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We have shown previously that the AKT2 pathway is essential for cell survival and important in malignant transformation. In this study, we demonstrate elevated kinase levels of AKT2 and phosphatidylinositol-3-OH kinase (PI3K) in 32 of 80 primary breast carcinomas. The majority of the cases with the activation are estrogen receptor alpha (ERalpha) positive, which prompted us to examine whether AKT2 regulates ERalpha activity. We found that constitutively activated AKT2 or AKT2 activated by epidermal growth factor or insulin-like growth factor-1 promotes the transcriptional activity of ERalpha. This effect occurred in the absence or presence of estrogen. Activated AKT2 phosphorylates ERalpha in vitro and in vivo, but it does not phosphorylate a mutant ERalpha in which ser-167 was replaced by Ala. The PI3K inhibitor, wortmannin, abolishes both the phosphorylation and transcriptional activity of ERalpha induced by AKT2. However, AKT2-induced ERalpha activity was not inhibited by tamoxifen but was completely abolished by ICI 164,384, implicating that AKT2-activated ERalpha contributes to tamoxifen resistance. Moreover, we found that ERalpha binds to the p85alpha regulatory subunit of PI3K in the absence or presence of estradiol in epithelial cells and subsequently activates PI3K/AKT2, suggesting ERalpha regulation of PI3K/AKT2 through a nontranscriptional and ligand-independent mechanism. These data indicate that regulation between the ERalpha and PI3K/AKT2 pathway (ERalpha-PI3K/AKT2-ERalpha) may play an important role in pathogenesis of human breast cancer and could contribute to ligand-independent breast cancer cell growth.

Successful breast conservation therapy with optimal cosmesis requires adequate tumor excision and negative tumor margins. Therefore, more sensitive techniques are being developed to identify lumpectomy margins intraoperatively with greater accuracy. Unidentified microscopic disease is seemingly responsible for a local recurrence rate of up to 25 per cent 3 to 5 years after lumpectomy and radiotherapy for breast cancer patients. As a result, Moffitt Cancer Center has routinely used an intraoperative touch preparation cytology (TPC) protocol to evaluate the entire resected surface of all lumpectomies. In addition, resection margins were also evaluated by gross examination and by standard histology. In rare instances frozen sections were used to evaluate tumor margins. In this study 701 consecutive lumpectomy specimens were evaluated by TPC during the period of 9 years with a mean follow-up of 3.5 years. Local cancer recurrence was 2.7 per cent (mean recurrence, 2.53 years), in women whose lumpectomy margins were evaluated by TPC. Of interest, a local recurrence rate of 14.6 per cent was observed in patients who had referral lumpectomies evaluated by conventional histopathology. This study suggests that accurate margin assessment with TPC plays an important role in the control of local recurrence after breast conservation therapy. Therefore, we conclude the routine use of intraoperative TPC provides rapid, reliable, topographically accurate identification of residual microscopic disease at lumpectomy margins.

Background: Breast conservation therapy is controversial for ductal carcinoma in situ (DCIS) due to recently reported high recurrence rates. We believe that cytologic evaluation of lumpectomy margins improves efficiency and leads to a lower recurrence rate following lumpectomy for DCIS., Methods: A prospectively accrued database of 1255 breast cancer patients at the H. Lee Moffitt Cancer Center and Research Institute was found to have 218 patients with DCIS (17.4%). Of those 218 cases, 114 were treated with lumpectomy, axillary dissection, and radiation therapy; the remaining 104 patients were treated with mastectomy with or without reconstruction. Imprint cytology was used to evaluate all lumpectomy margins. Permanent sections and imprint cytology were reviewed by the same pathologist., Results: All lumpectomy specimens (116 tumors in 114 patients) were evaluated. The median follow up was 57.5 months (range 2-110 months). One hundred and three patients with 104 tumors were selected on the basis of pure DCIS (with or without microinvasion), and treated with lumpectomy, axillary dissection and radiation therapy. Of the 104 tumors utilizing attempted breast conservation therapy, 7 (6.6%) required mastectomy. There were 6 recurrences (6.1%) with a median time for recurrence of 47.5 months (range 27-85 months); four recurrences were comedo and two were noncomedo at original diagnosis., Conclusions: The determination of lumpectomy margins in DCIS patients using imprint cytology leads to an overall recurrence rate of 6.1% with reduction in operative time, and re-excision rate. Significant recurrence rates were associated with microinvasion and multifocal tumors (28%) versus simple DCIS at 5 years. Breast conservation therapy and surgical margin determination with imprint cytology for DCIS is a cost-effective and reliable method of treatment for simple DCIS.

Background: Diagnostic breast biopsy (DxBx) requires an effective strategy for successful treatment of breast cancer by lumpectomy or mastectomy. Clearance of margins is required to achieve local control., Methods: We reviewed 844 malignant diagnostic biopsies. The strategy was to perform DxBx on all nonpalpable lesions and fine-needle aspiration (FNA) on all palpable lesions. When FNA was equivocal, DxBx was performed. After positive DxBx, either the biopsy cavity or FNA-positive breast mass was excised, and margins were documented with touch preparation cytology analysis (TPC) and frozen section (FS) as necessary to achieve negative margins., Results: Outside institutions referred 430 excisional biopsies. Two hundred twenty-five (52.3%) were found to have residual cancer at surgical excision. Our institution performed 414 biopsies: 169 were performed on nonpalpable lesions in which 58% had residual tumor at resection; 245 were diagnosed by FNA of palpable lesions. Residual disease was found in 12 (5%)., Conclusions: Of patients who undergo DxBx, > 50% have residual breast cancer. It is recommended that (a) FNA be performed on all palpable masses or DxBx of nonpalpable masses; when cancer is diagnosed, proceed to surgical excision. (b) When lumpectomy is the option, margins should be reexcised and intraoperatively evaluated with TPC and FS at the time of axillary dissection.

Lumpectomy with axillary dissection followed by radiotherapy is a frequently utilized technique for the treatment of selected breast carcinomas. Following this procedure, patients often present within a variable period of time with either clinical or mammographic abnormalities at the lumpectomy site suggesting recurrent cancer. In order to evaluate the utility of fine needle aspiration biopsy (FNAB) in the follow-up of such patients, aspirates were obtained 1-73 months after lumpectomy from 72 patients. A spectrum of cytologic changes was seen as a consequence of surgery and irradiation at the site of the lumpectomy as well as in the axilla and lumpectomy scar. The majority of these changes (94%) were benign and generally resolved during follow-up visits. Recurrent malignancy was detected in 4 of 72 patients. These results show that FNAB can reliably differentiate benign from neoplastic changes in postlumpectomy patients and indicate that FNAB may play an important role in the nonsurgical management of clinically alarming but benign postlumpectomy lesions.

Intraoperative evaluation of nonpalpable breast lesions by frozen section frequently is not performed because of potential tissue loss, sampling errors and frozen section artifacts. In a retrospective review we found that only 50 of 503 needle-localized breast biopsies (NLBBs) were evaluated at our institution by frozen section (FS), with a sensitivity of 88%. However, intraoperative evaluation of such lesions may be essential to single-stage and cost-effective management. To this end, this study evaluated the diagnostic accuracy and potential clinical role of intraoperative imprint cytology (IIC) in NLBB. A total of 88 lesions were evaluated by IIC and permanent histology. Fifteen of them were also concomitantly evaluated by FS. All but one deferred cytologic diagnoses were correct. Twelve of 15 FS diagnoses were correct, 2 were deferred, and 1 was false negative. The data suggest that IIC may circumvent some of the difficulties associated with FS, thereby allowing immediate clinical management and later definitive histologic typing and evaluation of prognostic parameters in nonpalpable breast lesions.

Fine needle aspiration biopsy (FNAB) is an increasingly accepted method for investigating palpable breast nodules. We reviewed our experience with a consecutive series of 1875 FNABs performed using 21 or 23 gauge butterfly needles. Correlation was made with histology (524 cases) or clinical follow-up (2-70 months). Cytological diagnoses utilized histopathological terminology and were categorized as: unsatisfactory (93 or 4.96%); no evidence of malignancy (1295 or 69.07%); atypical (183 or 9.76%); suspicious (42 or 2.24%); malignant (262 or 13.97%). Of the 1571 benign aspirates, 220 or 14% were followed by excisional biopsy because of clinical suspicion, atypia or hypocellularity. Of these aspirates, 198 were benign while 22 proved to be malignant (one in situ ductal, one intracystic papillary, two tubular, one cribriform, seven ductal nos, three in situ and five infiltrating lobular carcinomas, two large cell lymphomas). Malignancy was detected histologically in 12.9% of unsatisfactory, 3.06% of benign but often hypocellular, 8.16% of atypical, 97.62% of suspicious and 100% of malignant aspirates. Considering only histologically verified breast aspirates and including suspicious cytodiagnoses, FNAB had a sensitivity of 93.23%, a specificity of 99.50%, positive and negative predictive values of 99.62% and 90%, and an overall diagnostic accuracy of 95.61%. This series clearly shows that FNAB effectively evaluates palpable breast lesions when careful consideration is given to clinical judgement, specimen procurement, diagnostic criteria and a clinically relevant reporting style.

Argyrophilic nucleolar organizer regions (AgNOR) have been correlated with proliferative activity of neoplasms. Increased AgNOR may reflect increased proliferative activity of cells or ploidy. To explore this hypothesis, 41 breast carcinomas were processed for AgNOR silver staining and DNA flow cytometry. AgNOR counts were expressed as mean AgNOR/nucleus and percentage of tumor cells with more than five AgNOR/nucleus. The first count was designated mean AgNOR or mAgNOR, and the second count was designated AgNOR proliferative index or pAgNOR. Using Mantel-Haensel statistical analysis, carcinomas that exhibited mAgNOR of 2.4 or more had a high likelihood of aneuploidy (P less than 0.0001), an S-phase fraction of more than 5.8% (P less than 0.003), or a diameter greater than 2 cm (P less than 0.007). In addition, tumors with pAgNOR of 8% or more showed a statistically significant correlation with aneuploidy (P less than 0.004), tumor grade (P less than 0.04), and a more significant one with high S-phase fraction (P less than 0.0001). No significant correlation was obtained between pAgNOR and tumor size or lymph node status. These data indicate that AgNOR quantitation reflects changes in DNA ploidy and cell proliferation. They also suggest that the mean AgNOR counts correlate best with the DNA mass or ploidy and that the frequency of cells with higher AgNOR count best reflects proliferative activity or S-phase fraction.

Residual microscopic disease after lumpectomy may cause significant local recurrence. This study evaluated the use of touch preparation cytology to assess lumpectomy margins. For 90 specimens, the findings on Diff-Quik-stained touch preparations (rendered intraoperatively within 15 minutes after the lumpectomy) were correlated with the gross findings, frozen-section results and, later, the findings in the permanent histologic sections. Three specimens were cytologically unsatisfactory, 68 yielded benign findings, and 19 were suspicious or diagnostic for malignancy. The margins showed tumor involvement in 5 lumpectomy samples by gross examination, in 13 by frozen-section evaluation and in 17 by the study of permanent sections. Touch preparation cytology was putatively falsely positive in two cases while frozen-section evaluation was falsely negative in four cases. Cytology had a sensitivity of 100%, a specificity of 97.1% and a diagnostic accuracy of 97.7%. These results demonstrate that touch preparation cytology rapidly and reliably evaluates lumpectomy margins and can overcome some sampling errors and artifacts related to frozen-section analysis. Touch preparation cytology is now being used to complement frozen-section evaluation of lumpectomy margins as part of a protocol aimed at reducing the local recurrence rate.

Residual microscopic disease after lumpectomy for breast cancer may cause significant local recurrence. We evaluated one hundred fourteen consecutive breast lumpectomy margins in this study by touch preparation cytology. Cytologic preparations were intraoperatively correlated with gross and frozen section results and subsequently with permanent histologic sections of representative margins. Three specimens were cytologically unsatisfactory and 86 yielded benign findings, while material suggestive or diagnostic of malignancy was obtained from 25 specimens. Gross, frozen section, and permanent histologic margins were positive in 10, 17, and 22 cases, respectively. There were three false-positive touch preparation cytologic results, while frozen section specimens were false-negative in five cases. Sensitivity and specificity of touch preparation cytology were 100% and 96.6%, respectively, with a diagnostic accuracy of 97.3%. Touch preparation cytologic examination rapidly and reliably evaluates lumpectomy margins and overcomes sampling errors and artifacts related to frozen section evaluation. This technique currently complements frozen section evaluation of lumpectomy margins as part of a protocol aimed at reducing local recurrence of breast cancer.