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.

Breast conservation therapy for early stage breast carcinoma with outstanding 10-year locoregional control rates: a case for aggressive therapy to the tumor bearing quadrant.

PURPOSE: Between 1982 and 1988 233 American Joint Committee on Cancer Stage I and II invasive breast carcinomas were prospectively treated in 225 women with conservative tumor excision, careful assessment of histopathological margins, and dose-adjusted irradiation to maximum doses of 70 Gy to the tumor bearing quadrant of the breast. METHODS AND MATERIALS: The pathological stages at presentation were T1N0 and T1N1 in 57% and 13% and T2N0 and T2N1 in 19% and 10% of the patients, respectively. All patients were irradiated according to a policy that, beyond the 50 Gy to the whole breast and draining lymphatics, the tumor-bearing quadrant was boosted in adjustment to the histopathological margin. Normal tissue margins of < 2 mm were considered positive, margins 2-5 mm close, and margins > 5 mm negative and were boosted with 20, 15, and 10 Gy, respectively. Patients in whom the margin could not be assessed were re-excised or boosted to 20 Gy. Re-excisions with no residual carcinoma were not boosted. Most patients boosted to 20 Gy to the tumor-bearing quadrant received interstitial 192-Ir implantations. RESULTS: The actuarial local control rates in the treated breast were 97.5% at 10 years with three recurrences having occurred at a median of 4.5 years after completion of radiotherapy. An additional two patients failed regionally outside the irradiation portals. The overall and disease-free survival of the whole group is 87.5% and 77%, respectively. CONCLUSION: The approach to breast conservation therapy followed in this study has resulted in outstanding local control rates and suggests that there may be a subset of patients that could be irradiated to the tumor bearing quadrant only.

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.

The value of breast lumpectomy margin assessment as a predictor of residual tumor burden.

PURPOSE: Margin assessment is commonly used as a guide to the relative aggressiveness of therapy for breast conserving treatment (BCT), though its value as a predictor of the presence, type, or extent of residual tumor has not been conclusively studied. Controversy continues to exist as to what constitutes a margin that is "positive," "close," or "negative." We attempt to address these issues through an analysis of re-excision specimens. PATIENTS AND METHODS: As part of an institutional prospective practice approach for BCT, 265 cases with AJCC Stage I/II carcinoma with an initial excision margin that was < or = 2 mm or indeterminate were subjected to re-excision. The probability of residual tumor (+RE) was evaluated with respect to tumor size, histopathologic subtype, relative closeness of the measured margin, the extent of margin positivity graded as focal, minimal, moderate, or extensive, and the extent of specimen processing as reflected in the number of cut sections per specimen volume (S:V ratio). The amount of residual tumor was graded as microscopic, small, medium, or large. The histopathologic subtype of tumor in the re-excision specimen was classified as having an invasive component (ICa) or pure DCIS (DCIS). RESULTS: The primary excision margin was positive, > 0 < or = 1 mm, 1.1-2 mm, and indeterminate in 60%, 18%, 5%, and 17%, respectively. The predominant histopathologies in the initial excision specimens were invasive ductal (IDC) (50%) and tumors with an extensive intraductal component (EIC) (43%). The histopathology of the initial excision specimen was highly predictive of the histopathology of tumor found on re-excision, as residual DCIS was found in 60% of +RE specimens with initial histopathology of EIC compared to 26% for IDC (p = 0.001). Neither the extent of margin positivity nor the extent of tumor in the re-excision were significantly related to the initial histopathologic subtype; however, a +RE was seen in 59% of EIC, 43% of IDC, and 32% of invasive lobular ILC cases (p = 0.01). The extent of margin positivity was significantly related to the size of the tumor such that tumor size < or = 20 mm was associated with a greater probability of focal or minimal margin involvement. Positive margins graded as focal, minimal, moderate/extensive were associated with a +RE in 26%, 58%, and 84%, respectively (p = 0.001). Further, the extent of positivity was significantly correlated with the extent of residual tumor such that focal/minimal positivity was more commonly associated with micro/small +RE, whereas moderate/extensive positivity was associated with medium/large +RE. When the closest margin of the initial excision specimen was positive, > 0 < or = 1 mm, or 1.1-2 mm, a +RE was found in 56%, 41%, and 17%, respectively (p = 0.01) but did not correlate with the amount of residual tumor. The extent of specimen processing as reflected in the S:V ratio did not correlate with the probability of defining a measured margin as positive nor the probability of a +RE. In a univariate model, the extent of tumor in the re-excision and the histologic type of tumor in the re-excision were significantly associated with margin status and initial histopathology, respectively. The probability of finding a +RE, based on a multivariate model, was associated with the closeness and extent of margin involvement and initial histopathology of an EIC. CONCLUSION: The relative closeness of tumor to the specimen edge and the extent of margin positivity are predictive for residual tumor, though with an error consistent with its limitations as a sampling procedure. The histopathology of tumor in the initial excision is predictive of the type of residual tumor and the extent of margin positivity was correlated with the amount of residual tumor.

Factors determining outcome for breast-conserving irradiation with margin-directed dose escalation to the tumor bed.

PURPOSE: A prospectively applied treatment policy for breast-conserving therapy used margin assessment as the exclusive guide to the intensity of therapy directed at the tumor-bearing quadrant. METHODS AND MATERIALS: From 1982-1994, there were 509 treated Stage I and II breast carcinomas with a median follow-up of 72 months. For operational purposes, tumor excision margins were prospectively defined as: > 5 mm, 2.1-5 mm, > 0 < or = 2 mm, and positive. If a margin was assessed as < or = 2 mm or indeterminate, and it was deemed cosmetically feasible, a reexcision of the tumor bed would be performed. All patients received whole breast irradiation to 50-50.4 Gy. The following scheme for tumor bed boost irradiation as a function of final margin status (FMS) was observed: (a) Minimal risk = no tumor found on reexcision, no boost performed; (b) low risk = FMS > 5 mm, boost of 10 Gy; intermediate risk = FMS 2.1-5 mm, boost to 14 Gy; high risk = FMS < or = 2 mm or positive, boost to 20 Gy. Cases were analyzed for local failure (LF) with respect to histology (invasive ductal (IDC), IDC with associated DCIS (IDC/DCIS), invasive lobular (ILC)), age, tumor size, total excision volume, reexcision, total dose, tamoxifen therapy, and chemotherapy. RESULTS: There were 19 breast recurrences for a Kaplan-Meier local failure rate for all cases at 5 and 10 years of 2.7% and 7.1%, respectively. Local failure in the first 4 years of follow-up was rare, with a mean annual incidence rate of 0.25% that rose to a mean of 1.1% in subsequent years. Univariate results of Cox proportional hazards regression survival models found positive FMS (p = 0.02), IDC/DCIS (p = 0.04) and age (0.0006) as significantly associated with local failure. In a multivariable model of FMS and IDC/DCIS, FMS retained significance (p = 0.01) but IDC/DCIS was borderline (p = 0.06). When FMS and age were included in a multivariable model, there was a significant interaction (p = 0.01) between the two variables. There was a significant increase in the relative risk of LF for age < or = 45 years (range 11.1-17.4), irrespective of FMS category. Although excellent overall control rates were achieved for patients > 45 years, for younger patients LF rates appeared to remain proportional to the relative closeness of the FMS, despite rigorous dose escalation. CONCLUSIONS: Graded tumor-bed dose escalation in response to FMS results in an exceptionally low risk of "early" local recurrence within the first 5 years of follow-up. However, this strategy is unable to completely overcome the longer term adverse influence of young age and positive FMS.

The influence of age and extensive intraductal component histology upon breast lumpectomy margin assessment as a predictor of residual tumor.

PURPOSE: Young age and extensive intraductal component (EIC) histology have been shown to be associated with increased local recurrence in women treated with breast conservation therapy. This study was conducted to determine if the status of the lumpectomy specimen margin consistently predicted for residual tumor burden risk irrespective of these variables. METHODS AND MATERIALS: As part of an institutional prospective approach for breast conservation therapy (BCT), 265 cases with AJCC Stage I/II carcinoma with an initial excision margin that was < or =2 mm or indeterminate were subjected to reexcision. The probability of residual tumor (+RE) was evaluated with respect to tumor size, histopathologic subtype (invasive ductal carcinoma, invasive ductal carcinoma with an EIC, and invasive lobular carcinoma), relative closeness of the measured margin, and the extent of margin positivity graded as focal, minimal, moderate, or extensive. The amount of residual tumor was graded as microscopic, small, medium, or large. All variables were analyzed for patient age < or =45 or >45 years. RESULTS: There was no significant difference in the incidence of a +RE according to age < or =45 versus >45 years when the margin was >0 < or =2 mm. Of the patients aged < or =45 years, the incidence of a +RE with a margin that was positive as compared to >0 < or =2 mm was 71% vs. 23%, respectively (p = 0.002). For women >45 years old, the difference in the incidence of +RE comparing margins that were positive or >0 < or =2 mm was not significant at 50% vs. 40%, respectively (p = 0.23). For all cases in aggregate, age < or =45 years was associated with a greater incidence of +RE as compared to patients aged >45 years with the discrepant incidence of a +RE by age strata most pronounced for focally positive margins (60% vs. 18%;p< or =0.05). In a logistic regression analysis, age (per year, as a continuous variable) and an EIC histology were significantly associated with the probability of a +RE (odds ratio [OR] = 0.80, p = 0.05 and OR = 1.9, p = 0.01, respectively). Tumor size was not significant (p = 0.23). In patients with an EIC histology, margin status is generally less predictive for differences in the incidence of a +RE. Further, the overall magnitude of difference in the incidence of a +RE related to age appears to be minimized when an EIC histology is present. In contrast, for cases classified as having non-EIC histology, there is a near-linear relationship for both age strata with respect to margin status and the incidence of a +RE. When histology is classified as non-EIC, age < or =45 years is consistently associated with a greater risk of residual tumor for all margin status categories. When the extent of margin positivity was graded as focal or minimal, residual tumor was semiquantitatively estimated as a medium/large amount in 33% versus 26% of cases aged < or =45 or >45 years, respectively (p = 0.62). CONCLUSION: For positive lumpectomy specimen margins, younger age is associated with an increased residual tumor risk. An EIC histology appears to be associated with an elevated risk of residual tumor irrespective of age and may undermine the predictive utility of margin status. Therefore, age and an EIC histology should be factored into risk assessments for residual tumor that rely upon margin assessment.

Alpha1-antitrypsin and alpha-fetoprotein. Protein markers in endodermal sinus (yolk sac) tumors.

A combined immunocytochemical and quantitative serum and tissue study was performed on a group of endodermal sinus (yolk sac) tumors, localizing and measuring both alpha1-antitrypsin (AAT) and alpha-fetoprotein (AFP) in tumor tissue and patient sera. Utilizing indirect immunofluorescent and triple-sandwich immunoperoxidase methods, both proteins were demonstrated within intra- and extracellular periodic acid-Schiff-positive hyaline globules characteristic of the tumor, as well as within the cytoplasm of tumor epithelial cells lining endodermal sinuses, where AAT deposition predominated. Tumor tissue extracts confirmed the presence of significant quantities of both proteins, and pretreatment serum elevations of both showed a parallel decline during therapy. In this study, AAT is characterized as a tumor protein marker for the first time, and a parallelism between AAT and AFP is demonstrated in both serum and tumor tissue. These findings represent additional supportive evidence for the yolk sac origin of endodermal sinus tumors in man.

Mixed gonadal dysgenesis without virilization.

A 19-year-old phenotypic female with primary amenorrhea and 45X,0/46X,Y chromosomal mosaicism was found to have asymmetrical gonadal dysgenesis. Her lack of virilization precludes her precise categorization in the current nomenclature of gonadal dysgenesis and she is considered a unique variant of the syndrome of mixed gonadal dysgenesis.

Development of DES-associated clear-cell carcinoma: the importance of regular screening.

Because the rate of malignancies in young women exposed in utero to diethylstilbesterol (DES) is low, appropriate population screening methods have not been established. A case is presented that is believed to represent the first reported instance of a DES-exposed daughter who developed clear cell adenocarcinoma of the vagina after initially negative examinations. The patient was followed with Papanicolaou smears, pelvic examinations, and colposcopy every 6 months for 2 years prior to the discovery of malignancy. Initially negative, Papanicolaou smears successfully predicted the presence of an early adenocarcinoma. Palpation aided by colposcopy allowed directed biopsy of the small asymptomatic lesion. This case underscores the necessity for frequent vaginal cytologic smears and pelvic examinations at intervals no greater than 6 months. Colposcopy is indicated to direct biopsies when an abnormal cytologic smears is reported or when abnormal bleeding or discharge occurs. Biopsy of any palpable lesion is mandatory.

PIP: The value of frequent routine screening of in utero diethylstilbestrol-exposed women has not been established because of lack of documented reports of patients with initially negative examinations who later developed malignancy. LB, a 19-year-old nulligravida with a history of drug exposure in utero, was 1st evaluated for vaginal adenosis at age 15. The patient was followed with Papanicolaou (Pap) smears, pelvic examination, and colposcopy every 6 months for 2 years before malignancy was discovered. After initially negative Pap smears, the patient's smears were classified as Class III 2 years later, and she was referred to the hospital where colposcopy revealed a cervical collar and extensive columnar ectopy covering the entire cervix. A transformation zone, extending onto the upper third of the vagina, showed epithelial and vascular changes characteristic of immature squamous metaplasia. A directed biopsy (aided by palpation) of the area uncovered a clear cell carcinoma. Reactive hyperplasia was present in multiple lymph nodes, and surgery (radical hysterectomy, total vaginectomy, and vaginal reconstruction) was performed. At 24 weeks postoperation, the patient was sexually active and orgasmic, and 1 year postoperation was clinically free of disease, asymptommatic, and enrolled in college, where follow-up continues. This case underscores the necessity for frequent vaginal cytologic smears and pelvic examinations at intervals no greater than 6 months. Biopsy of any palpable lesion is mandatory.

Malignant transformation of vaginal endometriosis.

A well-documented case of extraovarian endometriosis undergoing malignant transformation is presented. A vaginal focus of endometriosis was biopsied over a 13-year interval during which time progression from benign to malignant disease was observed. Exogenous estrogen replacement was administered throughout the interval of transformation. The pathology, pertinent literature, and implications of the present case are discussed.

Immediate effect of prostaglandin F2alpha during the luteal phase of the menstrual cycle.

Serum luteinizing hormone, estradiol, 17-hydroxyprogesterone, and progesterone determinations were performed in five normally ovulating women prior to, during, and after prostaglandin F2alpha (PGF2alpha was infusion. A total of 75 mg of PGF2alpha was infused over 48 hours, beginning on the 4th day of the luteal phase. Endometrial biopsies were obtained at the completion of the infusion. In all patients, transient decreases in serum progesterone and 17-hydroxyprogesterone levels were observed during PGF2alpha infusion. Subsequent rises in the serum progesterone and 17-hydroxyprogesterone levels were observed 24 hours after completion of the infusion. There was no significant change in serum luteinizing hormone levels. Premature menstrual bleeding occurred with 24 to 72 hours after the termination of the infusion, while progesterone and 17-hydroxyprogesterone levels were still elevated. Endometrial histology revealed focal stromal necrosis in the presence of the normal secretory endometrium. It is concluded that, in the human, PGF2alpha at this dose and time schedule is not luteolytic.

Testicular response to exogenous gonadotropins in the syndrome of feminizing testes.

The steroid response of testes to exogenous gonadotropin administration was studied before castration in a patient with the complete form of the testicular feminization syndrome. Urinary steroid determination were made along with measurements of plasma testosterone during adrenal suppression with dexamethasone and after stimulation with human menopausal gonadotropin (HMG) and HMG plus human chorionic gonadotropin (HCG). Our data provide evidence that the gonads in the testicular feminization syndrome synthesize steroids normally. Estrogens and testosterone are secreted by the testes, and the Leydig cells of the testes are capable of responding to exogenous HMG and HCG stimulation. These findins are consistent with the hypothesis that there is no abnormal testicular steroidogenesis in these genotypic males. Additional evidence is provided that these gonads secrete testosterone in amounts comparable to those of normally functioning testes. Failure of virilization in the presence of androgen secretion as indicated by previous authors is probably the result of a deficiency of androgenic action at the end organs. The presence of sparse pubic and axillary hair in the mother and the finding of the same type of karyotype (46,XY) in a sibling suggest that hereditary factors play a role in this disorder.

Prebiopsy needle localization. Methods, problems, and expected results.

The use of mammography to screen patients for the presence of early breast cancer results in the detection of nonpalpable lesions. As screening becomes more widespread, the number of needle localizations performed will continue to increase. This procedure requires involvement of the radiologist, surgeon, and pathologist. Close cooperation between these disciplines will ensure optimal patient care.

Accuracy and cost-effectiveness of fine needle aspiration biopsy.

Two hundred eighty-six fine needle aspiration biopsies were reviewed. The sensitivity for the diagnosis of malignancy was 90 percent for lymph node specimens, 93 percent for skin and soft tissue masses, and 74 percent for breast cancer. The overall accuracy of the technique was 82 percent for breast lesions, and 90 to 92 percent for soft tissue and lymph node lesions. Traditional open biopsy in an outpatient setting for these tumors is twice as costly as fine needle aspiration biopsy. Further refinement in the use of the cytocentrifuge and immunohistochemical techniques will result in fewer inadequate fine needle aspiration specimens and an increase in the diagnostic information available with this technique. Fine needle aspiration is recommended as the first biopsy technique of choice for localized solid tumors.

Immunocytochemical localization of human papilloma virus and cytomorphologic correlation in smears and biopsies of cervical flat condylomata.

The cytomorphologic features in cervical biopsies and smears associated with human papilloma virus antigen (Ag) expression as demonstrated by immunoperoxidase staining techniques are presented. There was good concordance between cytology and biopsy results with respect to immunoperoxidase staining for human papilloma virus Ag. Cytomorphologic scoring of low-grade lesions (cervical intraepithelial neoplasias, grades O and I) showed atypical mitoses, macronucleated koilocytes, superficial cell keratohyaline granules, and multinucleation to be more frequent in Ag+ biopsy specimens. Chronic inflammation was more evident in Ag- biopsy specimens (P less than 0.05). Parakeratosis was more frequent in Ag+ cytologic specimens. Koilocytosis was not a reliable indicator of Ag expression in either preparation. Human papilloma virus (HPV) Ag testing by immunoperoxidase techniques appears to be a useful adjunct for screening low-grade atypias of the cervix.

Margin width as a determinant of local control with and without radiation therapy for ductal carcinoma in situ (DCIS) of the breast.

In order to assess the utility of margin width in relation to other histopathologic features as a determinant of local control in ductal carcinoma in situ (DCIS) of the breast, we retrospectively examined the treatment of 109 breasts treated with (n = 54) or without adjuvant radiotherapy (n = 55). Median follow-up was 49 and 54 months for patients treated with excision alone (E) or excision plus adjuvant radiotherapy (E+XRT), respectively. Cases treated with E+XRT were significantly larger and had a trend towards closer surgical margins than those treated with E alone. For all cases, margin width < or = 1 mm and lesion diameter >15 mm were significantly associated with increased local recurrence. Lesion size < or = 15 mm was associated with no cases of local failure regardless of treatment arm. For lesions >15 mm in diameter, there was a significant decrease in 5-year local failure with E+XRT compared to E alone (21% vs. 36%, P = 0.03). Tumor margin >1 mm was associated with a low rate of 5-year local failure for either E alone or E+XRT (10.9% vs. 4.6%, P = NS). Tumor margin < = 1 mm had a high rate of local failure that was not significantly decreased by the addition of adjuvant radiotherapy. These results show that large diameter (>15 mm) and close surgical margins (< or = 1 mm) are the dominant risk factors for local recurrence in DCIS. E+XRT significantly decreased local failure risk compared to E alone for large lesions but not for those with close margins.