Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches.

BACKGROUND: Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT-PCR), but are not routinely used. We evaluated the relevance of Q-RT-PCR for HER2 status determination. METHODS: We compared IHC and Q-RT-PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection. RESULTS: We observed 97.3% concordance between Q-RT-PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT-PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones. CONCLUSION: Q-RT-PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT-PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

[Sentinel node biopsy in breast cancer]. / La technique du ganglion sentinelle dans le cancer du sein: quelques considérations récentes.

As compared to conventional axillary dissection, the sentinel node technique is accompanied by reduced morbidity and shorter hospital stay. Based on available data, the use of this technique does not seem to yield higher rates of axillary recurrence. A combination of both radioisotope detection and blue dye increases the identification rate, while also reducing false-negative rate. Surgical results are optimized when preoperative lymphoscintigraphy mapping is obtained in addition to peroperative probe detection. Considering the site of injection, the subareolar injection can be easy to apply even in case of non-palpable tumours, and gives higher count rates. However, the intraparenchymal, peritumoral, injection is necessary to evidence cases of extra-axillary drainage (internal mammary, infra- or supraclavicular) that is present in about 20% of patients. With the advent of hybrid cameras (SPECT-CT), the topography of these extra-axillary nodes can be given with high precision. Use of the sentinel node technique has been accompanied by an increase in the percent of patients with node involvement, due to an increased detection of micrometastases inferior or equal to 2 mm. Following an overview of basic principles, and of the main results with the sentinel node technique we focus the discussion on several points that are still open to debate, such as: 1) which group of patients can benefit from the sentinel node technique? 2) What is the optimal methodology? 3) What is the prognostic significance of micrometastases and of isolated tumour cells? 4) What attention should be given to extra-axillary drainage?

[Invasive lobular carcinoma of the breast: specific diagnosis and evolution]. / Cancer lobulaire infiltrant du sein: particularités diagnostiques et évolutives.

Invasive lobular carcinoma accounts for 4 to 10% of breast cancers. The clinical and radiological diagnosis is difficult to make. Its progression is slower than that of ductal cancer, and the prognostic factors are more favourable. Its metastases are more frequently located in the digestive tract and the ovaries. It is more frequently bilateral. Its prognosis is not different from that of infiltrating ductal carcinomas. The choice of therapies depends on the individual characteristics of each patient and of the biological features of each tumour. However, lobular carcinomas seem to be less responsive to chemotherapy.

[Breast lobular carcinoma in situ: diagnosis and evolution]. / Cancer lobulaire in situ du sein. Particularités diagnostiques et évolutives.

The incidence of lobular cancers in situ is increasing, especially in post-menopausal women. Whereas this form of disease was regarded for a long time as nothing but a risk factor of the occurrence of later infiltrating carcinoma, it now tends to represent a precancerous state whose progression to subsequent infiltrating carcinoma does not inevitably occur. The clinical and radiological diagnosis remains difficult and the choice of therapies varies according to teams, ranging from mere surveillance to mastectomy.

Immunocytochemical detection of bone marrow micrometastases in colorectal carcinoma patients, using a monoclonal antibody to villin.

The search continues to find methods to more effectively distinguish colorectal carcinoma patients who could be separated into high-risk and low-risk categories. Investigators have reported on the detection of occult micrometastases in bone marrow using antibodies to cytokeratin, which is a marker of epithelial cells but which has no tissue specificity, as opposed to villin, a cytoskeletal protein that is specifically involved in the formation of brush-border microvilli in the small intestine and colon epithelium. Specificity and sensitivity of antibody to villin (ID2C3) and antibody to cytokeratin (A45-B/B3) were first studied in normal bone marrow and in a test system in which cancer cell lines were mixed in normal bone marrow. In a preliminary study including 16 colorectal carcinoma patients, we compared the number of villin-positive cells with cytokeratin-presenting cells. As A45-B/B3, ID2C3 was determined to be sensitive enough to detect one cancer cell in 10(6) hematopoietic cells. Staining of hematopoietic cells with irrelevant antibody and a light staining of megakaryocytes with ID2C3 limited the specificity of the method. In colorectal carcinoma patients, correlation between ID2C3 and A45-B/B3 was 94%. Sensitivity and specificity of ID2C3 antibody to villin were satisfactory. Its clinical relevance must be investigated in further studies.

Primary chemotherapy for operable breast cancer: incidence and prognostic significance of ipsilateral breast tumor recurrence after breast-conserving surgery.

PURPOSE: To determine the incidence and the prognostic value of ipsilateral breast tumor recurrence (IBTR) in patients treated with primary chemotherapy and breast-conserving surgery. PATIENTS AND METHODS: Between January 1985 and December 1994, 257 patients with invasive T1 to T3 breast carcinoma were treated with primary chemotherapy, lumpectomy, and radiation therapy. The median follow-up time was 93 months. To evaluate the role of IBTR in metastase-free survival, a Cox regression multivariate analysis was performed using IBTR as a time-dependent covariate. RESULTS: The IBTR rates were 16% (+/- 2.4%) at 5 years and 21.5% (+/- 3.2%) at 10 years. Multivariate analysis showed that the probability of local control was decreased by the following independent factors: age < or = 40 years, excision margin < or = 2 mm, S-phase fraction more than 4%, and clinical tumor size more than 2 cm at the time of surgery. In patients with excision margins of more than 2 mm, the IBTR rates were 12.7% at 5 years and 17% at 10 years. Nodal status, age < or = 40 years, and negative estrogen receptor status were predictors of distant disease in the Cox multivariate model with fixed covariates. The contribution of IBTR was highly significant (relative risk = 5.34) when added to the model, whereas age < or = 40 years was no longer significant. After IBTR, 31.4% (+/- 7.0%) of patients developed metastases at 2 years and 59.7% (+/- 8.1%) at 5 years. Skin involvement, size at initial surgery, and estrogen receptor status were predictors of metastases after IBTR. CONCLUSION: IBTR is a strong predictor for distant metastases. There are implications for conservative surgery after downstaging of the tumor and therapy at the time of IBTR.

Familial invasive breast cancers: worse outcome related to BRCA1 mutations.

PURPOSE: Although all studies confirm that BRCA1 tumors are highly proliferative and poorly differentiated, their outcomes remain controversial. We propose to examine, through a cohort study, the pathologic characteristics, overall survival, local recurrence, and metastasis-free intervals of 40 patients with BRCA1 breast cancer. PATIENTS AND METHODS: A cohort of 183 patients with invasive breast cancer, treated at the Institut Curie and presenting with a familial history of breast and/or ovarian cancer, were tested for BRCA1 germ-line mutation. Tumor characteristics and clinical events were extracted from our prospectively registered database. RESULTS: Forty BRCA1 mutations were found among the 183 patients (22%). Median follow-up was 58 months. BRCA1 tumors were larger in size (P =.03), had a higher rate of grade 3 histoprognostic factors (P =.002), and had a higher frequency of negative estrogen (P =.003) and progesterone receptors (P =.002) compared with non-BRCA1 tumors. Overall survival was poorer for carriers than for noncarriers (5-year rate, 80% v 91%, P =.002). Because a long time interval between cancer diagnosis and genetic counseling artificially increases survival time due to unrecorded deaths, the analysis was limited to the 110 patients whose diagnosis-to-counseling interval was less than 36 months (19 BRCA1 patients and 91 non-BRCA1 patients). The differences between the BRCA1 and non-BRCA1 groups regarding overall survival and metastasis-free interval were dramatically increased (49% v 85% and 18% v 84%, respectively). Multivariate analysis showed that BRCA1 mutation was an independent prognostic factor. CONCLUSION: Our results strongly support that among patients with familial breast cancer, those who have a BRCA1 mutation have a worse outcome than those who do not.

Detection of MUC1-expressing mammary carcinoma cells in the peripheral blood of breast cancer patients by real-time polymerase chain reaction.

We have prospectively analyzed blood samples of 122 patients with breast disease for the presence of circulating expressing MUC1 cells before and after treatment. Among them, 28 patients had histologically confirmed benign breast disease (group 1), 34 patients had operable breast cancer (group 2), and 60 patients had advanced breast cancer (group 3). Circulating epithelial cells were isolated with BerEP4-coated immunomagnetic beads. Total RNA was extracted and reverse transcribed before analysis by real-time PCR of a MUC1-specific cDNA sequence. The sensitivity of the reverse transcription-PCR tested with blood spiked with MCF7 cells was one cell in 5 ml of blood. The immunomagnetic separation step was mandatory to obtain the maximum specificity. Control samples from healthy donors never displayed cycle threshold (Ct) values for MUC1 lower than 38. Circulating cells (Ct, <38) were detected in 3 of 28 (11%) cases in group 1, in 8 of 34 (24%) cases in group 2, and in 27 of 60 cases (45%) in group 3. A semiquantitative estimate of blood-borne cells could be derived from the Ct value when below 32 (the lowest was 28) or by the number of positive aliquots of the same blood sample. Thus, immunomagnetic separation, followed by MUC1-specific RT-PCR, allows the semiquantitative detection of circulating mammary cells. A significant correlation between the presence of MUC1-positive cells and the group of breast tumors was observed. The clinical significance of blood-borne cells in breast cancer, especially at the operable stage, may be investigated by following these patients.

[Retrospective multivariate analysis of radio-pathological correlations of nonpalpable breast lesions. Experience of the Hospital Saint-Louis]. / Analyse rétrospective multivariée de la corrélation radio-anatomopathologique de lésions infracliniques du sein. Expérience de l'hôpital Saint-Louis.

PURPOSE: Several studies have demonstrated that systematic breast cancer screening increases overall survival. We report our experience regarding diagnosis of breast lesions detected using mammography. METHODS: Case reports of patients operated on in either 1992 or 1993 were retrospectively reviewed. A multivariate analysis of the clinico-pathological correlation was performed. RESULTS: Four hundred fifty seven patients representing on total 544 procedures, were included in the study. Mean age was 50.5 years (range 19-80 years). Most of the patients had no previous history of mammary lesion. Mammography was performed with prophylactic intent in more than 60% of the cases. Four hundred twelve (75.7%) benign lesions were diagnosed. Main lesions were: adenofibroma (15.7%), fibrocystic mastopathy (66.3%), adenosis (26.2%), ductal hyperplasia (23.9%), lobular hyperplasia (10.7%), and combined ductal and lobular hyperplasia (8.5%). Hyperplasia accompanied by cytonuclear atypia was observed in 49 (11%) cases. One hundred thirty two (24.3%) malignant lesions were reported, including 69 (52.3%) invasive carcinomas and 63 (47.7%) in situ carcinomas. Only nine axillary lymph node dissections were positive and 75 minimal breast cancers were diagnosed. The multivariate analysis showed that only radiological signs are a risk factor for cancer. The relative risk for cancer when focus of irregular and vermicular microcalcifications are diagnosed is 4.2 (2.0-8.5). It is 5.6 (2.5-12.5) in case of spiculated opacity. CONCLUSION: Exeresis following radiological prophylactic screening allows diagnosis of high-risk benign lesions and low-stage breast cancer. Radiological parameters are the most powerful predictive factors for malignancy.

Illegitimate villin transcripts in normal bone marrow precludes detection of colon cancer micrometastases.

Villin is a specific marker for normal and tumoral colon tissue. We have developed a highly sensitive assay using reverse transcription (RT) and real-time PCR to detect villin transcripts. The sensitivity of detection is one colon cancer cell. However, high levels of illegitimate villin transcripts were observed in normal bone marrow, precluding the use of villin RT-PCR for routine detection of colon cancer cells in bone marrow of patients with colon cancer.

Is p53 a protein that predicts the response to chemotherapy in node negative breast cancer?

The role of p53 in modulating apoptosis has suggested that it may affect efficacy of anti cancer agents. For this reason, we have evaluated p53 alterations in 282 consecutive patients with infiltrating node-negative breast cancer who underwent primary surgery and were randomized either to CMF (Cyclophosphamide 400 mg/m2, Fluorouracil 400 mg/m2, and Methotrexate 40 mg/m2) or control arm (no adjuvant therapy) from 1980 to 1989. p53 alterations were analyzed by immunohistochemistry using DO7 MoAb, revealed by immunoperoxidase technique, and quantitated in term of percentage of positive cells. We observed a positive staining in 24% of the tumors. Among them, 10% had a positive staining in more than 75% of the cells. There was a highly significant association between the proportion of positive cells and histologic grade of the infiltrating ductal carcinomas (p<0.004). However, there was no association with age, tumor size, hormone receptor content, or vascular embolism. There was a trend but no significant relationship between positive staining and overall survival either in each arm of the trial or in the overall population. Interestingly, we observed a higher relative risk of local relapse after conservative therapy in the boosted area in the group of mutated p53 (RR=4.41; p<0.0005). We conclude that, in this node-negative breast tumor population, alteration of p53 cannot predict the response to the chemotherapy. However, it may represent a useful marker of risk of local relapse and of radio resistance.

[Inflammatory cancer of the breast: review and experience at Saint-Louis hospital]. / Cancer du sein inflammatoire: revue et expérience de l'Hôpital Saint-Louis.

Inflammatory breast cancer represents about 1 to 6% of all breast cancers. It has a sudden onset and affects women of the same age as other breast cancers. Its particular feature is the almost constant development of metastases. The 5-year survival after surgery or surgery and radiotherapy varies between 0 and 28% with a median survival of 18 months. The use of anthracycline-based chemotherapy has doubled the median survival, but has not achieved cure. Clinical research is directed towards therapeutic intensification with or without autologous bone marrow transplantation. The authors report their experience at Hôpital Saint-Louis.

p53 mutations and overexpression in locally advanced breast cancers.

Alterations in the p53 gene were analysed in 39 patients with locally advanced breast cancers (LABCs) (stage III-IV) with inflammatory signs in most cases (UICC stage T4d = 32 patients) by molecular and immunohistochemical (IHC) approaches. All patients were included in the same therapy protocol. Using polymerase chain reaction (PCR) and a single-strand conformational polymorphism migration technique (SSCP), the presence of mutations in exons 2-11, covering the entire coding sequence of the p53 gene, was evaluated. Using the mouse specific anti-human p53 monoclonal antibody (PAb 1801), we also looked for overexpression of the p53 protein in tissue sections. In 16 cases shifted bands were reproducibly identified by PCR-SSCP, and all but one (localised to exon 10) were in exons 5-8, the usual mutational hotspots. Fifteen of these 16 samples were sequenced and 14 of the suspected mutations (36%) were confirmed. Most of them (12) were single nucleotide substitutions, and transitions were more frequent (eight cases) than transversions (four cases). Fourteen of the tumour samples were positively stained with the monoclonal antibody PAb 1801, 11 with nuclear staining only, two with mixed cytoplasmic and nuclear staining and one with cytoplasmic staining only. Staining patterns were very heterogeneous in terms of the percentage of positive cells (10-75%) and their distribution in the tissue section (isolated foci or dispersed cells). In 11 of the 14 mutated cases a positive immunostaining was observed. The presence of a p53 mutation was significantly associated with larger tumour diameter (chi 2 = 7.490, P = 0.0062) and the presence of clinical metastases (stage IV) (chi 2 = 10.113, P = 0.0015). A non-statistically significant trend of association was observed between p53 mutation, negative oestrogen receptors and lower response rate to therapy. Our results in this group of patients and the heterogeneity of the staining of tumour cells in tissue sections suggest that p53 mutations could be a late event in this non-familial form of breast cancer.

[Promising results of bimonthly chemotherapy with high dose cyclophosphamide and epirubicin in induction treatment of inflammatory cancer of the breast]. / Résultats prometteurs d'une chimiothérapie bimensuelle avec cyclophosphamide et épirubicine à forte dose dans le traitement d'induction du cancer du sein inflammatoire.

Forty-eight patients with inflammatory breast cancer were treated by an induction regimen with cyclophosphamide (1200 mg/m2), and epirubicin (75 mg/m2). Four to six courses were given every two weeks, whatever the absolute leukocyte counts, before mastectomy, loco-regional radiation, and conventional, or reinforced, for selected patients, chemotherapy. Main toxicity was leukopenia, with grade IV in 25% of cases. However, febrile neutropenia was rare, and relative dose intensity was 102% after four courses, and 97% after six courses. Complete disappearance of inflammatory signs was observed in 90% of patients. Three patients had a pathological complete response, and ten patients had negative axillary lymph nodes. Furthermore, 13 patients had a major pathological response. With a median follow-up of 42 months, progression-free survival and overall survival are 48% and 66%, respectively.

Splenectomy in human immunodeficiency virus-related thrombocytopenia.

To evaluate the efficacy and safety of splenectomy in patients with human immunodeficiency virus (HIV)-related thrombocytopenia, 30 HIV-infected patients with thrombocytopenia (platelet count < 50 x 10(9)/l) who underwent splenectomy were followed prospectively for a mean period of 42 months. There were no perioperative deaths and morbidity was minimal. Twenty-one patients had a persistent complete response, six had a partial response and were asymptomatic after splenectomy, and only three showed no response. Three patients developed acquired immune deficiency syndrome during follow-up, an incidence that was no different from that expected. Splenectomy is a safe and effective treatment in HIV-infected patients with severe symptomatic thrombocytopenic purpura resistant to medical therapy.

Surgical restaging of advanced Hodgkin's disease after first line chemotherapy.

47 patients with stages IIIB and IV Hodgkin's disease underwent laparotomy with splenectomy as restaging procedure after first-line chemotherapy (CT) which included 4 cycles of CT ABV/IMEP/PCAV/ABV. After surgical restaging (SR), all patients were scheduled to receive 2 additional cycles IMEP/PCAV followed by TNI 20 Gy and patients with residual clinical abnormality or positive restaging surgery received a 20 Gy boost to these areas. 11 patients (23.4%) were found to have active disease in the spleen (10 patients) and/or the lymph nodes (3 patients) after SR. In the spleen, foci of active disease had a size in millimeters and confirm the limits of clinical restaging. According to the response to CT, 5 of the 35 patients (14%) clinically restaged as good responders had active disease; 6 of the 12 patients (50%) clinically restaged as poor responders had active disease. SR is of interest in selected patients with slowly responding disease to determine the indication of an extended field RT for responding patients and salvage therapy for patients resistant to CT.

[Splenectomy for chronic thrombocytopenic purpura]. / Splénectomie pour purpura thrombopénique chronique.

During the period 1985-1989, 49 patients underwent splenectomy for chronic idiopathic thrombocytopenic purpura in our department. A correct follow-up was available for 39 patients. All patients had been previously treated with corticosteroids. We evaluated the platelet count one year after the operation and the relation between this count and several preoperative and postoperative factors. We observed a favourable prognosis when the preoperative study of platelet sequestration showed splenic destruction, when the duration of symptoms was less than one year and when there was a rapid increase in the platelet count during the first week after the operation.