DSS1 is required for the stability of BRCA2.

DSS1 is an evolutionarily conserved acidic protein that binds to BRCA2. However, study of the function of DSS1 in mammalian cells has been hampered because endogenous DSS1 has not been detectable by Western blotting. Here, we developed a modified Western blotting protocol that detects endogenous DSS1 protein, and used it to study the function of DSS1 and its interaction with BRCA2 in mammalian cells. We found that essentially all BRCA2 in human cell lines is associated with DSS1. Importantly, we found that RNAi knockdown of DSS1 in human cell lines led to dramatic loss of BRCA2 protein, mainly due to its increased degradation. Furthermore, the stability of BRCA2 mutant devoid of the DSS1-binding domain is unaffected by the depletion of DSS1. Most notably, like BRCA2 depletion, DSS1 depletion also led to hypersensitivity to DNA damage. These results demonstrated that the stability of BRCA2 protein in mammalian cells depends on the presence of DSS1. Deletion or mutation of DSS1 or suppression of its expression by other mechanisms are therefore potential causative mechanisms for human breast and ovarian cancer. Such mechanisms may be relevant to sporadic as well as familiar breast cancer where BRCA1 and BRCA2 mutations are not present.

The E6 oncoproteins of high-risk papillomaviruses bind to a novel putative GAP protein, E6TP1, and target it for degradation.

The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.

Abrogation of wild-type p53-mediated transactivation is insufficient for mutant p53-induced immortalization of normal human mammary epithelial cells.

The p53 protein has become a subject of intense interest since the discovery that about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancer, suggesting a critical role for p53 protein in normal mammary epithelial cell (MEC) growth control. We previously demonstrated that abrogation of the p53 function by a cancer-derived p53 mutant, del239, was sufficient to induce immortalization of normal MECs. To further extend these findings and to examine the mechanism of mutant p53-induced immortalization of MECs, we tested the immortalizing ability of four selected p53 mutants (R175H, R248W, R249S, and R273H), which involve residues that cluster close to N239 in the three-dimensional structure and which are critical for the DNA-binding function of p53. Interestingly, two of these mutants (R175H and R249S) reproducibly immortalized 76N normal MECs, whereas the other two mutants (R248W and R273H) induced an extension of life span but not immortalization. These results further substantiate that selective ablation of p53 function with dominant-negative mutants is sufficient for immortalization of MECs. To determine whether abrogation of the transactivation function of endogenous p53 was important for the differential immortalizing ability of p53 mutants, we measured the effects of mutant p53 on the endogenous wild-type p53-mediated transactivation of a chloramphenicol acetyltransferase reporter linked to a consensus p53 binding DNA sequence in transiently transfected 76N MECs. All of the mutants, regardless of their immortalizing phenotype, abrogated the endogenous wild-type p53-mediated transactivation to a similar extent. Thus, abrogation of transactivation function is not sufficient for mutant p53-induced immortalization of normal MECs. The p53-immortalized MECs showed substantial telomerase activity; however, induction of telomerase activity occurred at late passages and was undetectable in mutant p53-expressing cells prior to immortalization. We suggest that mechanisms other than abrogation of transactivation and induction of telomerase activity determine the differential MEC-immortalizing behavior of various p53 mutants.

E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway.

Rb protein is a critical regulator of entry into the cell cycle, and loss of Rb function by deletions, mutations, or interaction with DNA viral oncoproteins leads to oncogenic transformation. We have shown that the human papilloma virus (HPV)-16 E7 gene is sufficient to induce the immortalization of mammary epithelial cells (MECs). Surprisingly, the steady-state level of Rb protein in these immortal cells was drastically decreased. Here, we used pulse-chase analysis to show that the in vivo loss of Rb protein in E7-immortalized MECs is a consequence of enhanced degradation. Expression of HPV16 E7 in a cell line with a temperature-sensitive mutation in the E1 enzyme of the ubiquitin pathway demonstrated that degradation of Rb was ubiquitin dependent. Treatment of E7-immortalized MECs with aldehyde inhibitors of proteasome-associated proteases led to a marked stabilization of Rb protein, particularly the hypophosphorylated form. Taken together, our results provide evidence for HPV-16 E7-induced enhanced degradation of Rb protein via a ubiquitin-proteasome pathway and suggest a second mechanism of oncogenic transformation by E7, in addition to its previously identified ability to sequester Rb from E2F. Our analyses also show that normal Rb levels are regulated by the ubiquitin-proteasome degradation pathway.

Identification of a novel serine protease-like gene, the expression of which is down-regulated during breast cancer progression.

In an effort to isolate genes with down-regulated expression at the mRNA level during oncogenic transformation of human mammary epithelial cells (MECs), we performed subtractive hybridization between normal MEC strain 76N and its radiation-transformed tumorigenic derivative 76R-30. Here, we report the isolation of cDNA clones corresponding to a 1.4-kb mRNA species that is abundantly expressed in 76N cells but is drastically reduced in 76R-30 cells. Based on its selective expression in MECs compared with fibroblasts, the corresponding gene is designated NES1 (normal epithelial cell-specific 1). Sequence analysis of the full-length NES1 cDNA clones revealed it to be a novel gene with a predicted polypeptide of 30.14 kilodaltons; in vitro transcription and translation confirmed this prediction. Database searches revealed a 50-63% similarity and 34-42% identity with several families of serine proteases, in particular the trypsin-like proteases, members of the glandular kallikrein family (including prostate-specific antigen, nerve growth factor gamma, and epidermal growth factor-binding protein) and the activators for the kringle family proteins (including the human tissue plasminogen activator and human hepatocyte growth factor activator). Importantly, all of the residues known to be crucial for substrate binding, specificity, and catalysis by the serine proteases are conserved in the predicted NES1 protein, suggesting that it may be a protease. An antipeptide antibody directed against a unique region of the NES1 protein (amino acids 120-137) detected a specific 30-kilodalton polypeptide almost exclusively in the supernatant of the mRNA-positive MECs, suggesting that NES1 is a secreted protein. The 1.4-kb NES1 mRNA was expressed in several organs (thymus, prostate, testis, ovary, small intestine, colon, heart, lung, and pancreas) with highest levels in the ovary; a 1.1-kb transcript was found in the pancreas. Although expression of the NES1 mRNA was observed in all normal and immortalized nontumorigenic MECs, the majority of human breast cancer cell lines showed a drastic reduction or a complete lack of its expression. The structural similarity of NES1 to polypeptides known to regulate growth factor activity and a negative correlation of NES1 expression with breast oncogenesis suggest a direct or indirect role for this novel protease-like gene product in the suppression of tumorigenesis.

Radiation-enhanced expression of major histocompatibility complex class I antigen H-2Db in B16 melanoma cells.

Exposure of eukaryotic cells to ionizing radiation induces several cellular responses including DNA repair, arrest of DNA synthesis, and increased synthesis of specific cellular proteins. We derived from the murine melanoma cell line B16-F10 a clonal isolate (M1) that was exposed to a total dose of 5000 cGy in 25 fractions, according to a protocol that reflects the standard for current radiotherapeutic regimens. We measured, by flow cytometry of fluorescence-stained cells, the surface expression of the two major histocompatibility complex class I antigens H-2Db and H-2Kb in irradiated M1 cells and untreated M1 controls. We found that after 2000 cGy, expression of H-2Db antigen was enhanced in irradiated cells versus controls. Radiation-induced expression of H-2Db antigen appeared to be selective, since no up-regulation of the H-2Kb antigen was detectable, and persisted for at least 5 weeks following the last irradiation. Enhanced H-2Db antigen expression correlated with increased steady-state levels of H-2Db mRNA in irradiated cells. These results are consistent with the notion that enhanced expression of major histocompatibility complex class I antigens is part of a long-lasting stress response elicited in cells by radiation.

CpG methylation as a basis for breast tumor-specific loss of NES1/kallikrein 10 expression.

The normal epithelial cell-specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary epithelial cells, but its expression is dramatically decreased in breast cancer cell lines. Now, we have cloned and characterized the active promoter region of NES1. Using a luciferase reporter system, we demonstrate that most tumor cell lines are able to support full or partial transcription from the NES1 promoter, suggesting a role for promoter-independent cis-acting mechanisms of loss of NES1 expression. We show that hypermethylation of the NES1 gene represents one such mechanism. Using methylation-specific PCR and sequence analysis of sodium bisulfite-treated genomic DNA, we demonstrate a strong correlation between exon 3 hypermethylation and loss of NES1 mRNA expression in a panel of breast cancer cell lines and in primary tumors. Treatment of NES1-nonexpressing cells with a demethylating agent led to reexpression of NES1, suggesting an important role of hypermethylation in the loss of NES1 expression. We suggest that hypermethylation is responsible for tumor-specific loss of NES1 gene expression. Our results also suggest that hypermethylation of the NES1 gene may serve as a potential marker for breast cancer.

The role for NES1 serine protease as a novel tumor suppressor.

Previously (Liu et al, Cancer Res., 56: 3371-3379, 1996), we isolated a novel serine protease-like gene--Normal Epithelial Cell Specific-1 (NES1)--that is expressed in normal mammary epithelial cells but is down-regulated in most breast cancer cell lines. Here, we demonstrate that stable expression of NES1 in the NES1-negative MDA-MB-231 breast cancer cell line suppressed the oncogenicity as revealed by inhibition of the anchorage-independent growth and tumor formation in nude mice. Fluorescence in situ hybridization localized the NES1 gene to chromosome 19q13.3, a region that contains genes for related proteases (including the prostate-specific antigen) and is rearranged in human cancers. Similar to breast cancer cell lines, prostate cancer cell lines also lacked NES1 mRNA and protein expression. Together, these results strongly suggest a tumor-suppressor role for NES1 in breast and prostate cancer.

Mutant p53-induced immortalization of primary human mammary epithelial cells.

Mutations of the p53 gene are the most frequent genetic lesions in breast cancer, suggesting a critical role for p53 protein in normal mammary cell growth control. Indeed, the p53-targeting human papillomavirus oncogene E6 induces efficient immortalization of normal human mammary epithelial cells (MECs). To assess whether selective loss of p53 is sufficient for MEC immortalization, we introduced seven missense mutants and one single-amino acid deletion mutant (del239) of p53 into the 76N normal MEC strain. Although the missense mutants failed to immortalize MECs, the del239 mutant reproducibly immortalized these cells. The immortal cells were anchorage dependent and nontumorigenic, indicating a preneoplastic transformation. Gamma-irradiation of these cells failed to induce G1 cell cycle arrest and did not lead to an increase in WAF1 and mdm-2 mRNA levels, demonstrating a loss of the endogenous p53 function. These results demonstrate that selective ablation of p53 function by a dominant-negative mutant is sufficient for immortalization of MECs. Availability of an immortalizing as well as several nonimmortalizing p53 mutants should help identify functions critical for cell growth control by p53 in mammary epithelial cells.

Boron neutron capture therapy for murine malignant gliomas.

Boron neutron capture therapy (BNCT) involves administration of a boron compound followed by neutron irradiation of the target organ. The boron atom captures a neutron, which results in the release of densely ionizing helium and lithium ions that are highly damaging and usually lethal to cells within their combined track length of approximately 12 microns. Prior to Phase I clinical trials for patients with malignant gliomas, mice with glioma 261 intracerebral tumors were fed D,L-3-(p-boronophenyl)alanine and irradiated with total tumor doses of 1000-5000 RBE-cGy of single fraction thermal neutrons to determine the maximum tolerated dose and effect on survival. These mice were compared to mice that received D,L-3-(p-boronophenyl)alanine alone, neutron irradiation alone, photon irradiation alone, or no treatment. Additional normal mice received escalating doses of neutron irradiation to determine its toxicity to normal brain. BNCT caused a dose-dependent, statistically significant prolongation in survival at 1000-5000 RBE-cGy. At 3000 RBE-cGy, median survival rates of the BNCT and untreated control groups were 68 and 22 days, respectively, with a long-term survival rate of 33%. At 4000 RBE-cGy, median survival was 72 and 21 days, respectively, with a long-term survival rate of 43%. At lower radiation doses, the extended survival was comparable between the BNCT and photon-irradiated mice; however, at 3000 and 4000 RBE-cGy the median survival of BNCT-treated mice was significantly greater than photon-irradiated mice. The maximum tolerated single fraction dose to normal brain was approximately 2000 RBE-cGy.

Factors influencing cosmetic outcome and complication risk after conservative surgery and radiotherapy for early-stage breast carcinoma.

PURPOSE: The study was undertaken to assess the relationship among cosmesis and complications to factors related to disease presentation, surgical and radiotherapeutic technique, and adjuvant systemic therapy in conservative treatment for early-stage breast carcinoma. PATIENTS AND METHODS: Between 1982 and 1988, 234 women with stage I/II breast carcinoma were treated with conservation therapy by a highly standardized protocol of limited excision and radiotherapy. Radiation boost and/or reexcision were determined by careful quantitation of the normal tissue margin around the primary tumor. Boosts to 20 Gy were preferentially performed with interstitial iridium-192 (192Ir) implants. Axillary node dissections were performed in all patients aged less than 70 years. Adjuvant therapy consisted of cyclophosphamide, methotrexate, (doxorubicin), and fluorouracil (CM[A]F) six to eight times for node-positive premenopausal women and tamoxifen for node-positive or -negative postmenopausal women. Median follow-up was 50 months (range, 20 to 80 months). Cosmesis was graded by defined criteria, and complications were individually scored. RESULTS: Factors found to impact cosmesis adversely were palpable tumors (P = .046), volume of breast tissue resected (P = .027), reexcision of the tumor bed (P = .01), number of radiation fields (P = .03), radiation boost (P = .01), and chest wall separation (P = .01). There was a trend toward worse cosmesis (P = .062) in patients receiving tamoxifen. Cosmesis was not adversely affected by interstitial implant in spite of a higher prescribed dose. Factors influencing complication risk were axillary node dissection (P = .02), number of lymph nodes harvested (P = .05), and chemotherapy (P = .03). CONCLUSIONS: Optimal cosmesis and minimal complication risk require careful attention to the technical details of surgery and radiotherapy. The impact of systemic therapies needs to be more thoroughly examined.

Analysis of normal epithelial cell specific-1 (NES1)/kallikrein 10 mRNA expression by in situ hybridization, a novel marker for breast cancer.

PURPOSE: Normal epithelial cell specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary and prostate epithelial cells, but the expression of NES1 mRNA and protein is markedly reduced in established breast and prostate cancer cell lines although the NES1 gene is intact. Here, we wished to assess whether NES1 expression is down-regulated in primary breast cancers. EXPERIMENTAL DESIGN: We developed and used an in situ hybridization technique with an antisense NES1 probe to detect NES1 mRNA in sections of normal breast specimens, typical and atypical ductal hyperplasia, ductal carcinoma in situ, and infiltrating ductal carcinoma. RESULTS: All of the 30 normal breast specimens showed high NES1 expression. Notably, 18 (75%) of 24 typical and atypical breast hyperplasia specimens showed high NES1 expression, with weak-to-moderate expression in 6 (25%). Significantly, 13 (46%) of 28 ductal carcinoma in situ specimens lacked NES1 expression, and the remaining 15 (54%) showed weak-to-moderate expression. Finally, 29 of 30 (97%) infiltrating ductal carcinoma grades I-III samples lacked NES1 mRNA, with weak expression in the remaining one sample. CONCLUSIONS: Our results demonstrate that NES1 mRNA is expressed in normal breast tissue and benign lesions, with loss of NES1 expression during tumor progression. We suggest that NES1 expression may serve as a molecular tool in the study of breast cancer progression. Studies with larger series of specimens should help assess whether NES1 expression can be a diagnostic and/or prognostic marker in breast and other cancers.

Optimal radiotherapy for prostate cancer: predictions for conventional external beam, IMRT, and brachytherapy from radiobiologic models.

PURPOSE: To determine, on the basis of radiobiological models, optimal modalities of radiotherapy for localized prostate cancer, and to provide a rational basis for therapeutic decisions. METHODS AND MATERIALS: An algorithm based on extensions to the linear-quadratic (LQ) cell survival model is constructed for fractionated and protracted irradiation. These radiobiological models include prostate tumor cell line-derived LQ parameters, clonogen repopulation, repair of sublethal damage, hypoxia, and radioisotope decay. In addition, dose inhomogeneities for both IMRT and brachytherapy (125I and 103Pd) from patient-derived Dose Volume Histograms (DVH), as well as dose escalation, are incorporated. Three risk groups are defined in terms of sets of biologic parameters tailored to correspond to clinical risk groups as follows: Favorable-iPSA <10 and bGS < or =6 and stage T2; Intermediate-one parameter increased; and Unfavorable-two or more parameters increased. Tumor control probabilities (TCP) are predicted for conventional external beam radiotherapy (EBRT, including 3D-CRT), intensity modulated radiotherapy (IMRT), and permanent brachytherapy. RESULTS: Brachytherapy is less susceptible to variations in alpha/beta than EBRT and more susceptible to variations in clonogen potential doubling time (Tp). Our models predict TCP consistent with the bNED results from recent dose escalation trials and long-term outcomes from brachytherapy. TCP from IMRT are systematically superior to those from conventional fractionated RT, and suggests its possible use in dose escalation without additional dose to surrounding normal tissues. For potentially rapidly dividing tumors (Tp < 30 days) 103Pd yields superior cell kill compared with 125I, but for very slowly proliferating tumors the converse is suggested. Brachytherapy predicts equivalent or superior TCP to dose escalated EBRT. For unfavorable risk tumors, combined 45 Gy EBRT+brachytherapy boost predicts superior TCP than with either modality alone. CONCLUSIONS: The radiobiological models presented suggest a rational basis for choosing among several radiotherapeutic modalities based on biologic risk factors. In addition, they suggest that IMRT may potentially be superior to 3D-CRT in allowing dose escalation without increased morbidity, and that brachytherapy, as monotherapy or as boost, may achieve superior tumor control compared with dose escalation 3D-CRT. The latter conclusion is supported by clinical data.

Alterations in growth phenotype and radiosensitivity after fractionated irradiation of breast carcinoma cells from a single patient.

PURPOSE: To investigate growth regulation and radiosensitivity in surviving clonogens after fractionated irradiation. METHODS AND MATERIALS: Four breast carcinoma cell lines isolated from the primary tumor (21NT, 21PT) and metastases (21MT-1, 21MT-2) of a single patient were exposed to cumulative radiation doses of 30 Gy yielding cell lines designated-IR with respect to their parent. The irradiated lines were then compared to their parent for serum-and growth factor-requirements under defined media conditions, ability to proliferate in soft agar, concentration of TGF-alpha in conditioned medium, and radiosensitivity. RESULTS: The irradiated lines showed no change in proliferative doubling times under serum- and growth factor-supplemented media conditions. A single line, 21MT-1-IR, acquired a limited ability to proliferate in serum-and growth factor-deplete medium with a day 2-4 doubling time of 44.5 hr. Three lines, 21MT-1-IR, 21MT-2-IR, and 21NT-IR, formed colonies in soft agar in contrast to none of the unirradiated parent lines. There were significant 6-8 fold increases in conditioned media TGF-alpha concentrations for 21MT-2-IR and 21NT-IR cells. The 21MT-1-IR and 21NT-IR cells were significantly less radiosensitive than their respective parent lines. This decrease in radiosensitivity appeared to be at least partially mediated by a released factor as the radiosensitivity of 21MT-1 cells was significantly decreased by pre-incubation with conditioned medium from 21MT-1-IR cells. CONCLUSION: Radiation-induced changes in growth phenotype vary with respect to clonal origin of the cell line and may influence the radiosensitivity of surviving clonogens after fractionated treatment.

The influence of quantitative tumor volume measurements on local control in advanced head and neck cancer using concomitant boost accelerated superfractionated irradiation.

PURPOSE: Current methods to clinically define head and neck tumor bulk are qualitative and imprecise. Although the American Joint Committee on Cancer (AJCC) staging system is important for this purpose, limitations exist. This study will investigate the prognostic value of computed tomography (CT) derived tumor volume measurements in comparison to AJCC stage and other significant variables. MATERIALS AND METHODS: Seventy-six patients with advanced head and neck squamous cell carcinoma were treated with concomitant boost accelerated superfractionated irradiation. Doses ranged from 68.4-73.8 Gy (median 70.2 Gy). Good quality pretherapy CT scans were available in 51 patients. Total tumor volume (TTV) estimates were derived from these scans using digital integration of primary tumor and metastatic lymphadenopathy. Actuarial and multivariate statistical techniques were applied to analyze local control. RESULTS: Thirty-six-month local control was 63%. TTV ranged from 5-196 cm3 (median 35 cm3) for all cases, 5-142 cm3 (median 17 cm3) for those controlled, and 16-196 cm3 (median 47 cm3) for local failures. There was a significant increase in failures above 35 cm3. Univariate analysis found that TTV, T-stage, N-stage, and primary site were each significant prognostic variables. Local control for TTV < or = 35 cm3 was 92% at 36 months vs. 34% for TTV > 35 cm3 (p = 0.0001). Multivariate analysis, however, found that TTV, primary site, and sex were important as independent variables; T and N stage were not independently significant unless TTV was removed from the model. CONCLUSIONS: This study demonstrates the prognostic significance of TTV in advanced carcinoma of the head and neck. This variable appears to be a more predictive than AJCC clinical stage. Quantitative tumor volume measurements may prove to be a useful parameter in future analyses of head and neck cancer.

Factors determining outcome in patients treated with interstitial implantation as a radiation boost for breast conservation therapy.

PURPOSE: To evaluate the relative utility of interstitial implant as a technique for tumor bed dose escalation and assess technical factors related to outcome. METHODS AND MATERIALS: From 1982-1994, a prospectively applied institutional policy of margin-directed boost dose escalation to the tumor bed was followed whereby interstitial implantation was commonly employed for a final margin status (FMS) < or = 2 mm. There were 509 treated breasts, of which 127 received an implant boost. For purposes of comparison, cases were broadly classed as "implant" (all FMS < or = 2 mm) and "nonimplant" (FMS < or = 2 mm or FMS > 2 mm). The implant target volume was determined at completion of whole breast irradiation by clinical assessment. All implants were constructed in accordance with a preplanning algorithm designed to maximize dose homogeneity within a prescription isodose goal of 0.50 Gy/h for 40 h. Local control and cosmetic outcome were evaluated with respect to extent of tumor, histopathology, FMS, extent of surgery, and systemic adjuvant therapy. Implant quality was assessed using four calculated parameters: strand separation quotient (SSQ), planar separation quotient (PSQ), global separation quotient (GSQ), and dose homogeneity index (DHI). The mean implant volume was 48.3 +/- 20 cc, the mean prescribed dose rate was 0.46 +/- 0.08 Gy/h, and the mean total implant dose was 19.94 +/- 1.52 Gy. RESULTS: Cosmetic outcome was good/excellent in 90% of implant and 83% of all nonimplant cases, which was not statistically different. Cosmesis was significantly superior with implant when compared to nonimplant cases receiving an external boost of 20 Gy. Logistic regression analyses of implant cases revealed that reexcision volume and decreased DHI were associated with adverse cosmesis. There were 10 local failures in the implanted patients (4 within the prescribed isodose volume, 5 at the periphery, and 1 elsewhere in the breast). The local failure rate at 5 and 7 years in the implanted group was 3.9 and 9.0%, respectively, compared to nonimplant cases with a margin < or = 2 mm of 3.2 and 3.2%, respectively. These differences were not significant. The crude local failure rate in patients with an associated DCIS component was 12% a compared to 3% in patients with pure invasive histology (p = 0.06). A proportional hazards survival model revealed a significant association of local failure with the performance of a reexcision and young age. CONCLUSION: We conclude that interstitial implant boost for breast conserving irradiation is associated with cosmesis that is superior than the same nominal dose of external beam boost, although this is highly dependent upon the technical quality of the source position and the relative uniformity of dose deposition. Breast implantation results in a rate of local control no better than dose-matched external beam boost in patients with a final margin < or = 2 mm. Local control with implantation might be further enhanced by increasing implant volume and/or improved target localization.

Comparison of radiobiologic modeling for one- and two-isocenter dose distributions applied to ellipsoidal radiosurgery targets.

PURPOSE: RTOG protocol 90-05 determined the maximum acutely tolerated dose of single-fraction radiosurgery in patients receiving prior fractionated large volume cranial irradiation. Data from 90-05 have suggested that patients treated with a gamma unit, compared to linac-based therapy, have a tumor control advantage and lower rates of severe complications. This study was performed to investigate the radiobiologic effect of using one vs. two isocenters in single-fraction radiosurgery of ellipsoidal targets. METHODS AND MATERIALS: For a series of ellipsoidal targets that varied by volume and radiosensitivity, single and two-isocenter treatment plans were generated to approximate those typically employed for gamma unit and linac radiosurgery. Tumor control probabilities (TCP) and normal tissue complication probabilities (NTCP) were generated automatically by the treatment planning system based on established parameter values. RESULTS: The modeling data showed that multiple-isocenter plans resulted in improved TCP with equivalent or lesser NTCP, particularly for larger, radioresistant targets. Multiple-isocenter plans reduce the amount of normal tissue that receives high dose. Also, areas within the tumor receive significantly higher doses than the prescription dose, which contributes to increased tumor cell inactivation. CONCLUSION: For ellipsoidal targets, radiobiologic modeling data are consistent with the clinical findings of the RTOG 90-05 trial, as they predict improved outcome with a multiple-isocenter plan relative to a single-isocenter plan. The benefit is most apparent with increasing target volume and decreasing tumor radiosensitivity.

Clinically evident fat necrosis in women treated with high-dose-rate brachytherapy alone for early-stage breast cancer.

PURPOSE: To investigate the incidence of and variables associated with clinically evident fat necrosis in women treated on a protocol of high-dose-rate (HDR) brachytherapy alone without external-beam whole-breast irradiation for early-stage breast carcinoma. METHODS AND MATERIALS: From 6/1997 until 8/1999, 30 women diagnosed with Stage I or II breast carcinoma underwent surgical excision and postoperative irradiation via HDR brachytherapy implant as part of a multi-institutional clinical Phase I/II protocol. Patients eligible included those with T1, T2, N0, N1 (< or = 3 nodes positive), M0 tumors of nonlobular histology with negative surgical margins, no extracapsular lymph-node extension, and a negative postexcision mammogram. Brachytherapy catheters were placed at the initial excision, re-excision, or at the time of axillary sampling. Direct visualization, surgical clips, ultrasound, or CT scans assisted in delineating the target volume defined as the excision cavity plus 2-cm margin. High activity (192)Ir (3-10 Ci) was used to deliver 340 cGy per fraction, 2 fractions per day, for 5 consecutive days to a total dose of 34 Gy to the target volume. Source position and dwell times were calculated using standard volume optimization techniques. Dosimetric analyses were performed with three-dimensional postimplant dose and volume reconstructions. The median follow-up of all patients was 24 months (range, 12-36 months). RESULTS: Eight patients (crude incidence of 27%) developed clinically evident fat necrosis postimplant in the treated breast. Fat necrosis was determined by clinical presentation including pain and swelling in the treated volume, computed tomography, and/or biopsy. All symptomatic patients (7 of 8 cases) were successfully treated with 3 to 12 months of conservative management. Continuous variables that were found to be associated significantly with fat necrosis included the number of source dwell positions (p = 0.04), and the volume of tissue which received fractional doses of 340 cGy, 510 cGy, and 680 cGy (p = 0.03, p = 0.01, and p = 0.01, respectively). Other continuous variables including patient age, total excised tissue volume, tumor size, number of catheters, number of days the catheters were in place, planar separation, dose homogeneity index (DHI), and uniformity index (UI) were not significant. Discrete variables including the presence/absence of DCIS, sentinel versus full axillary nodal assessment, receptor status, presence/absence of diabetes, and the use of chemotherapy or hormone therapy were not found to have a significant association with the risk of fat necrosis. CONCLUSIONS: In this study of HDR brachytherapy of the breast tumor excision cavity plus margin, treatment was planned and delivered in accordance with the dosimetric parameters of the protocol resulting in a high degree of target volume dose homogeneity. Nonetheless, at a median follow-up of 24 months, a high rate of clinically definable fat necrosis occurred. The overall implant volume as reflected in the number of source dwell positions and the volume of breast tissue receiving fractional doses of 340, 510, and 680 cGy were significantly associated with fat necrosis. Future dosimetric optimization algorithms for HDR breast brachytherapy will need to include these factors to minimize the risk of fat necrosis.

Tumor margin assessment as a guide to optimal conservation surgery and irradiation in early stage breast carcinoma.

Between 1982 and 1985, 108 women with AJC Stage I and II invasive mammary carcinoma were treated to 115 breasts with conservative surgery and irradiation. The irradiation dose was adjusted to the histopathological normal tissue margin around the carcinoma in the tumor excision specimens. Margins were arbitrarily determined negative, close, and positive with normal tissue margins in the inked tumor excision specimens of greater than 5 mm, 2-5 mm, and less than 2 mm, respectively. Negative, close, and positive tumor margin patients were treated to radiation doses of 60, 65, and 70 Gy, respectively. The boost in excess of 50 Gy was directed to the tumor bearing quadrant of the breast using interstitial Ir-192 implants for doses greater than or equal to 70 Gy. The draining lymphatics were irradiated to 50 Gy except in patients with tumor in the lateral half of the breast and no axillary lymph node metastases. Histopathological evaluation of re-excision specimens revealed the difficulty of obtaining negative margins for tumors greater than 2 cm. By our criteria, 54% of the patients had a positive resection margin. None of the patients experienced a local recurrence at 60 months median follow-up. Three patients failed regionally, two in un-irradiated lymph node areas, one in the skin of the contralateral breast; five patients failed systemically. Overall and disease-free survival for Stages T1/N0, T1/N1, T2/N0 was 100 and 95%, respectively, and for T2/N1, 90 and 80%, respectively. The cosmesis was excellent in 66% of the patients with minimal treatment related complications. Carefully planned standardized irradiation with assessment of resection margins yields both excellent local control rates and cosmetic results.