Nita Williams

Analysis of the Molecular Quality of Human Tissues An Experience From the Cooperative Human Tissue Network

The scientific usefulness of the data obtained from tissue analysis is related to specimen quality, which may be affected by conditions that may contribute to the degradation of the specimen before processing and analysis. We determined the usability of nucleic acids extracted from banked human tissues for further molecular analyses. We assayed 151 tissue specimens, storedfor various times at 4 divisions of the Cooperative Human Tissue Network, National Cancer Institute, Bethesda, MD, for DNA and RNA degradation. Simple electrophoresis, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, and Northern blot analysis were compared to determine the optimal quality control procedure. In addition, a time course degradation procedure was performed on human lung tissue. Gel electrophoresis was as informative as PCR, RT-PCR, and Northern blot analysis in determining the molecular usefulness of the human tissues. Overall, 80% of the stored human tissues had good-quality DNA, and 60% had good-quality RNA. Electrophoresis procedures for DNA and RNA offer a quick and valuable measure of the molecular quality of stored human tissues. The DNA and RNA degradation of one tissue type (lung) was stable for both nucleic acids for up to 5 hours after excision.

A Founding Locus within the RET Proto-Oncogene May Account for a Large Proportion of Apparently Sporadic Hirschsprung Disease and a Subset of Cases of Sporadic Medullary Thyroid Carcinoma

Hirschsprung disease (HSCR) is a common congenital disorder characterized by aganglionosis of the gut. The seemingly unrelated multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant disorder characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Yet, germline mutations in the RET proto-oncogene are associated with both MEN 2 and HSCR. In the former, gain-of-function mutations in a limited set of codons is found, whereas, in the latter, loss-of-function mutations are found. However, germline RET mutation is associated with only 3% of a population-based series of isolated HSCR, and little is known about susceptibility to sporadic MTC. We have found previously that specific haplotypes comprising RET coding single-nucleotide polymorphisms (SNPs) comprising exon 2 SNP A45A were strongly associated with HSCR, whereas haplotypes associated with exon 14 SNP S836S were associated with MTC. In this study, we describe three novel intron 1 SNPs, and, together with the coding SNP haplotypes, the data suggest the presence of distinct ancestral haplotypes for HSCR and sporadic MTC in linkage disequilibrium with a putative founding susceptibility locus/loci. The data are consistent with the presence of a very ancient, low-penetrance founder locus approximately 20-30 kb upstream of SNP A45A, but the failure of the SNPs to span the locus presents challenges in modeling mode of transmission or ancestry. We postulate that this founding locus is germane to both isolated HSCR and MTC but also that different mutations in this locus would predispose to one or the other.

Pilot Study of Rosiglitazone Therapy in Women with Breast Cancer: Effects of Short-term Therapy on Tumor Tissue and Serum Markers

Purpose: Peroxisome proliferator-activated receptor γ (PPARγ) is a steroid nuclear receptor that is activated by natural compounds such as specific fatty acids and synthetic drugs such as thiazolidinedione antidiabetic agents. Expressed in normal and malignant mammary epithelial cells, activation of PPARγ is associated with antiproliferative effects on human breast cancer cells in preclinical studies. The purpose of this study was to test the hypothesis that PPARγ ligand therapy might inhibit tumor growth and progression in human breast cancer. Experimental Design: We conducted a pilot trial of short-term (2-6 weeks) treatment with the thiazolidinedione rosiglitazone in 38 women with early-stage (Tis-T2, N0-1, M0) breast cancer, administered between the time of diagnostic biopsy and definitive surgery. Results: Short-term treatment with rosiglitazone (8 mg/d) did not elicit significant effects on breast tumor cell proliferation using Ki67 expression as a measure of cell proliferation and surrogate marker of tumor growth and progression. In pretreatment tumors notable for nuclear expression of PPARγ by immunohistochemistry, down-regulation of nuclear PPARγ expression occurred following rosiglitazone administration (P = 0.005). No PPARG mutations were identified, and the incidence of P12A and H446H polymorphisms did not differ relative to U.S. controls (P = 0.5). Treatment with rosiglitazone resulted in increased serum adiponectin (P < 0.001), decreased insulin levels (P = 0.005), and increased insulin sensitivity (P = 0.004). Rosiglitazone was well tolerated without serious adverse events. Conclusion: Our data indicate that short-term rosiglitazone therapy in early-stage breast cancer patients leads to local and systemic effects on PPARγ signaling that may be relevant to breast cancer.

Variability in organ-specific EGFR mutational spectra in tumour epithelium and stroma may be the biological basis for differential responses to tyrosine kinase inhibitors

Organ-specific differences in epidermal growth factor receptor (EGFR) mutational spectra and frequencies were found in lung cancer and sporadic and BRCA 1/2-related breast cancers. Additionally, we found a high frequency of EGFR mutations in the tumour stroma of these invasive breast carcinomas. Those organ-specific mutational spectra and potential targets in the cancer-associated stroma might influence the efficacy of TKI therapy.

A novel mutation in the tyrosine kinase domain of ERBB2 in hepatocellular carcinoma

BackgroundSeveral studies showed that gain-of-function somatic mutations affecting the catalytic domain of EGFR in non-small cell lung carcinomas were associated with response to gefitinib and erlotinib, both EGFR-tyrosine kinase inhibitors. In addition, 4% of non-small cell lung carcinomas were shown to have ERBB2 mutations in the kinase domain. In our study, we sought to determine if similar respective gain-of-function EGFR and ERBB2 mutations were present in hepatoma and/or biliary cancers.MethodsWe extracted genomic DNA from 40 hepatoma (18) and biliary cancers (22) samples, and 44 adenocarcinomas of the lung, this latter as a positive control for mutation detection. We subjected those samples to PCR-based semi-automated double stranded nucleotide sequencing targeting exons 18–21 of EGFR and ERBB2. All samples were tested against matched normal DNA.ResultsWe found 11% of hepatoma, but no biliary cancers, harbored a novel ERBB2 H878Y mutation in the activating domain.ConclusionThese newly described mutations may play a role in predicting response to EGFR-targeted therapy in hepatoma and their role should be explored in prospective studies.

Molecular Classification of Parathyroid Neoplasia by Gene Expression Profiling

The current classification of sporadic parathyroid neoplasia, specifically the distinction of adenoma from multiple gland neoplasia (double adenoma and nonfamilial primary hyperplasia) is problematic and results in a relatively high rate of clinical error. Oligonucleotide microarrays (Affymetrix U133A) were used to evaluate parathyroid samples from 61 patients; 35 adenomas, 10 nonfamilial multiple gland neoplasia, 3 familial primary hyperplasia, 8 renal-induced hyperplasia, and 5 from patients without parathyroid disease (normals). A multiclass comparison using supervised clustering identified distinct gene signatures for each class of parathyroid samples. We developed a predictor model that correctly identified 34 of 35 cases of adenoma, 9 of 10 cases of nonfamilial multiple gland neoplasia, and identified a minimum set of 11 genes for the distinction of adenoma versus multiple gland neoplasia. All methods of unsupervised clustering showed two related but different types of parathyroid adenomas that we have arbitrarily designated as type 1 and type 2 adenomas. Multiple gland parathyroid neoplasia, which represents either synchronous or asynchronous autonomous growth in two, three, or all four parathyroid glands, is a distinct molecular entity and does not represent the molecular pathogenesis of adenoma occurring in multiple glands.

Lenalidomide and vorinostat maintenance after autologous transplant in multiple myeloma

Single‐agent post‐autologous transplant maintenance therapy with lenalidomide is standard of care for patients with multiple myeloma. The tolerability and effectiveness of combination post‐transplant maintenance therapy is unknown, so we investigated lenalidomide and vorinostat (suberoylanilide hydroxamic acid) in this setting, hypothesizing that the regimen would be well tolerated and associated with an improved post‐transplant response. This trial followed a standard 3 × 3 dose escalation phase 1 design. Vorinostat was administered beginning day +90 post‐haematopoietic stem cell transplantation for days 1–7 and 15–21, and lenalidomide was started at 10 mg days 1–21, both on a 28‐d cycle. The primary endpoint was maximum tolerated dose and dose limiting toxicities were assessed during the first cycle. Treatment was well tolerated in 16 enrolled patients. During Cycle 1, the most common toxicities included cytopenias, gastrointestinal complaints and fatigue. Seven patients improved their transplant response after starting combination therapy. The median follow‐up was 38·4 months, and the median progression‐free survival and overall survival have yet to be reached. This oral post‐transplant maintenance regimen was well tolerated. This is the first trial to publish results on the use of a histone deacetylase inhibitor in the maintenance setting, and it provides rationale for the ongoing randomized trial in maintenance (ISRCTN 49407852).

Associations of high‐dose melphalan pharmacokinetics and outcomes in the setting of a randomized cryotherapy trial

High‐dose melphalan followed by autologous stem cell transplantation remains the standard of care for eligible patients with multiple myeloma, but disease response and toxicity, including severe mucositis, varies among patients. Our randomized trial investigated duration of cryotherapy (2 and 6 h) for reduction of mucositis prevalence and severity and explored factors associated with variability in pharmacokinetics and outcomes from melphalan therapy. The results demonstrate that 2‐h is at least as effective as 6‐h cryotherapy in decreasing severe mucositis. From a population pharmacokinetic model, we identified that fat‐free mass, hematocrit, and creatinine clearance were significant covariates, as reported previously. Furthermore, we observed the rs4240803 SLC7A5 polymorphism was significantly associated with pharmacokinetic variability, and pharmacokinetics was associated with both mucositis and neutropenia. However, melphalan exposure was not associated with progression‐free or overall survival in our dataset. These findings contribute to ongoing efforts to personalize melphalan dosing in transplant patients.

Ninety-minute daratumumab infusion is safe in multiple myeloma

Daratumumab is a first-in-class anti-CD38 moantibody approved for relapsed and refractory multiple myeloma [1, 2] and being tested in both smoldering [3] and newly diagnosed myeloma [4]. CD38 is expressed on airway smooth muscle cells, and infusion related reactions (IRRs) were marked by symptoms (cough, wheezing, and rhinorrhea) similar to those of allergic rhinitis. Of all patients treated at 8 and 16 mg/kg in two pivotal trials, the proportion of patients that suffered an IRR was 65% (grade 1–2) and 3% (grade 3–4) in the GEN501 trial [1], and 59% (grade 1–2) and 4% (grade 3–4) in the SIRIUS trial [2]. A pooled analysis of SIRIUS and GEN501 data demonstrated that most IRRs (95.8%) occur during the first infusion, with a decreased incidence of IRRs during second (7%) and subsequent infusions (7%) [5]. There is no relationship between daratumumab serum levels and the development of IRRs [6]. The incidence and severity of IRRs has led to the recommended administration rates which result in infusion times for the first, second, and subsequent infusions of 6.5, 4.5, and 3.5 h, respectively [7]. To decrease the IRR rate, one trial tested the addition of 10 mg of montelukast as a premedication prior to the first daratumumab infusion and found the IRR rate was one-third lower in patients who received this leukotriene inhibitor [8]. Beyond slowing the infusion rate, no other clinical trial has successfully reported any intervention to decrease the IRR rate [9]. Because of the low incidence of infusion reactions after the first infusion, we hypothesized that increasing the infusion rate of the third and subsequent doses of daratumumab would not affect the safety profile. Previously this concept was studied with rituximab in which the second or subsequent infusion was given at a rapid 1 h infusion rate [10]. This led to a rapid rituximab infusion protocol, where patients would be switched if they met certain rapid infusion criteria. With this experience, we developed an accelerated daratumumab infusion protocol with the hypothesis that increasing the infusion rate would not increase the IRR rate beginning with the third dose of daratumumab. This was a prospective, single-center, and open-label safety study of an accelerated daratumumab infusion in multiple myeloma patients. To be eligible, patients had to have received two or more doses of daratumumab at standard infusion rates; prior IRR’s with daratumumab did not exclude patients. The infusion rate was calculated to deliver 20% of the dose over 30 min (200 mL/hr), and then the rate was increased to deliver the remaining 80% over 60 min (450 mL/hr). This resulted in a 90 min estimated infusion time (total volume 550 mL). Per institutional policy, the total volume accounts for manufacturer overfill, and the tubing is primed with drug. Standard vital signs were collected prior to infusion start, every 15 min for 1 h and finally at the end of the infusion. For the first accelerated infusion, patients were observed in the infusion suite for 30 min after infusion completion to assess for a delayed IRR. Existing premedication regimens were not altered for study purposes. Simon’s two-stage optimal design was utilized based on the null hypothesis that the IRR-free rate was at most 85% and the alternative hypothesis that it is at least 98%. With a type I error rate of 0.05 and 80% power, the design allowed seven patients to be treated in the first stage, and if no patient experienced ≥grade 3 IRR, an additional 21 patients would be treated. At the end of the study, if none or only one patient out of the total 28 experienced ≥grade 3 IRR, the regimen will be declared safe. IRRs and their respective symptoms were graded as per the National Cancer Institute (NCI) Common Toxicity Criteria for Adverse Events (CTCAE version 4.03). The protocol was * Hallie Barr Hallie.Barr@osumc.edu

G-CSF improves safety when you start the day after autologous transplant in multiple myeloma

Douglas W. Sborov , Yu Kyoung Cho , Francesca Cottini , Erinn M. Hade, Misty Lamprecht, Karen Tackett, Nidhi Sharma, Nita Williams, Junan Li, Steven Devine, Ming Poi, Mitch A. Phelps and Craig C. Hofmeister Division of Hematology, Department of Internal Medicine, College of Medicine, the Ohio State University, Columbus, OH, USA; Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, the Ohio State University, Columbus, OH, USA; Internal Medicine Residency Program, Department of Internal Medicine, College of Medicine, the Ohio State University, Columbus, OH, USA; Center for Biostatistics, Department of Biomedical Informatics, the Ohio State University, Columbus, OH, USA; Ohio State University Wexner Medical Center, the Ohio State University, Columbus, OH, USA; Division of Pharmacy Practice and Administration, College of Pharmacy, the Ohio State University, Columbus, OH, USA