately to strongly positive in the tumor epithelial cells by central immunohistochemistry (IHC). 16 patients underwent pre-treatment biopsy, of whom 4 also had a second biopsy prior to cycle 2. Biopsy samples and paired primary tumor samples (when available) were subjected to multi-color immunofluorescence and/or immune-FISH for leukocyte markers, pSTAT3, and JAK2. RNA sequencing was performed on available on-study frozen biopsy specimens. 17 patients had plasma collected with cell-free DNA (cfDNA) extracted and subjected to low coverage whole-genome sequencing. Results: Of 217 patients who consented to archival tumor testing, T-score for pSTAT3 was 'high' (>5) in 69 patients (31.8%), demonstrating frequent activation of the JAK/STAT pathway in metastatic TNBC or IBC. 23 pSTAT3 high patients were enrolled. Ruxolitinib was generally well-tolerated. The most commonly observed adverse events (any grade) were anemia, neutropenia, thrombocytopenia, constipation, nausea, and increased AST/ALT. Grade 3 or higher toxicities were uncommon. No objective responses were seen among 21 evaluable patients, therefore the study was closed to accrual based on study design. Intensive correlative analyses revealed important insights regarding ruxolitinib effects. Pharmacodynamic analyses of baseline versus cycle 2 biopsies demonstrate downregulation of JAK2 target genes, STAT3 signatures, and JAK/STAT gene ontology gene sets, suggesting on-target activity. There was evidence of immune microenvironment modulation: gene set enrichment analysis implicated reduced macrophage/myeloid phenotypes after treatment and CIBERSORT analysis of inferred immune cell subsets demonstrated reduced monocyte/macrophage proportion after treatment (t-test p=0.013). Multi-color immunofluorescence analyses of immune microenvironment are ongoing and will be reported. 17 patients underwent cfDNA analysis with 8 patients (47%) demonstrating gain or amplification of JAK2. Conclusions: Ruxolitinib, as a single agent, did not meet the primary efficacy endpoint in this refractory patient population. Correlative studies demonstrate evidence of on-target activity and immune microenvironment modulation. Frequent JAK/STAT pathway activation and JAK2 locus chromosomal gains in this cohort suggest that the JAK/STAT pathway remains a potential therapeutic target in BC. Citation Format: Stover DG, Gil Del Alcazar CR, Tolaney SM, Bardia A, Guo H, Balko JM, Overmoyer BA, Gelman RS, Lloyd M, Wang V, Brock JE, Winer EP, Polyak K, Lin NU. Phase 2 study and correlative analyses of ruxolitinib, a selective JAK1/2 inhibitor, in patients with metastatic, triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-10.

P184 Next generation sequencing of small bowel biopsy reveals unresolved donor alleles and eliminates further augmented immunosuppression for antibody mediated rejection

Aim A young pediatric small bowel recipient was transplanted with a donor who was typed only at low resolution by an outside center. For 6 years, the patient received multiple blood transfusions and was monitored by IgG and C1q using LabScreen SAB (One Lambda). High MFI DSA to DQ2 was treated by IVIG desensitization; however, based on the low resolution typing, DQ2 DSA remained. To determine whether this was real DSA or whether the donor had a different DQ2 heterodimer, a fresh frozen biopsy sample was obtained to try to identify the donor HLA alleles by Next Generation Sequencing (NGS). Methods Full length genomic DNA from the patient and biopsy was sequenced for 11 loci (A,B,C,DRB1/3/4/5,DQA1/B1,DPA1/B1) using TruSight HLA v2 Sequencing Panel (Illumina), and analyzed by Assign v.2.1 (Illumina). Two to 3 field resolution was obtained. Results At each locus on the biopsy DNA, the sequence showed discontinuous alignments and much higher numbers of mismatched nucleotide positions than usual due to the mixture of both patient and donor. Individual sequenced reads containing mismatched nucleotides compared with the two predominant alleles were applied to BLAST using both the NCBI and IMGT websites. Once multiple alleles of the first field were obtained, polymorphic positions among exons were compared with other mismatched positions of the sequenced reads to determine the second and third allelic fields. Among all loci, more than two alleles were observed. By eliminating the recipient HLA alleles from the output, the donor’s HLA typing became clear. Typing of the donor by NGS from a biopsy sample revealed that the donor did not have the target heterodimer that had been giving high MFI values by both IgG and C1q. The DQ2 that had been followed was, in fact, not a DSA. Since a prior weak DR53 DSA had already resolved, continued treatment for AMR (Antibody-Medicated Rejection) was not indicated and augmented immunosuppression was discontinued. Conclusions This case decisively demonstrates the power of NGS technology to identify 2–3 field allele level HLA typing from a chimeric specimen using a frozen biopsy specimen. It also highlights the power of the technology to inform clinical decision making.

Fractional laser-assisted percutaneous drug delivery via temperature-responsive liposomes

Liposomes are used for transdermal delivery of drugs and vaccines. Our objective was to develop temperature-responsive (TR) liposomes to achieve temperature-dependent, controlled release of an encapsulated drug, and use fractional laser irradiation to enhance transdermal permeability of these liposomes. TR-liposomes prepared using a thermosensitive polymer derived from poly-N-isopropylacrylamide, N,N-dimethylacrylamide, egg phosphatidylcholine, and dioleoylphosphatidylethanolamine, delivered fluorescein isothiocyanate-conjugated ovalbumin (OVA-FITC) as a model drug. Effect of temperature on liposome size and drug release rate was estimated at two temperatures. Transdermal permeation through hairless mouse skin, with and without CO2 fractional laser irradiation, and penetration into Yucatan micro-pig skin were investigated using Franz cell and fluorescence microscopy. Dynamic light scattering showed that mean liposome diameter nearly doubled from 190 to 325 nm between 37 and 50 °C. The rate and amount of OVA-FITC released from TR-liposomes were higher at 45 °C that those at 37 °C. Transdermal permeation of OVA-FITC across non-irradiated skin from both TR- and unmodified liposomes was minimal at 37 °C, but increased at 45 °C. Laser irradiation significantly increased transdermal permeation of both liposome groups at both temperatures. Fluorescence microscopy of frozen biopsy specimens showed deeper penetration of FITC from unmodified liposomes compared to that from polymer-modified liposomes. Rhodamine accumulation was not observed with polymer-modified liposomes at either temperature. Temperature-dependent controlled release of an encapsulated drug was achieved using the TR-liposomes. However, TR-liposomes showed lower skin permeability despite higher hydrophobicity. Fractional laser irradiation significantly increased the transdermal permeation. Additional studies are required to control liposome size and optimize transdermal permeation properties.

Proteomic Analysis Directs Effective Drug Selection in Relapsed AML By Quantifying Drug Targets

Introduction:Standard therapy for acute myeloid leukaemia (AML) generally includes intense induction with daunorubicin (D) on days 1-3 and cytarabine (A) on days 1-7, followed by consolidation should complete remission (CR) be achieved. Assessment of bone marrow morphology, including percentage of blasts, remains the standard approach to gauge treatment response, however more sensitive molecular based approaches are capable of detecting subclinical levels of leukemic blasts (minimal residual disease, MRD). MRD often remains during and after standard treatment and is the main cause of relapse, a major problem in the management of AML. Resistance of the residual blasts to treatment can be attributed to the activity of pro-survival enzymes, some of which can be pharmacologically inhibited, however, finding the right inhibitor for the right patient presents a major challenge due to the plethora of different enzymes and combinations thereof. Liquid chromatography - tandem mass spectrometry (LC-MS/MS) proteomics enables global and unbiased quantification of protein expression and enzymatic activity in samples. We applied this technology to AML blasts at relapse compared to diagnosis, and in cell lines treated with standard chemotherapy to detect modulated biochemical pathways that contribute to resistance. Thorough investigation into the expression and activity of the protein drug targets enabled selection of inhibitors which proved effective when cells were treated in culture. This approach represents an effective way to better understand the biochemistry of cells following chemotherapy and identify suitable drug targets in biopsies to guide effective inhibitor selection.Methods:LC-MS/MS proteomics and phosphoproteomics was used to investigate global protein expression and kinase activity in primary AML samples at diagnosis and matched relapse (18 cases), and in 3 AML/APL cell lines before and after chemotherapy. Briefly, we collected frozen biopsy specimens from the Barts tissue bank and after thawing the AML blasts were incubated in media for 2 hr at 37oC. Cell lines (HL60, MV411 and P31/FUJ) were treated ± D and/or A (2, 6, or 24 hr). After incubation, cells were centrifuged and washed in PBS, then proteins extracted in urea lysis buffer. Proteins were digested with trypsin, and resulting peptides analysed directly by LC-MS/MS for proteomics or subjected to phosphopeptide enrichment using TiO2 for phosphoproteomics. Commercial (Mascot) and in-house (Pescal, KSEA) software were utilised to identify and quantify proteins, determine kinase activities and investigate intracellular signalling. Cell Viability of blasts ± treatments were recorded using the Guava ViaCount Reagent and Cytometer.Results:On average, >3000 proteins and >9,000 phosphorylation sites were identified per sample. One of the drug targets that correlated strongest with % blasts was CD99 (r=0.79). Blasts showed high abundance & activity of enzymes involved in DNA repair (e.g. PARP1, ATR and PRKDC) at diagnosis and relapse, several significantly increasing in relapse (e.g. PLK3 and APEX1). We observed significant increase in phosphorylation of signalling proteins, such as KIT and STAT5, in relapse. Other signalling pathways regulating survival, apoptosis and metabolism were modulated after relapse but these were patient specific. AML cell lines were more sensitive to D than A. HL60 was the most sensitive cell line while P31/FUJ were least sensitive. Chemotherapy significantly increased the activity of ATM, ATR, PRKDC and MAPKAPK2. Phosphorylation of HSPB1 increased significantly in the presence of D and/or A, and inversely correlated with sensitivity of cells to these drugs. Simultaneous inhibition of ATM & ATR significantly reduced P31/FUJ & MV411 cell viability ± A, while MAPKAPK2 inhibition increased sensitivity of MV411 cells to A.Conclusion:We identified the most abundant and active protein drug targets in AML primary samples and cell lines. Investigating primary AML at diagnosis and relapse uncovered changes in biochemical pathways that regulate DNA repair, survival, apoptosis and metabolism, some of them being modulated by chemotherapy in AML cell lines. These changes were often patient specific, suggesting that to effectively implement targeted therapies, a personalised approach is required and we demonstrate drug selection can be directed by LC-MS/MS proteomics. DisclosuresNo relevant conflicts of interest to declare.

Prostate Cancer in Deceased Liver Donors

BackgroundProstate cancer is the second most common malignant tumor (13%) among male subjects in Poland. The aim of this study was to assess the prevalence of prostate cancer in a group of deceased liver donors. MethodsA total of 784 liver procurement attempts from deceased donors were performed in the Department of General, Transplant and Liver Surgery, Medical University of Warsaw, from January 1, 2012, to April 1, 2015; 700 grafts were actually used in a liver transplant. A retrospective analysis was performed based on these data. Among male donors (n = 486 [62%]), there were 30 (6.2%) cases of a frozen biopsy of the prostate performed before making the decision regarding liver graft utilization. ResultsIn the group of 30 donors who underwent prostate examination, 3 (10%) were diagnosed as having prostate cancer of a moderate invasive stage. In 2 other cases, fresh frozen section suggested prostate cancer; however, this fact was not confirmed in routine section. liver transplantation was not performed in these cases of suspicion of prostate cancer (5 of 30 [17%]) in the frozen biopsy specimens. The difference between groups of donors with prostate cancer and benign pathology of the prostate gland according to prostate-specific antigen serum concentration (P = .578) or age (P = .730) was not statistically significant. ConclusionsIncreased prostate-specific antigen serum concentrations without a diagnosis of prostate cancer in histopathologic examinations should not be an independent contraindication for performing organ transplantation. Nevertheless, for recipient safety, even when prostate cancer is only suspected in the frozen biopsy sample, the procured organ should not be used for transplantation.

Feasibility of Nipple-Sparing Mastectomy with Immediate Breast Reconstruction in Breast Cancer Patients with Tumor-Nipple Distance Less Than 2.0cm.

Debate continues concerning the oncological risk of nipple-sparing mastectomy (NSM) with immediate breast reconstruction (IBR) if the tumor-nipple distance (TND) is less than 2.0cm. In this retrospective study, we analyzed oncological outcomes after NSM with IBR for the treatment of breast cancer to determine the risk posed by NSM in cases in which magnetic resonance imaging (MRI) showed a TND <2.0cm but intraoperative frozen biopsy results were negative for tumor cells at the nipple base. We conducted a retrospective review of patients with breast cancer who underwent NSM with IBR at Samsung Medical Center between 2008 and 2014. Preoperative MRI was done in all cases to define the TND, and frozen biopsy specimens were obtained intraoperatively. Among the 266 NSMs performed, TND was <2.0cm in 145 cases (54.5%) and ≥2.0cm in 121 cases (45.5%). Median follow-up was 25.6months. There were no significant differences between the two patient groups with respect to disease-free survival or local recurrence-free survival. Our results suggest that NSM can be a feasible treatment option when the intraoperative frozen biopsy is negative for tumor cells even with a TND <2.0cm in MRI.

Abstract 3826: A reversible DNA methylation signature precedes sporadic mutations in the colorectal adenoma-dysplasia-cancer development

Abstract Background and aims: New generation sequencing and array technologies now allow the systematic investigation and comparison of genetic or epigenetic alterations in AD-CRC. The colorectal adenoma-dysplasia-cancer (AD-CRC) development is characterised by sporadic (&amp;lt;45% frequency) mutations in a limited number of genes. Recent epigenetic investigations showed that methylation of selected genes occurs in high frequency (&amp;gt;95%) in early stages of cancers already. Aims: Evaluation of the role of epigenetic and genetic alterations in the AD-CRC development using new generation sequencing and DNA methylation arrays. Materials and methods: DNA was isolated from fresh frozen biopsy specimens (20 normal; 33 adenomas; 17 CRCs). First, a multiplex PCR panel was designed to amplify mutation hot spots of 12 selected genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, MSH6, NRAS, PIK3CA, SMAD2, SMAD4, TP53). Further DNA methylation analysis of 94 genes was performed on Human Colon Cancer EpiTect Methyl II Signature PCR Array (Qiagen) from the same DNA specimen. After RNA isolation whole genome expression analysis was performed with HGU133plus2 microarrays (Affymetrix) from 49 normal, 49 adenoma and 49 CRC specimens and on HT-29 cells with and without 5-aza-deoxycytidin treatment. Immunohistochemistry confirmation of expression changes on protein level was performed using tissue microarrays. Results: Mutations were found in 76% of adenomas and 78% of the cancer cases. The average number of mutations found in mutated samples was 1; 1,8; 1,9 and 2,3 in low grade adenomas, high grade adenomas, carcinomas and serrated adenomas, respectively. The APC suppressor gene was mutated in adenomas more frequently than in carcinomas (36% vs. 24%). The most frequently mutated genes were APC, TP53 and KRAS with 36%, 18% and 26% frequencies in adenomas and 24%, 47% and 45% frequencies in carcinomas. DNA methylation was found in 100% of the investigated colorectal adenoma and cancer specimens. Eight genes were found to be methylated in all of the cases. This gen set included SFRP1, MAL, SLIT2, SST, VIM; another set of genes (DKK1, SLI3, TMEFF2) was found to be hypermethylated in adenomas and cancers in &amp;gt;75% of the cases. In adenomas 56 genes, in dysplasias 40 in cancer 37 genes were methylated (&amp;gt;50% of the cases). The effect of methylation could be confirmed by decreased mRNA and protein expression. Demethylation treatment successfully restored the expression profile of the top methylated genes. Conclusion: Methylation of gene sets occurred in early premalignant stages followed by somatic mutations in increasing number through the AD-CRC development. Demethylation treatment could reverse the systematic, robust methylation alterations. Epigenetic alterations precede the somatic mutations of the AD-CRC and show higher significance in CRC development than genetic. Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Barnabas Wichmann, Arpad V. Patai, Zsolt Tulassay. A reversible DNA methylation signature precedes sporadic mutations in the colorectal adenoma-dysplasia-cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3826. doi:10.1158/1538-7445.AM2015-3826

Correlation between ERK1 and STAT3 expression and chemoresistance in patients with conventional osteosarcoma.

BackgroundThe standard therapy regimen of conventional osteosarcoma includes neoadjuvant chemotherapy followed by surgical resection and postoperative chemotherapy. The percentage of necrotic tissue following induction chemotherapy is assessed by using the Huvos grading system, which classifies patients as “poor responders” (PR) and “good responders” (GR). The aim of this study was to identify molecular markers expressed differentially between good and poor responders to neoadjuvant chemotherapy in order to predict the response to chemotherapy in conventional osteosarcomas before beginning treatment.MethodsSuppression Substractive Hybridization (SSH) was performed by using cDNA from frozen biopsy specimens. Expression of selected relevant genes identified by SSH was validated by using QRT-PCR. Immunohistochemistry (IHC) on tissue microarray (TMA) sections of 52 biopsies was performed to investigate protein expression in an independent cohort.ResultsERK1 and STAT3 mRNA level were significantly different between PR and GR in an independent cohort. Phosphorylated STAT3 and ERK1 expressions by IHC on TMA were correlated with poor response to chemotherapy.ConclusionsOur results suggest that ERK1 and STAT3 expression are good predictive markers for chemotherapy response and that inhibitors might be used in combination with common chemotherapeutic drugs in conventional osteosarcomas.

Prognostic Value and Clinical Pathology of MACC-1 and c-MET Expression in Gastric Carcinoma

This study was to assess the expression of MACC-1 and c-MET in gastric cancer, and to correlate this expression with clinicohistological parameters and patient prognosis. Total RNA was extracted from cancer tissue and adjacent normal mucosa from frozen biopsy specimens of 30 patients with gastric cancer, and MACC-1 expression was assessed by RT-PCR. MACC-1 and c-MET protein expression were also assessed in paraffin-embedded tissues obtained from 436 tumor mucosa and 92 normal mucosa specimens by immunohistochemistry. The correlation between MACC-1 and c-MET expression and clinicopathological factors (age, sex, histology, tumor depth, lymph node status and vessel invasion) were also evaluated. RT-PCR analysis revealed that MACC-1 expression was significantly higher in cancerous mucosa compared with normal tissue. Immunohistochemical analysis indicated that MACC-1 and c-MET were moderately or strongly expressed in gastric cancer tissue, whereas expression was weak or absent in non-cancer tissue. Expression of MACC-1 or c-MET was significantly associated with larger tumor size, deeper tumor invasion, presence of lymph node metastasis, lymphatic involvement, venous invasion, distant metastasis and advanced clinical stage. However, only MACC-1 exhibited significantly greater expression in carcinomas from the higher age group. The intensity of MACC-1 and c-MET expression was also positively correlated. Survival analysis of the 436 gastric cancer patients revealed that patients in clinical stages I, II and III exhibiting lower MACC-1 and c-MET expression had a higher 5-year survival rate compared with patients expressing high levels of these proteins. Multivariate analysis revealed that MACC-1 and c-MET may be independent prognostic indexes of gastric carcinoma (P < 0.01). Our findings confirm that MACC-1 and c-MET expression is strongly related to gastric cancer stage and degree of malignancy, and is inversely correlated to patient prognosis. Thus, MACC-1 and c-MET may interact to promote tumorigenesis and their expression may be used as independent prognostic markers in gastric cancer.

Histopathologic findings in the sacrocaudalis dorsalis medialis muscle of horses with vitamin E–responsive muscle atrophy and weakness

To characterize clinical findings, outcomes, muscle characteristics, and serum or muscle concentrations of α-tocopherol for horses with vitamin E-responsive signs of muscle atrophy and weakness consistent with signs of equine motor neuron disease (EMND). Retrospective case-control study. 8 affected (case) adult horses with acute (n = 3) or chronic (5) gross muscle atrophy that improved with vitamin E treatment and 14 clinically normal (control) adult horses with adequate (within reference range; 8) or low (6) muscle concentrations of α-tocopherol. Medical records were reviewed, serum and muscle concentrations of α-tocopherol were measured, and frozen biopsy specimens of sacrocaudalis dorsalis medialis muscle and gluteal muscle were histologically evaluated for pathological changes. Fiber type composition and fiber diameters were assessed in gluteal muscle specimens. A myopathy that was histologically characterized by redistribution of mitochondrial enzyme stain (moth-eaten appearance) and anguloid atrophy of myofibers was evident in sacrocaudalis dorsalis medialis muscle fibers of the 8 affected horses that had low serum (6/8) or skeletal muscle (5/5) concentrations of α-tocopherol; these histopathologic changes were not found in muscle specimens of control horses with low or adequate muscle concentrations of α-tocopherol. All affected horses regained strength and muscle mass within 3 months after initiation of vitamin E treatment and dietary changes. A vitamin E-deficient myopathy characterized histologically by a moth-eaten appearance in the mitochondria and anguloid myofiber atrophy in frozen sections of sacrocaudalis dorsalis medialis muscle biopsy specimens was found in horses with clinical signs of EMND that were highly responsive to vitamin E treatment. This myopathy may be a specific syndrome or possibly precede the development of neurogenic muscle fiber atrophy typical of EMND.

Abstract P3-04-01: Protein pathway activation mapping of the I-SPY 1 biopsy specimens identifies new network focused drug targets for patients with triple negative tumors

Abstract Background: Identification of new therapies and predictive biomarkers is needed for patients with triple negative (TN) breast cancer. Elucidating pathway activation in cancer is critical as these proteins represent targets for many of the new molecular therapeutics and companion diagnostic markers. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens collected 1–4 days after first chemotherapy treatment (T2). Reverse Phase Protein Microarray was used to measure the activation of 39 and 100 signaling proteins in the T1 and T2 biopsies, respectively, including drug targets for Phase I-III and FDA cleared therapeutics. Associations between pathway activation and response to chemotherapy (RCB 0/1 vs. 2/3) and relapse-free survival (RFS) were evaluated for the triple-negative (TN) patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p &amp;lt; 0.05; Cox proportional hazard model, likelihood ratio p &amp;lt; 0.05. Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of receptor tyrosine kinases such as MET and ALK in the T2 biopsies from patients that recurred, and VEGFR activation was seen in the T1 relapsed tumors. Activation in the AURORA-PLK1 cell cycle signaling network was seen with increased activation in the T2 biopsy from patients who did not respond to therapy and had poor RFS. Dynamic changes in ERK activation were revealed with decreased ERK activation between T1 and T2 time points associated with recurrence. Conclusions: Our analysis has revealed activation and increased expression of proteins involved in proliferation (Ki67), DNA repair (phosphorylated p53) and protein kinases in patients with TN disease who had poor clinical response. These events, if validated, point to potential treatment options that could be considered for non-responding TN breast cancer patients. VEGFR, ALK and MET, have FDA cleared therapeutics that could be rationally proposed for rapid clinical investigation, and there are numerous investigational agents that target AURORA and PLK1 in the clinic. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with TN tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-01.

Abstract P3-04-02: Protein pathway activation mapping of I-SPY 1 biopsy specimens identifies new network focused drug targets for patients with HR+/HER2− tumors

Abstract Background: Profiling protein signaling activation in cancer is critical as these proteins represent targets for new molecular therapeutics. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane- based chemotherapy that longitudinally collected biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens (T2, 1–4 days post-chemotherapy). Reverse Phase Protein Microarray technology was used to quantitatively measure the activation of 39 and 100 key signaling proteins in the T1 and T2 biopsies, respectively, including numerous drug targets for Phase I-III and FDA-cleared therapeutics. Associations between protein activation and response to chemotherapy (RCB 0/1 vs 2/3) and relapse-free survival (RFS) were evaluated for the HR+/HER2− patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p &amp;lt; 0.05; Cox proportional hazard model, likelihood ratio p &amp;lt; 0.05. Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of HER family signaling and downstream AKT activation in tumors from patients who do not respond (RCB2/3) and/or whom recurred. Increased ERBB2 and ERBB4 levels were observed at T1 and T2, respectively, from patients who did not respond to neoadjuvant therapy. Dynamic changes in ERBB3 and AKT phosphorylation/activation were revealed between the T1 and T2 time points, which were associated with recurrence. Increased phosphorylation of MARCKS, a known PKC substrate, was seen in the T1 biopsy in the non-response cohort. Conclusions: Our analysis revealed activation/increased expression of HER family proteins along with downstream AKT activation in HR+/HER2− patients who have poor clinical response. These events, if validated, point to potential treatment options that could be rationally considered for non-responding HR+/HER2− patients with a number of investigational agents that target ERBB3 and AKT signaling in the clinic today. Increasing HER2 protein expression in a HER2− cohort may appear contradictory, however these findings may point to important subtle increases in HER2 that have important clinical considerations. Given the known clinical benefit from HER2 directed therapy in HER2− patients seen in other studies, our findings may have clinical relevance if validated. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with HR+/HER2− tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-02.

Proteomic approach to human kidney glomerulus prepared by laser microdissection from frozen biopsy specimens: Exploration of proteome after removal of blood‐derived proteins

Abundance of blood-derived proteins in glomeruli prepared by laser microdissection from human kidney biopsy specimens has hampered in-depth proteomic analysis of glomeruli. We attempted to establish experimental platform for in-depth proteomic analysis of glomeruli by removal of blood-derived proteins from frozen biopsy samples. Frozen sections of biopsy samples were exposed to repeated PBS washes prior to laser microdissection to remove blood-derived proteins, and glomerular dissectants were analyzed by MS. The depth of proteomic analysis was evaluated by dynamic range of identified proteins and detection of low-abundance proteins. Two times PBS washes of frozen sections effectively eliminated blood-derived proteins in laser-microdissected glomeruli and gave an increased number of identified proteins. Analysis of glomeruli from single specimens by a linear ion trap-Orbitrap mass analyzer generated nonredundant, high-confidence datasets of more than 400 identified proteins with high reproducibility, which attained to a considerable depth of the glomerulus proteome as revealed by a wide dynamic range and identification of low-abundance proteins. Implementation of washing of frozen section with PBS successfully removed blood-derived proteins and resulted in an in-depth proteomic analysis of laser-microdissected glomeruli, suggesting applicability to clinical study.

Can cryopreservation improve diagnostic utility of frozen biopsy specimens? Analysis of tissue morphology and antigenicity upon cryopreservation

Can cryopreservation improve diagnostic utility of frozen biopsy specimens? Analysis of tissue morphology and antigenicity upon cryopreservation

In Situ Detection of HY-Specific T Cells in Acute Graft-versus-Host Disease–Affected Male Skin after Sex-Mismatched Stem Cell Transplantation

HY-specific T cells are presumed to play a role in acute graft-versus-host disease (aGVHD) after female-to-male stem cell transplantation (SCT). However, infiltrates of these T cells in aGVHD-affected tissues have not yet been reported. We evaluated the application of HLA-A2/HY dextramers for the in situ detection of HY-specific T cells in cryopreserved skin biopsy specimens. We applied the HLA-A2/HY dextramers on cryopreserved skin biopsy specimens from seven male HLA-A2(+) pediatric patients who underwent stem cell transplantation with confirmed aGVHD involving the skin. The dextramers demonstrated the presence of HY-specific T cells. In skin biopsy specimens of three male recipients of female grafts, 68% to 78% of all skin-infiltrating CD8(+) T cells were HY-specific, whereas these cells were absent in biopsy specimens collected from sex-matched patient-donor pairs. Although this study involved a small and heterogeneous patient group, our results strongly support the hypothesis that HY-specific T cells are actively involved in the pathophysiology of aGVHD after sex-mismatched stem cell transplantation.

Integrin expression in oral hairy leukoplakia and normal tongue epithelium.

To describe the expression of integrins in the epithelium of oral hairy leukoplakia (HL) and compare to that of normal lateral tongue epithelium. Immunohistochemistry to identify integrins (alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1) was performed, using a standard biotin-streptavidin-peroxidase technique on five clinically and histologically confirmed frozen biopsy specimens of HL and five normal lateral tongue control tissues. Expression of integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 was seen both in HL epithelium and in normal control tissue. alpha 5 expression was not seen in HL or in control tissue epithelium. alpha 2 and alpha 3 were expressed mainly in the basal and suprabasal layers; alpha 6 expression was most intense on the basal surface of the basal cells, alpha v was expressed in the basal and suprabasal layers with more expression seen in the higher differentiated cell layers than the other integrins. beta 1 expression was seen in the basal and suprabasal layers only. No apparent difference between HL and normal oral mucosa was noted in the staining pattern of the various integrins. Integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 are expressed in HL and the expression pattern is not different from that of normal oral mucosa. alpha 5 is not expressed in HL or in normal oral epithelium.

Hyperinsulinemic hypoglycemia due to diffuse nesidioblastosis in adults: A case report

Persistent hyperinsulinemic hypoglycemia is caused most commonly by an insulinoma in adults or by nesidioblastosis in neonates. In adults, nesidioblastosis is a rare disorder characterized by diffuse or disseminated proliferation of islet cells. We recently encountered a case of nesidioblastosis in an adult. A 71-year-old man was admitted due to intermittent general weakness, abdominal pain, and mild dyspnea. The patient underwent a subtotal gastrectomy for a gastric adenocarcinoma two years ago. After 5 d of admission, the patient showed symptoms of cold sweating, chilling, and hypotension 30 min after eating. Thereafter, he frequently showed similar symptoms accounting for hypoglycemia regardless of food consumption. Laboratory findings revealed a low fasting blood glucose level (25 mg/dL), and a high insulin level (47 muIU/mL). Selective intra-arterial calcium stimulation with hepatic venous sampling (ASVS) was performed to localize a mass and revealed an increased insulin level about four-fold that of the normal fasting level at 60 s in the splenic artery, which suggested the presence of an insulinoma in the tail of pancreas. A distal pancreatectomy was performed. Neither intraoperative exploration nor a frozen biopsy specimen detected any mass-forming lesion. On the histological examination, many of the islets were enlarged and irregularly shaped in all specimens, the arrangement of which was a lobulated islet pattern. Cytologically, a considerable subpopulation of endocrine cells showed enlarged and hyperchromatic nuclei. By immunohistochemistry, the cells were identified as beta-cells. These clinical, radiological, microscopic and immuno-histochemical findings are consistent with diffuse nesidioblastosis in adults.

Helicobacter pylori and Helicobacter heilmannii in untreated Bulgarian children over a period of 10 years

The aims of the study were to evaluate the incidence of Helicobacter pylori and Helicobacter heilmannii in untreated Bulgarian children from 1996 to 2006, to analyse the performance of diagnostic tests, and to look at H. pylori density in specimens by culture. Antral specimens from children with chronic gastritis (n=513), peptic ulcers (n=54) and other diseases (n=91) were evaluated by direct Gram staining (DGS), in-house rapid urease test (RUT) and culture. The living environment and semi-quantitative H. pylori density were assessed in 188 and 328 children, respectively. H. pylori infection was found in children with ulcers (77.8 %), chronic gastritis (64.5 %) and other diseases (36.3 %). Half (51.4 %) of patients aged 1-5 years and 77.4 % of those aged 16-17 years were H. pylori-positive. Of all children, 328 (49.8 %) showed positive DGS, 184 (28 %) had a positive RUT, and 386 (58.7 %) were culture-positive. Unlike gastric mucus specimens, frozen biopsy specimens provided reliable diagnosis. H. heilmannii was observed in two (0.3 %) children. High H. pylori density (growth into all quadrants of plates) was found in 18 % of 328 children evaluated, involving 31 % of ulcer and 16.7 % of non-ulcer patients. H. pylori infection was more common in rural children with chronic gastritis (91.3 %) than in the remainder (66.7 %). In conclusion, H. pylori infection was common in symptomatic Bulgarian children. The infection prevalence was >77 % in patients aged 16-17 years, in children with a duodenal ulcer, and in rural patients. H. heilmannii infection was uncommon. The performance of the bacterial culture was good. The impact of H. pylori density on the clinical expression and eradication of the infection requires further evaluation. The results highlight the need for routine H. pylori diagnosis in rural children with chronic gastritis.