The role of frozen section analysis of margins during breast conservation surgery.

PURPOSE: The role of frozen section analysis during breast conservation surgery is undefined. Assessment of margins using permanent section evaluation is the standard method of ensuring complete tumor excision. If the margin is positive, however, surgical re-excision is necessary to reduce the likelihood of subsequent local recurrence. Therefore, biopsy of the surgical cavity with immediate pathological evaluation during lumpectomy was performed to evaluate the effect on local recurrence, the number of re-excisions, and cosmesis. PATIENTS AND METHODS: One hundred sixty patients underwent attempted lumpectomy with frozen section margin determination. One hundred forty patients were available for long-term follow-up (mean = 57 months, median = 46 months). All patients underwent attempted breast conservation surgery, which consisted of tumorectomy with excision of a greater than 1-cm rim of grossly normal tissue. Tumor margins were obtained by intraoperative biopsy with frozen section analysis of the lumpectomy cavity walls. RESULTS: In 21 patients (15%), frozen section analyses (FSA) revealed positive margins, resulting in immediate re-excision. In seven of these patients (5%), margins were persistently positive, and these patients therefore underwent mastectomy. Fourteen patients were successfully re-excised to a negative margin. The sensitivity and specificity of FSA were 91% and 100%, respectively. Five percent of patients definitively managed by lumpectomy with FSA of margins recurred locally. The mean cosmesis score after radiotherapy was 7.0 out of a possible 10, correlating with a good to excellent result. DISCUSSION: The accuracy of FSA, low recurrence rate, avoidance of reoperation, and good cosmesis indicate that intraoperative frozen section analysis should be adopted as a safe and effective method of margin analysis during breast conservation surgery.

Heat shock protein 27 stimulates recovery of RNA and protein synthesis following a heat shock.

Constitutive expression of human hsp27 resulted in a 100-fold increase in survival to a single lethal heat shock in CHO cells without effecting the development of thermotolerance. A possible mechanism for the thermoprotective function of hsp27 may be increased recovery of protein synthesis and RNA synthesis following a heat shock. A lethal heat shock (44 degrees C, 30 min) results in a 90% reduction in the rate of protein synthesis in non-tolerant cells. Control transfected cells recovered protein synthesis to a pre-heat shock rate 10 h after the heat shock; while cell lines that constitutively express human hsp27 recovered 6 h after the heat shock. Thermotolerant cells had a 50% reduction in protein synthesis, which recovered within 7 h following the heat shock. The same lethal heat shock (44 degrees C, 30 min) reduced RNA synthesis by 60% in the transfected cell lines, with the controls recovering in 7 h; while the hsp27 expressing cell lines recovered within 5 h. Thermotolerant cells had a 40% reduction in RNA synthesis and were able to recover within 4 h. The enhanced ability of hsp27 to facilitate recovery of protein synthesis and RNA synthesis following a heat shock may provide the cell with a survival advantage.

Heat shock protein 27 overexpression in breast cancer lymph node metastasis.

BACKGROUND: Heat shock protein 27 (hsp-27) is overexpressed in approximately 67% pure ductal carcinoma in situ (DCIS), in approximately 50% DCIS associated with invasive ductal carcinoma (IDC), and in approximately 25% IDC alone. If this decrease in hsp-27 expression has a role in the progression of malignancy in IDC, we postulate a further reduction in expression in nodal metastasis. METHODS: To test this hypothesis, we evaluated the distribution of hsp-27 in primary IDC and in synchronous regional lymph node metastasis within the same patient by immuno-histochemistry. RESULTS: Nine of 30 primary IDCs (30%) and 22 of 30 lymph node metastases (73%) overexpressed hsp-27. Contrary to our hypothesis, of 21 IDCs with no or low hsp-27 expression, 13 (62%) had overexpression of this protein within nodal metastasis. CONCLUSIONS: hsp-27 appears to confer cytoprotection for metastatic cells, which may help explain why hsp-27 overexpression is associated with reduced disease-free survival in breast carcinomas.

Lack of association between tumor necrosis and hsp-27 expression in primary breast cancer.

Overexpression of heat shock protein 27 (hsp-27) is associated with reduced disease-free survival in early stage breast cancer. Histopathologic evidence of confluent necrosis within primary infiltrating ductal carcinoma (IDC) is similarly an indication of poor prognosis. We postulated that IDC evidencing confluent tumor necrosis (TN) might overexpress this protein, which would help explain why hsp-27 is associated with higher-risk cancers. To test this hypothesis, presence of TN (as opposed to individual cell apoptosis) and of hsp-27 expression by immunohistochemistry were evaluated independently in 48 specimens of IDC. Nineteen (40%) overexpressed hsp-27 and 10 (21%) displayed necrosis. IDCs with areas of TN are less likely to overexpress hsp-27, suggesting a lack of association between these histoprognostic variables. This negative correlation, however, supports hsp-27 as an independent predictor of high-risk disease.

Interleukin-2 does not sequester activated lymphocytes into lung lymph of sheep.

PURPOSE OF STUDY: Interleukin-2 (IL-2) is a potent activator of lymphocytes, but its effectiveness as an anti-cancer agent is compromised by several adverse side effects including pulmonary edema. One explanation for the pulmonary toxicity of IL-2 is that activated lymphocytes directly induce the pulmonary vascular endothelium to become more leaky. METHODS: To test this hypothesis the number of total lymphocytes, gamma delta T cells, and CD2-positive cells (alpha beta T cells and natural killer cells) in peripheral blood and lung lymph of sheep were compared before and after IL-2 infusion. Hemodynamic and lymph dynamic changes were also evaluated. RESULTS: IL-2 decreased mean aortic pressure, increased cardiac output, lowered systemic vascular resistance, and doubled lung lymph flow (P < or = 0.05), but had no effect on plasma or lymph oncotic pressure. The lymph protein concentration and the lymph-to-plasma protein concentration ratio were not different after IL-2 infusion. IL-2 had no effect on the number of total lymphocytes, gamma delta T cells, or CD2-positive cells in the peripheral blood. In contrast, the number of total lymphocytes, gamma delta T cells, and CD2-positive cells in lung lymph decreased significantly (P < or = 0.05). CONCLUSIONS: The lymphocyte populations decreased more than could be explained by the increase in lymph flow, demonstrating that lung lymphocytes were not reduced simply by dilution. These results imply that the pulmonary edema associated with IL-2 is not caused by activated lymphocytes.

Retroperitoneal masses, adenopathy, and adrenal glands.

The diagnosis of primary retroperitoneal masses is approached best by CT scan-guided transretroperitoneal core biopsy or by open biopsy. Excisional or wedge biopsy is the preferred method for undiagnosed lymphadenopathy. Resection without biopsy is indicated for large or enlarging nonfunctional primary adrenal masses.

Distribution of Hsp-27 and HER-2/neu in in situ and invasive ductal breast carcinomas.

BACKGROUND: The overexpression of heat shock protein 27 (hsp-27) in early-stage breast cancer is associated with histopathologic features of poor prognosis and clinically with an increased probability of disease recurrence. Hsp-27 is overexpressed in 25% of invasive ductal carcinomas (IDC); however, its distribution in ductal carcinoma in situ (DCIS) and DCIS associated with IDC has not been investigated. We postulated that hsp-27 might be detected and variably expressed in DCIS and, like HER-2/neu oncoprotein expression, might be a tumor-specific marker worthy of future clinical investigation. METHODS: To test these hypotheses, the distribution of hsp-27 in noncomedo and comedo DCIS, and DCIS associated with IDC, was evaluated by immunohistochemistry and compared with HER-2/neu expression within the same cancers. RESULTS: Hsp-27 was overexpressed in 28 of 47 (approximately 60%) cases of DCIS; expression in pure DCIS was 16 of 24 (67%), and 12 of 23 (approximately 50%) in DCIS associated with IDC. Hsp-27 expression by in situ and invasive components of the same neoplasm were concordant in 22 of 23 (approximately 95%) cases tested. Comedo variants appeared to have somewhat higher hsp-27 expression than noncomedo DCIS, whether or not there was an associated IDC. These results are reminiscent of HER-2/neu oncoprotein expression in DCIS and DCIS associated with IDC observed by others. However, although 4 of 22 (18%) cancers containing DCIS + IDC expressed HER-2/neu, no relationship with hsp-27 expression in the same cancers was observed. CONCLUSIONS: We found a high incidence of hsp-27 overexpression in DCIS and in DCIS associated with IDC. This rate is twice that previously observed in IDC alone. Hsp-27 expression is independent of HER-2/neu expression.

Hsp-27 has no diagnostic or prognostic significance in prostate or bladder cancers.

Heat shock protein 27 (hsp-27) is a cytosol protein of unknown function that is concentrated in many estrogen-sensitive normal target organs and is expressed to a varying degree in many tumors, including ductal carcinoma of the breast, malignant fibrous histiocytoma (MFH) of the soft tissues, adenocarcinoma of the prostate, and transitional cell carcinoma (TCC) of the urinary bladder. Overexpression of hsp-27 has independent prognostic significance in patients with breast cancer and MFH, but its potential predictive value with prostate and bladder cancers has not been evaluated. Differential expression of hsp-27 may occur between invasive cancer and host tissue that could aid in diagnosis, and varying expression among invasive cancers may have potential prognostic significance that could influence the use of adjuvant therapy. To test these hypotheses, hsp-27 expression was evaluated by immunohistochemistry in archival formalin-fixed paraffin-embedded sections of primary prostate and bladder carcinomas where the outcome of the patient was known. In 36 prostate cancer specimens from patients who had undergone radical prostatectomy (Stages T1, T2; N0; M0), no normal glandular elements or invasive cancers expressed this protein. In 24 bladder cancer specimens from patients who had undergone radical cystectomy (Stages T2, T3A, T3B, T4A; N0, N1; M0), 12 (50%) cancers overexpressed this protein. Hsp-27 did not correlate with degree of histologic differentiation, T-stage, nodal status, local recurrence, metastases, or survival. From these observations, we conclude that hsp-27 expression has neither diagnostic nor prognostic significance and will not serve as a predictive biologic marker with these important genitourinary cancers.

Overexpression of 27-kDa heat-shock protein in MCF-7 breast cancer cells: effects on lymphocyte-mediated killing by natural killer and gamma delta T cells.

Overexpression of the heat-shock protein hsp27 protein in primary breast cancers has been associated with early relapse in women with breast cancer. This study was designed to determine the role of the hsp27 protein in lymphocyte recognition of estrogen-receptor(ER)-positive breast cancer cells and to assess the effect of hsp27 expression on lymphocyte-mediated lysis. The hsp27 cDNA was inserted into the pHbAPr-1-neo plasmid expression vector and driven by the constitutive actin promoter. The ER-positive MCF-7 human breast cancer cell line was then transfected with this vector and the resulting clonal cell lines were confirmed to overexpress hsp27. hsp27-transfected clonal cell lines stimulated the proliferation of fresh peripheral blood lymphocytes (PBL) significantly better than control cells transfected with the expression vector alone. When clonal gamma delta T cell lines were utilized as effectors, hsp27-transfected cell lines were significantly better targets for lysis than a control-transfected MCF-7 cell line. In contrast, hsp27-transfected cell lines had no increase in susceptibility to lymphokine-activated-killer- or natural-killer-mediated lysis. These results suggest that overexpression of the hsp27 protein in ER-positive MCF-7 cells stimulated the proliferation of fresh PBL and the lysis of MCF-7 cells by gamma delta T cell clones.

Technical details of intraoperative lymphatic mapping for early stage melanoma.

The initial route of metastases in most patients with melanoma is via the lymphatics to the regional nodes. However, routine lymphadenectomy for patients with clinical stage I melanoma remains controversial because most of these patients do not have nodal metastases, are unlikely to benefit from the operation, and may suffer troublesome postoperative edema of the limbs. A new procedure was developed using vital dyes that permits intraoperative identification of the sentinel lymph node, the lymph node nearest the site of the primary melanoma, on the direct drainage pathway. The most likely site of early metastases, the sentinel node can be removed for immediate intraoperative study to identify clinically occult melanoma cells. We successfully identified the sentinel node(s) in 194 of 237 lymphatic basins and detected metastases in 40 specimens (21%) on examination of routine hematoxylin-eosin-stained slides (12%) or exclusively in immunohistochemically stained preparations (9%). Metastases were present in 47 (18%) of 259 sentinel nodes, while nonsentinel nodes were the sole site of metastasis in only two of 3079 nodes from 194 lymphadenectomy specimens that had an identifiable sentinel node, a false-negative rate of less than 1%. Thus, this technique identifies, with a high degree of accuracy, patients with early stage melanoma who have nodal metastases and are likely to benefit from radical lymphadenectomy.

Cellular effects of piezoelectric versus electrohydraulic high energy shock waves.

High energy shock waves produced by a piezoelectric lithotripter, (EDAP LT.01) and an electrohydraulic lithotripter, (Dornier HM3) were examined for their effects on Chinese hamster ovary cells in suspension. The EDAP caused acute lactate dehydrogenase release, consistent with severe membrane disruption in a proportion of cells, with the remaining proportion of cells replicating normally as measured by clonogenic assay. Similarly, the Dornier also caused lactate dehydrogenase release. However, a significant proportion of cells which remained "viable" after Dornier treatment, (intact to lactate dehydrogenase), did not replicate by clonogenic assay. The Dornier HM3 lithotripter has been reported to produce free radicals in aqueous solution. In the current investigation, we could not detect significant free radical formation from the EDAP LT.01. Chinese hamster ovary cell killing by the Dornier HM3 was significantly augmented by radiosensitizers, 5-iodo-2-deoxyuridine or buthionine sulfoximine, while radioprotectors cysteamine and WR-1065 had no protective effect. EDAP cell killing was not influenced by either radioprotectors or radiosensitizers. The mechanism of in vitro cytotoxicity differs between piezoelectric and electrohydraulic high energy shock wave delivery.

Pulmonary and systemic fluid filtration after continuous versus bolus interleukin-2 infusion.

Interleukin-2 has been widely investigated as adjuvant therapy for advanced cancer and is administered by either bolus or continuous infusion. We compared the effects of bolus and continuous interleukin-2 infusion on pulmonary (QL) and systemic microvascular fluid filtration in 11 adult sheep prepared with chronic lung and soft-tissue lymph fistulas. Interleukin-2 was administered as a bolus infusion (100,000 units/kg) every 8 hours for 3 days or as a continuous infusion at the same dose for 3 days. No significant changes in pulmonary hydrostatic pressures or pulmonary vascular resistance were noted after either bolus or continuous interleukin-2 infusion. However, significantly decreased (p less than or equal to 0.05) systemic vascular resistances were observed in both groups. QL increased steadily throughout the infusion period in both groups, peaking at three times baseline on the third infusion day. The plasma/interstitial protein clearance (QL X lymph/plasma protein ratio) rose similarly in both groups, indicating increased barrier permeability. Increased lymphocyte clearance into lung lymph occurred by day 3 but was not associated with lymphocytic sequestration in the lung interstitium. We conclude that pulmonary and systemic microvascular fluid and protein flux exhibit similar changes after bolus or continuous interleukin-2 infusion. These changes are associated with increased clearance of lymphocytes into lung lymph that are not sequestered in the pulmonary interstitium after infusions of shorter duration.

Diagnosis and management of retroperitoneal soft-tissue sarcoma.

Retroperitoneal soft-tissue sarcomas are locally invasive tumors that remain occult for long periods and grow quite large due to the abdominal cavity's remarkable ability to accommodate these slowly expanding masses with a paucity of attendant symptoms. An open biopsy is required to establish diagnosis definitively. Despite improved imaging techniques and preoperative and intraoperative patient management, resectability has not changed significantly in the past 20 years. Even with an aggressive operative approach, only one half the tumors can be resected completely, and of those, more than 90% recur locally and result in the death of the patient. The addition of adjuvant radiotherapy or chemotherapy has not altered this pattern of local failure, in contrast to promising results with extremity soft-tissue sarcoma. Because of the rarity of these tumors, there is an urgent need to establish a national retroperitoneal sarcoma registry and to form cooperative intergroup studies to evaluate, treat, and apply innovative multimodality combination therapies to these otherwise lethal tumors.

Evaluation of hypoxia and acidosis on the cytotoxicity of the stereoisomers of tetraplatin at normal and hyperthermic temperatures.

Tetraplatin is a second-generation platinum analogue that will be introduced into clinical trials during 1990. We have evaluated the cytotoxicity of tetraplatin and its stereoisomers under a variety of conditions in a human solid tumour cell line (A549). The d-trans stereoisomer of tetraplatin is the most cyutotoxic form of the drug under all conditions examined. Simultaneous treatment with hyperthermia (43 degrees C) resulted in a greater than 1 log increase in cytotoxicity over tetraplatin (10 micrograms/ml) alone. Lowering the pH of the media from 7.4 to 6.4 did not significantly modify the cytotoxicity of tetraplatin at 37 degrees C or at 43 degrees C. Under acidic pH (6.4) hypoxia did not alter the cytotoxicity of tetraplatin at 37 degrees C or 43 degrees C. These results indicate a potential therapeutic advantage of combining tetraplatin chemotherapy with hyperthermia, which appears to be uneffected by acidic and/or hypoxic conditions.

An unusual presentation of metastatic squamous cell carcinoma of the vulva.

Recurrent squamous cell cancer of the vulva metastasized via regional lymphatics. Hematogenous spread is late and unusual. A patient with metastatic disease presented with soft tissue mass in her thigh musculature. We believe this to be the first reported case of noncontiguous skeletal metastasis for this tumor.

Induction of transient hyperglycaemia in cancer patients.

Serum glucose levels up to 700 mg/dl were achieved and maintained for 3 h in patients with advanced metastatic cancer by infusion of concentrated glucose solutions. Even with infusions of large amounts of glucose, the maximum sustainable level of serum glucose did not exceed 700 mg/dl. The apparent ceiling of serum glucose level appeared to be due to a large and rapid loss of glucose from the kidneys. With adequate fluid volume replacement there was no rebound hypoglycaemia and there was no significant changes in haematocrit or electrolytes.

In vitro and in vivo cytotoxicity of rhodamine 123 combined with hyperthermia.

Because both Rhodamine 123 (R123) and hyperthermia have been shown to be cytotoxic, we examined their effect, independently and in combination, on five different human malignant cell lines in vitro and on cultured melanoma cells grown intradermally in nude mice. The cell lines examined include two human melanomas, UCLA-SO-M14 and UCLA-SO-M21, the colon cancer cell line HT29, the human lung cancer cell line P3, and the human breast cancer cell line B231. R123 and hyperthermia, when used in combination, were found to be cytotoxic for these five different human malignant cell lines in vitro. The two agents together appear to enhance the cytotoxic effect of each alone, as documented by synergistic ratios ranging from 2.31 to 45 for the different cell lines. In the "nude" mouse model, animals were treated with a combination of R123 and hyperthermia (43 degrees C for 90 min). A statistically significant (P = 0.04) decrease in tumor growth rate was observed when compared with the rate of tumor growth in untreated animals. The results suggest a potential role for R123 in combination with hyperthermia in the treatment of malignant cells.