Harvey J. Mamon

Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing

PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

Oncogenes, growth factors, and signal transduction

HOW external growth signals are transmitted from the surface of the cell to the nucleus is one of the fundamental problems of cell biology. Research in the field of signal transduction has been mot...

Adjuvant Treatment of Colorectal Cancer

Colorectal cancer is the fourth most common noncutaneous malignancy in the United States and the second most frequent cause of cancer‐related death. Approximately three quarters of patients are diagnosed with disease limited to the bowel wall or surrounding lymph nodes. Over the past decade, significant progress has been made in the treatment of localized colorectal cancer due to advances in surgery, radiotherapy, and chemotherapy. For patients with Stage III colon cancer, an overall survival benefit for fluorouracil‐based chemotherapy has been firmly established, and recent data have shown further efficacy through the inclusion of oxaliplatin into adjuvant treatment programs. For patients with Stage II colon cancer, the use of adjuvant chemotherapy remains controversial, but may be appropriate in a subset of individuals at high risk for disease recurrence. In the treatment of patients with rectal cancer, improved outcomes have been noted with the use of total mesorectal excision and preoperative concurrent chemoradiotherapy. Current randomized clinical trials in the adjuvant therapy of colorectal cancer are examining the value of adding agents known to be active in metastatic disease, including those that modify specific molecular targets.

High Risk of Brain Metastases in Surgically Staged IIIA Non–Small-Cell Lung Cancer Patients Treated With Surgery, Chemotherapy, and Radiation

PURPOSE Lung cancer is the leading cause of cancer mortality in the United States. We sought to review our experience with surgically staged IIIA (N2) non-small-cell lung cancer (NSCLC), focusing on the patterns of failure in consecutively treated patients from 1988 to 2000. PATIENTS AND METHODS The records of 177 patients were reviewed. Collected data included stage, histology, use of chemotherapy and radiation, initial and subsequent sites of failure, and survival. One hundred twenty-four patients have died; follow-up time is 35 months among the remaining patients. RESULTS The median survival from the time of surgery was 21.0 months, with a 3-year overall survival (OS) of 34%. Nodal downstaging to N0 disease correlated with OS and progression-free survival (PFS; P < .001). The most common site of recurrence was the brain. Thirty-four percent of patients recurred in the brain as their first site of failure, and 40% of patients developed brain metastases at some point in their course. In patients with nonsquamous histology and residual nodal involvement after neoadjuvant therapy, the risk of brain metastases was 53% at 3 years. CONCLUSION Patients treated with neoadjuvant therapy for N2-positive stage IIIA NSCLC enjoy an advantage in both OS and PFS if their lymph node status is downstaged to N(0). Because brain metastases constitute the most common site of failure in these patients, future studies focusing on prophylaxis of brain metastases may improve the outcome in patients with stage IIIA NSCLC.

Raf-1: A kinase currently without a cause but not lacking in effects

Recent studies of the molecular mechanisms of signal transduction have highlighted four molecules as potential secondary signal transducers for receptor tyrosine kinases. These are phospholipase C-γ, GTPase activating protein, type I phosphoinositide kinase and Raf-1. Recent work on the daf-1 gene of Caenorhabditis elegans raises the possibility that, in at least some cases, extracellular signals can activate Raf-1 family members directly. In this minireview we will discuss what is not known concerning Raf-1 and related molecules, why the unknowns are important to our understanding of signal transduction, and how genetic studies in invertebrates may provide new meanings to investigate these issues.

Phase II and Pharmacodynamic Study of Autophagy Inhibition Using Hydroxychloroquine in Patients With Metastatic Pancreatic Adenocarcinoma

BACKGROUND Autophagy is a catabolic pathway that permits cells to recycle intracellular macromolecules, and its inhibition reduces pancreatic cancer growth in model systems. We evaluated hydoxychloroquine (HCQ), an inhibitor of autophagy, in patients with pancreatic cancer and analyzed pharmacodynamic markers in treated patients and mice. METHODS Patients with previously treated metastatic pancreatic cancer were administered HCQ at 400 mg (n = 10) or 600 mg (n = 10) twice daily. The primary endpoint was 2-month progression-free survival (PFS). We analyzed peripheral lymphocytes from treated mice to identify pharmacodynamic markers of autophagy inhibition that were then assessed in peripheral lymphocytes from patients. RESULTS Among 20 patients enrolled, 2 (10%) were without progressive disease at 2 months. Median PFS and overall survival were 46.5 and 69.0 days, respectively. Treatment-related grade 3/4 adverse events were lymphopenia (n = 1) and elevated alanine aminotransferase (n = 1). Tolerability and efficacy were similar at the two dose levels. Analysis of treated murine lymphocytes suggested that LC3-II expression by Western blot is a reliable marker for autophagy inhibition. Analysis of LC3-II in patient lymphocytes demonstrated inconsistent autophagy inhibition. CONCLUSION Mouse studies identified LC3-II levels in peripheral lymphocytes as a potential pharmacodynamic marker of autophagy inhibition. In patients with previously treated metastatic pancreatic cancer, HCQ monotherapy achieved inconsistent autophagy inhibition and demonstrated negligible therapeutic efficacy.

Multi-Institutional Phase II Study of High-Dose Hypofractionated Proton Beam Therapy in Patients With Localized, Unresectable Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma

PURPOSE To evaluate the efficacy and safety of high-dose, hypofractionated proton beam therapy for hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). MATERIALS AND METHODS In this single-arm, phase II, multi-institutional study, 92 patients with biopsy-confirmed HCC or ICC, determined to be unresectable by multidisciplinary review, with a Child-Turcotte-Pugh score (CTP) of A or B, ECOG performance status of 0 to 2, no extrahepatic disease, and no prior radiation received 15 fractions of proton therapy to a maximum total dose of 67.5 Gy equivalent. Sample size was calculated to demonstrate > 80% local control (LC) defined by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0 criteria at 2 years for HCC patients, with the parallel goal of obtaining acceptable precision for estimating outcomes for ICC. RESULTS Eighty-three patients were evaluable: 44 with HCC, 37 with ICC, and two with mixed HCC/ICC. The CTP score was A for 79.5% of patients and B for 15.7%; 4.8% of patients had no cirrhosis. Prior treatment had been given to 31.8% of HCC patients and 61.5% of ICC patients. The median maximum dimension was 5.0 cm (range, 1.9 to 12.0 cm) for HCC patients and 6.0 cm (range, 2.2 to 10.9 cm) for ICC patients. Multiple tumors were present in 27.3% of HCC patients and in 12.8% of ICC patients. Tumor vascular thrombosis was present in 29.5% of HCC patients and in 28.2% of ICC patients. The median dose delivered to both HCC and ICC patients was 58.0 Gy. With a median follow-up among survivors of 19.5 months, the LC rate at 2 years was 94.8% for HCC and 94.1% for ICC. The overall survival rate at 2 years was 63.2% for HCC and 46.5% ICC. CONCLUSION High-dose hypofractionated proton therapy demonstrated high LC rates for HCC and ICC safely, supporting ongoing phase III trials of radiation in HCC and ICC.

FDG-PET/CT tumor segmentation-derived indices of metabolic activity to assess response to neoadjuvant therapy and progression-free survival in esophageal cancer: correlation with histopathology results.

Purpose:To evaluate the diagnostic and prognostic abilities of PET tumor segmentation-derived indices of metabolic activity for the assessment of response to neoadjuvant chemoradiotherapy and progression-free survival in patients with esophageal cancer. Methods:Twenty-five patients with histologically confirmed esophageal cancer were retrospectively evaluated. The patients underwent PET-CT imaging before and after completion of neoadjuvant therapy. Images were evaluated visually and quantitatively with a three-dimensional threshold-based region-growing program, which calculates SUVm, SUVa of the entire tumor, metabolic tumor length (Lm) and volume (Vm) before and after therapy (SUVm1, SUVm2, SUVa1, SUVa2, Lm1, Lm2, Vm1, and Vm2, respectively). Percentage changes in these metabolic variables before and after therapy were also calculated (%SUVm, %SUVa, %Lm, %Vm, respectively). Results:SUVm1 (P = 0.018), SUVa1 (P = 0.019), Lm1 (P = 0.016), and Vm1 (P = 0.016) correlated with T-status. Advanced stage tumors (T3 + T4) had significantly higher glucose metabolism, metabolic length, and volume. Moreover, Lm1 >47.4 mm and Vm1 >29 cm3 were the best predictors of the level of tumor invasiveness. SUVm1 >12.7 and SUVa1 >5.9 could differentiate patients with positive lymph nodes from those without at presentation. %SUVa >32.3% and the SUVa1 >5.5 proved to be reliable predictors of pathologic response. SUVa2 >3.55 and SUVm2 >4.35 were the best predictors of disease progression during follow-up, with the latter having the best prognostic value. Conclusions:This study showed that FDG-PET tumor segmentation-derived indices of metabolic activity play a definite role in the evaluation of response to neoadjuvant chemoradiotherapy and progression-free survival in patients with esophageal cancer.

Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells.

To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication.

Allergic Skin Reactions to Anticonvulsant Medications in Patients Receiving Cranial Radiation Therapy

Summary: Purpose: Erythema multiforme and Stevens‐Johnson syndrome have been associated with anticonvulsant medications (AEDs) in patients with brain tumors receiving cranial irradiation. AEDs are also known to cause mild drug rashes. The incidence of these complications has not been well studied among patients with brain tumors. We reviewed the records of patients with brain tumors treated with cranial radiation and AEDs to assess the frequency of both severe and mild skin reactions.