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Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.

All patients with small intramural rectal cancers are at risk for lymph node metastasis

PURPOSE: Although local excision can be curative in patients with early-stage rectal cancer, approximately 20 percent of patients will develop local recurrence, many as a result of unrecognized and unresected regional lymph node metastases. Our objective was to determine if standard pathologic factors can predict lymph node metastases in small intramural rectal cancers and provide a basis for patient selection for nonradical surgery. METHODS: Between June 1986 and September 1996, 318 patients with T1 or T2 rectal cancers underwent radical resection at our institution. Of these, 159 patients (48 T1 and 111 T2) were potentially eligible for curative local excision (≤4 cm in size, ≤10 cm from the anal verge, no synchronous metastases), and the prevalence of lymph node metastases based on T stage and other pathologic factors was analyzed in this group. RESULTS: The overall frequency of lymph node metastasis was 15 percent (24/159 patients). T stage (T1, 10 percent; T2, 17 percent), differentiation (well-differentiated or moderately differentiated, 14 percent and poorly differentiated, 30 percent), and lymphatic vessel invasion (lymphatic vessel invasion-negative, 14 percent and lymphatic vessel invasion-positive, 33 percent) influenced the risk of lymph node metastasis. However, only blood vessel invasion (blood vessel invasion-negative, 13 percent and blood vessel invasion-positive, 33 percent) reached statistical significance as a single predictive factor (P=0.04). Tumors with no adverse pathologic features (low-risk group) had a lower overall frequency of lymph node metastasis (11 percent) compared with the remaining tumors (high-risk group, 31 percent;P=0.008). However, even in the most favorable group (T1 cancers with no adverse pathologic features) lymph node metastases were present in 7 percent of patients. CONCLUSION: In rectal cancer patients potentially eligible for local excision, the overall risk of undetected and untreated lymph node metastases is considerable (15 percent). The use of pathologic factors alone after local excision does not reliably assure the absence of lymph node metastases.

Stage I Rectal Cancer: Identification of High-Risk Patients

BACKGROUND Stage I rectal cancer (T1, T2 N0) is currently treated by surgical resection alone. Despite adequate surgical resection, approximately 10-15% of patients will develop recurrence. Identification of patients at high risk for recurrence could potentially lead to an improvement in outcome by selection of these patients for adjuvant therapy. METHODS Between June 1986 and September 1996, 211 patients with primary rectal cancer (stage I) were treated by radical surgical resection alone. The medical data of all patients were entered into a database and prospectively followed. The following 10 prognostic factors were correlated with recurrence and tumor-related mortality: patient factors: age, gender, and preoperative carcinoembryonic antigen level; tumor factors: location from the anal verge (< 6 cm vs. > or = 6 cm), T stage (T1 vs. T2), intratumoral blood vessel invasion (BVI), intratumoral lymphatic vessel invasion, presence of tumor ulceration, and histologic differentiation; and treatment-related factors: extent of surgical resection--abdominal perineal resection versus low anterior resection. Univariate analysis of the effect of the prognostic factors on recurrence and tumor-related mortality were performed by the method of Kaplan-Meier and log rank test. Independent prognostic factors were determined by a multivariate analysis performed using the Cox proportional hazards model. RESULTS The overall 5-year actuarial recurrence was 12% and tumor-related mortality was 10%. Independent predictors of recurrence were male gender and BVI. Independent predictors of tumor-related mortality were male gender, BVI, and poorly differentiated tumors. CONCLUSIONS Despite radical resection, patients with stage I rectal cancer with male gender, BVI, and poorly differentiated tumors should be considered high-risk patients.

Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells.

SummaryLectin binding specificities for carbohydrate allow phenotypic and functional characterization of membrane-associated glycoproteins expressed on cancer cells. This analysis examined wheatgerm agglutinin binding to pancreatic cancer cells in vitro and the resulting toxicity. Membrane preparations of nine human pancreatic carcinoma cell lines were studied for lectin binding using wheatgerm agglutinin (WGA), concanavalin A (ConA) and phytohaemagglutinin-L (PHA-L) in a lectin blot analysis. Cell proliferation in vitro was measured by thymidine incorporation in the absence or presence of lectins at various concentrations. Sialic acid binding lectins or succinyl-WGA (succWGA) served as controls. WGA toxicity was tested after swainsonine or neuraminidase pretreatment. Binding and uptake of fluorescein-labelled lectins was studied under fluorescence microscopy. All pancreatic cell lines displayed high WGA membrane binding, primarily to sialic acid residues. Other lectins were bound with weak to moderate intensity only. Lectin toxicity corresponded to membrane binding intensity, and was profound in case of WGA (ID50 at 2.5–5 μg ml–1). WGA exposure induced chromatin condensation, nuclear fragmentation and DNA release consistent with apoptosis. Important steps for WGA toxicity included binding to sialic acid on swainsonine-sensitive carbohydrate and lectin internalization. There was rapid cellular uptake and subsequent nuclear relocalization of WGA. In contradistinction to the other lectins studied, WGA proved highly toxic to human pancreatic carcinoma cells in vitro. WGA binding to sialic acid residues of N-linked carbohydrate, cellular uptake and subsequent affinity to N-acetyl glucosamine appear to be necessary steps. Further analysis of this mechanism of profound toxicity may provide insight relevant to the treatment of pancreatic cancer.

Sentinel Lymph Node Biopsy for Cutaneous Head and Neck Melanoma: Mapping the Parotid Gland

BackgroundSentinel lymph node biopsy (SLNB) for primary cutaneous head and neck melanoma (CHNM) has been shown to be successful and is the current standard of care for intermediate-thickness melanoma. We evaluated our experience with CHNM associated with SLNB mapping to the region of the parotid gland.MethodsRetrospective review of a prospectively collected melanoma database identified 1014 CHNMs. Two-hundred twenty-three patients underwent SLNB, and 72 (32%) had mapping in the region of the parotid gland between May 1995 and June 2003.ResultsThe mean number of SLNs per patient was 2.5. A sentinel lymph node (SLN) was successfully identified in 94% of patients, and in 12%, the SLN was positive for metastatic disease. Biopsy of intraparotid SLNs was performed in 51.4% and of periparotid SLNs in 26.4%, and a superficial parotidectomy was performed in 22.2%. Ten patients were found to have lymph nodes in the parotid region with metastatic disease (eight identified by SLNB), and two (20%) patients developed intraparotid lymph node recurrence in the setting of a negative SLNB. Same-basin recurrence in SLN-negative patients was 3.3% with a median follow-up of 26 months. Facial nerve dysfunction was identified in seven (10%) patients. Facial nerve function returned to preoperative status in all patients.ConclusionsSLNB for patients with primary CHNM mapping to the parotid gland can be performed with a high degree of accuracy and a low morbidity consisting of temporary facial nerve paresis.

Barriers to implementing a surgical beta blocker protocol: the Beth Israel cardiac risk reduction initiative

BACKGROUND Experience with a quality improvement (QI) program undertaken to increase the use of beta-adrenergic blockade in at-risk patients at both a major academic medical center and a community hospital suggests barriers to implementation. METHODS A retrospective and prospective cohort study was performed to establish the incidence and effectiveness of beta-blockade use pre- and postimplementation of a standardized screening tool and a major education program as part of a QI project. Data gathering involved a baseline phase pre-intervention; 6 weeks postintervention; and 3-6 months postintervention. RESULTS During phase I (baseline) 56% of eligible received beta-blockers, but targeted measures (a pre-induction heart rate < 70 or a systolic blood pressure [BP] < 110 mmHg) were achieved in only 11% of patients. Phase II saw a significant overall increase in beta-blocker administration (79%) and efficacy (50%). However, during phase III (3-6 months postimplementation), the rate of beta-blocker administration fell to 61% overall, while overall efficacy remained stable at 52%. Significant differences between the academic and community hospitals were observed throughout the study. CONCLUSION Implementation of a quality program for beta-blockade is significantly affected by the presence or absence of ongoing physician and staff education beyond the study period.