David C. Allison

Impact of axillary lymph node dissection on the therapy of breast cancer patients.

PURPOSE We studied a series of 283 breast cancer patients retrospectively to determine the actual benefits of axillary lymph node dissection (ALND) for these patients. PATIENTS AND METHODS The records of 283 women with invasive breast cancer treated between 1988 and 1990 were reviewed for histologic status of the axillary lymph nodes, tumor size, DNA content, hormone-receptor values, and actual adjuvant treatments received. RESULTS ALND was of possible therapeutic benefit for the 15% (43 of 283) of patients who had clinically positive nodes. Nodal metastases were found in 86% (37 of 43) of patients in this subgroup. ALND alone determined the indication for standard adjuvant therapy for a group of 31% (88 of 283) of patients who had favorable primary biopsy findings and clinically negative axillary nodes; ALND proved that 13% (11 of 88) of these latter patients had positive nodes. For 54% (152 of 283) of patients who had clinically negative nodes and unfavorable biopsies, ALND played no role in the decision as to whether standard adjuvant therapy was indicated. Only 5% (seven of 152) and 3% (four of 152) of these latter patients received radiation therapy and/or high-dose adjuvant chemotherapy, respectively, because of ALND. CONCLUSION The benefits of ALND vary greatly for different groups of breast cancer patients, and controlled studies may be needed to determine whether ALND is necessary for all breast cancer patients.

DNA content and other factors associated with ten‐year survival after resection of pancreatic carcinoma

Background and Objectives: The 5‐year survival rates after resection of pancreatic carcinoma have recently increased and are predicted by tumor size, DNA content, and lymph node metastases at the time of resection. However, whether the 10‐year survival rates have also increased and are similarly predicted by these factors is not known.

Pancreatic Cancer Cell Dna Content Correlates With Long-term Survival After Pancreatoduodenectomy

The DNA content of 47 adenocarcinomas arising in the head of the pancreas from patients who had undergone successful pancreatoduodenectomy was measured. The DNA measurements of each tumor were made without knowledge of the clinical course by absorption cytometry performed on Feulgen-stained nuclei that had been disaggregated from pancreatic cancer tissue blocks. Forty-seven evaluable DNA distributions were obtained from specimens taken between 1975 and 1988. Of the 47 tumors, 19 (40%) were diploid and 28 (60%) were aneuploid cancers. The 19 patients with diploid cancers had a median survival time of 25 months. Median survival of the 28 patients with aneuploid cancers was 10.5 months. This difference was statistically significant (p = 0.003). A multivariate life table regression analysis demonstrated that the ploidy and proliferative index as determined by absorption cytometry were independent prognostic factors, as strong as or stronger than the number of positive nodes and tumor size. Thus cellular DNA content appears to be one of the most important predictors of survival in patients with adenocarcinoma of the head of the pancreas who have successfully undergone a pancreaticoduodenectomy.

Use of a trypan blue assay to measure the deoxyribonucleic acid content and radioactive labeling of viable cells.

A simple method of determining cellular viability, DNA content, and isotopic labeling is described. Cells are sedimented onto slides and fixed in a mixture of trypan blue and paraformaldehyde which stains nonviable cells a dark blue color, whereas viable cells exclude the dye. The staining pattern is stable for at least 6 months and persists after the Feulgen reaction, allowing the selection of viable cells for cytophotometric DNA determinations. We ascertained cellular incorporation of a 3H-thymidine label by photomapping cells prior to cytophotometry and relocating these cells after preparation of autoradiographs. The method gives accurate quantitative DNA and labeling values of mouse thymocytes labeled in vitro and may be of value in studies of tumors containing a large proportion of necrotic cells.

A comparison of flow cytometric and absorption cytometric DNA values as prognostic indicators for pancreatic carcinoma.

Background. The DNA content of 30 adenocarcinomas of the head of the pancreas was measured by flow and absorption cytometric analysis.

Measurement of DNA content and of tritiated thymidine and bromodeoxyuridine incorporation by the same cells.

We tested a method of measuring DNA content (Feulgen) and tritiated thymidine ([3H]-T) and bromodeoxyuridine (BrdU) incorporation by the same cell. Initial experiments showed that Feulgen hydrolysis denatured the DNA of fixed cells sufficiently to allow detection of incorporated BrdU with monoclonal antibodies. MCa-11 cells were then double-labeled with [3H]-T and BrdU, placed on slides, and Feulgen stained. Next, absorption cytometry was performed to measure the DNA content of randomly selected cells. Feulgen staining and the development and removal of either the [3H]-T or the BrdU grains after DNA measurements did not interfere with subsequent detection of the grains from the other label, and BrdU and [3H]-T can be used reliably in combination for identification of S-phase cells. This method may eventually allow the use of microscope-based image analysis to selectively measure the DNA contents and the BrdU/[3H]-T labeling of non-transformed stromal and cancer cells in solid tumors, thereby providing new insights into the growth kinetics of these heterogeneous cell populations.

Neointimal Hyperplasia and Vasoreactivity Are Controlled by Genetic Elements on Rat Chromosome 3

Neointimal hyperplasia (NIH) can lead to restenosis after clinical vascular interventions. NIH results from complex and poorly understood interactions between signaling cascades in the extracellular matrix and the disrupted endothelium, which lead to vessel occlusion. Quantitative trait loci (QTLs) were reported previously on rat chromosomes 3 and 6 through linkage analysis of postinjury NIH in midiliac arterial sections. In the current study, substitution mapping validated the RNO3 NIH QTL but not the RNO6 NIH QTL. The SHR.BN3 congenic strain had a 3-fold increase in the percentage of NIH compared with the parental spontaneously hypertensive rat strain. A double congenic study of RNO3+RNO6 NIH QTL segments suggested less than additive effects of these 2 genomic regions. To test the hypothesis that changes in vessel dynamics account for the differences in NIH formation, we performed vascular reactivity studies in the Brown Norway (BN), spontaneously hypertensive rat (SHR), SHR.BN3, and SHR.BN6 strains. De-endothelialized left common carotid artery rings of the SHR.BN3 showed an increased vascular responsiveness when treated with serotonin or prostaglandin F2α, with significant differences in EC50 and maximum effect (P<0.01) values compared with the spontaneously hypertensive rat parental strain. Because both vascular reactivity and percentage of NIH formation in the SHR.BN3 strain are significantly higher than the SHR strain, we postulate that these traits may be associated and are controlled by genetic elements on RNO3. In summary, these results confirm that the RNO3 NIH QTL carries the gene(s) contributing to postinjury NIH formation.

Acid giemsa technique for rapid identification of mitotic cells

We developed a rapid technique for differential staining of compacted chromatin as a tool for screening of large tissue culture cell populations for mitotic cells. With a combination of acid Giemsa staining and counterstaining, differential staining of mitotic cells and classification according to stage of mitosis can be accomplished at magnifications as low as x 50-100 (objectives of x 5-10). The mapped and classified cells can then be de-stained and re-studied for DNA content by Feulgen staining and/or for uptake of radioactive DNA precursors by autoradiography. The staining and de-staining procedures outlined do not affect the reproducibility and accuracy of DNA content measurements or measurements of radioactive uptake. Therefore, this technique can be used for cell kinetic analysis by the percentage labeled mitoses method and for cytophotometric studies of mitotic segregation.

An improved method of preparing nuclei for absorption cytophotometry.

An improved method of isolating nuclei from tissue for Feulgen-DNA measurements has been developed. The optimal nuclear isolation medium (NIM) was found to be a solution of 2% polyethylene glycol (PEG) and 0.6% NP40 in phosphate-buffered saline. The disaggregation procedure consists of gentle mechanical disruption of the tissues, followed by a 10 min incubation in the NIM at room temperature. The mixture is then syringed four times through a 27-gauge needle, and the nuclei are placed onto slides with a cytocentrifuge. Nuclei prepared in NIM without PEG had obvious DNA leakage and tended to form clumps. Addition of PEG to the NIM led to separation of nuclei without any DNA leakage, thus greatly increasing the accuracy of the DNA cytophotometry results. G0/G1 nuclei at the appropriate ploidy levels were found for non-transformed and transformed tissues prepared with this technique. In addition, S-phase liver nuclei prepared in this manner showed the expected incorporation of (3H) thymidine after a 1/2 hr pulse in vivo.

Ligation of superior mesenteric vein and portal to splenic vein anastomosis after superior mesenteric-portal vein confluence resection during pancreaticoduodenectomy - Case report

62 year old Caucasian female with pancreatic head mass abutting the superior mesenteric vein (SMV) presented with fine needle aspiration biopsy confirmed diagnosis of ductal adenocarcinoma. CT scan showed near complete obstruction of portal vein and large SMV collateral development. After 3 months of neoadjuvant therapy, her portal vein flow improved significantly, SMV collateral circulation was diminished. Pancreaticoduodenectomy (PD) and superior mesenteric portal vein (SMPV) confluence resection were performed; A saphenous vein interposition graft thrombosed immediately. The splenic vein remnant was distended and adjacent to the stump of the portal vein. Harvesting an internal jugular vein graft required extra time and using a synthetic graft posed a risk of graft thrombosis or infection. As a result, we chose to perform a direct anastomosis of the portal and splenic vein in a desperate situation. The anastomosis decompressed the mesenteric venous system, so we then ligated the SMV. The patient had an uneventful postoperative course, except transient ascites. She redeveloped ascites more than one year later. At that time a PET scan showed bilateral lung and right femur metastatic disease. She expired 15 months after PD. Conclusion The lessons we learned are (1) Before SMPV confluence resection, internal jugular vein graft should be ready for reconstruction. (2) Synthetic graft is an alternative for internal jugular vein graft. (3) Direct portal vein to SMV anastomosis can be achieved by mobilizing liver. (4) It is possible that venous collaterals secondary to SMV tumor obstruction may have allowed this patients post-operative survival.