V. Distante

Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies ☆

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

Non-palpable lesions of the breast detected by mammography — Review of 1182 consecutive histologically confirmed cases

We report on 1182 consecutive histologically confirmed non-palpable breast lesions detected by mammography (infiltrating carcinoma 427, in situ carcinoma 121, benign 634). The proportion of cancer cases varied according to age (< 50 years = 33%; 50-59 years = 46%; > 59 years = 63%), mammographic pattern (regular opacities = 8%, parenchymal distortions = 20%, isolated calcifications = 42%, irregular opacities = 62%, stellate opacities = 73%), and calendar period (1970-1985 = 29%, 1986-1989 = 56%; 1990-1992 = 69%). A sharp decrease of the benign/malignant biopsy ratio was evident after routine fine-needle aspiration cytology (sonography-guided or stereotaxic) was introduced in 1986. The independent significant association of cancer frequency to age, calendar period and mammographic pattern was confirmed by multivariate analysis. A significant trend over time in favour of conservative surgery was also observed for cancer cases (1970-1979 = 6%, 1980-1985 = 41%, 1986-1992 = 83%). Among invasive cancers, node involvement was observed in 11.5% of cases, being associated with tumour size (pT1a = 0%, pT1b = 7%, pT1c = 13%, pT2a = 33%). Five-, ten- and fifteen-year overall survivals of invasive cancers were 98.1, 95.7 and 87.3%, respectively.

Breast Cancer in the Elderly: Treatment of 1500 Patients

Abstract:  There is a significant difference in the extent of treatment offered to the elderly with breast cancer; in the United States, while 98% of patients less than 65 years of age receive standard treatment, 81% of those older than 65 years were treated according to protocol. This studys goal was to evaluate disease‐specific survival and local‐regional recurrence in breast cancer patients more than 65 years of age at diagnosis. A total of 1500 patients with invasive breast carcinoma were treated consecutively from May 1971 to July 2002 at the University of Florence, Florence, Italy. All patients were more than 65 years of age. The median age was 70.6 years (range 65.1–87.3 years).The median follow‐up was 8.7 years (range 1–30 years). The crude probability of survival (or relapse occurrence) was estimated using the Kaplan–Meier method and survival (or relapse occurrence) comparisons were carried out using Cox proportional hazard regression models. The Cox regression model by stepwise selection showed as independent prognostic factors for disease‐specific survival (DSS), the occurrence of a local relapse (p < 0.0001), pN status (p < 0.0001), the type of surgery (p < 0.0001), and the use of radiotherapy (p < 0.0006) and chemotherapy (p = 0.01). For local disease‐free survival (LDFS), the Cox regression model by stepwise selection showed that mastectomy (p < 0.0001), histotype (p < 0.0001), pN status (p < 0.0001), and pT status (p = 0.001) were the only independent prognostic factors. Age was not a prognostic factor for DSS nor LDFS. We suggest treating patients with appropriate treatment for their prognostic factors. 

Intensive diagnostic follow-up after treatment of primary breast cancer: A randomised trial

Objective.\p=m-\Toevaluate the effectiveness of early detection of intrathoracic and bone metastases in reducing mortality in breast cancer patients. Design.\p=m-\Randomizedclinical trial allocating breast cancer patients to two alternative follow-up protocols (intensive vs clinical) for at least 5 years. Setting.\p=m-\Twelvebreast clinics (referral centers) in different areas in Italy. Patients.\p=m-\Atotal of 1243 consecutive patients (either premenopausal or postmenopausal) surgically treated for unilateral invasive breast carcinoma with no evidence of metastases. The two study groups were well balanced in terms of clinical and prognostic characteristics. Intervention.\p=m-\Patientsin both treatment groups had physical examination and mammography, while patients of the intensive follow-up group had, in addition, chest roentgenography and bone scan every 6 months. Main Outcome Measures.\p=m-\Vitalstatus at 5 years was the main outcome; information was available for all except five patients (0.4%). Relapse-free survival was also analyzed. Results.\p=m-\Overall,393 recurrences (104 local and 289 distant) were observed during the study. Increased detection of isolated intrathoracic and bone metastases was evident in the intensive follow-up group compared with the clinical follow-up group (112 vs 71 cases), while no difference was observed for other sites and for local and/or regional recurrences. The 5-year relapse-free survival rate was significantly higher for the clinical follow-up group, with patients in the intensive follow-up group showing earlier detection of recurrences. No difference in 5-year overall mortality (18.6% vs 19.5%) was observed between the two follow-up groups. Conclusions.\p=m-\Periodicchest roentgenography and bone scan allow earlier detection of distant metastases, but anticipated diagnosis appears to be the only effect of intensive follow-up, and no impact on prognosis is evident after 5 years. Periodic intensive follow-up with chest roentgenography and bone scan should not be recommended as a routine policy.

The Role of a New Monoclonal Antibody Assay in the Detection of Recurrent Breast Cancer

The immunoradiometric assay “CA 15-3”, recently developed to measure a breast tumor-associated antigen, gave a mean serum value of 13.8 U/ml (S.D. 6.2) for this antigen in 156 non-cancer controls (36 biopsies for a benign breast lesion and 120 healthy controls). Setting a cut-off value of 30 U/ml (specificity 99.3%), only 3 out of 58 primary breast cancer cases were positive. In metastatic breast cancer, 11 out of 33 cases with limited recurrence (33.3%) and 36 out of 56 cases with extensive recurrence (64.3%) gave abnormal values in this assay, above the cut-off point, with an overall sensitivity of 52.8%; the difference between the sensitivity values in the two groups of recurrent cases was statistically significant (P < 0.01). According to the findings of the present study, CA 15-3 has no role in the detection of primary breast cancer, but its usefulness in disease monitoring can be hypothesized, as circulating levels of the antigen seem to be dependent on the tumor mass.

Prognostic significance of estrogen receptor determination in primary breast cancer

The authors report on 767 consecutive primary Stage I-II breast cancer cases followed-up from 3 to 8 years. The estrogen receptor (ER) content was determined in all cases and did not influence the treatment choice. A correlation was attempted between ER and menstrual or pathological nodal status (N) or the 5-year disease-free survival (DFS). ER was correlated with menopausal status ER+ cases being more frequent in postmenopausal patients, whereas no correlation was observed between ER and nodal status. In absence of nodal involvement (N-) the prognosis was not influenced by the ER status. A significantly better DFS was evident for ER+ respect to ER- patients in the N+ series but such a correlation is questionable as the adjuvant treatment (hormone or chemotherapy) given to such patients may have influenced the DFS according to the ER status. According to the present study, ER determination should not be used as a discriminant in the performance of adjuvant postoperative treatment based on a prognostic judgment.

Diagnostic features of 225 consecutive cases of cancer recurrence in the conserved breast

Abstract The diagnostic features of 225 consecutive cases of cancer recurrence (186 true recurrences, 39 new primary cancers) in the conserved breast were reviewed. The sensitivity of palpation, mammography, ultrasonography or fine needle aspiration cytology was 69.8, 66.1, 73.3 and 77.8%, respectively. The sensitivity was not significantly influenced by previous radiotherapy, type of recurrence (new primary or true recurrence), or patient age. Although the sensitivity of mammography was lower (60.6 vs 69.3%) in the presence of dense compared with fatty breasts, this did not explain most of the mammographic false negatives, some of which were likely to be due to the masking effect of surgical scar and distortion. In the study period suspicion of breast recurrence following diagnostic evaluation resulted in 34 unnecessary benign biopsies, giving a positive predictive value for biopsy of 86.8%. Of 171 recurrences undergoing a triple diagnostic approach (palpation, mammography/ultrasonography, cytology), recurrence was suspected by at least one, two or three tests in 99.3, 81.8 or 40.3% of cases, respectively. A multimodal diagnostic approach is necessary to maximize the sensitivity of detecting breast cancer recurrence after conservation therapy, and should be employed routinely.

Polyamine modification in human breast tumors after short treatment with tamoxifen

The authors measured estrogen receptors, and polyamine (putrescine, spermidine and spermine) in breast tumors from patients (n = 23) who had received tamoxifen for a few days (5-10) before surgery. Women undergoing mastectomy without any preoperatory treatment were selected as the control group (n = 44). As already reported about in vitro experiments, the treatment resulted in a significant lowering of the spermidine to spermine ratio. Such a modification was larger in the ER positive tumors then in the ER negative ones and it seems to be related to the regression process of the drug-responsive tumors. On the basis of the experimental data the authors suggest the development of a in vivo tamoxifen-sensitivity test.

295 Follow-up in breast cancer

It is undeniable that an accurate follow-up supplies precise and detailed information about the modes and rapidity of development of metastasis, but it has not been clinically proven that follow-up examination effect the survival time. We can assume that early detection of cancer in the other breast or of a local recurrence can influence the prognosis, allowing a better rate of recovery in these two cases. However, there is no clinical evidence that early diagnosis of metastasis can influence the survival time. Thus clinical and mammographic examinations are a sufficient and necessary combination for the early diagnosis of local recurrences of the disease and for rehabilitation end full return of function, which periodic reassessment of performance status. The results of our randomised study (Jama, May 25, 1994) have confirmed that 6 monthly chest roentgenography and bone scan allow for early diagnosis of intrathoracic and bone metastasis, without influencing the 5 years-survival. It appears that recourse to diagnostic test only in the presence of symptoms is the most appropriate procedure for the follow-up of patients with breast cancer.