Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness.

BACKGROUND: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential. METHODS: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines. RESULTS: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population. CONCLUSION: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.

[2002 Standards, Options and Recommendations: good practice for the management and shipment of histological and cytopathological cancer specimens]. / Standards, Options et Recommandations 2002 bonnes pratiques de l'acheminement et de la prise en charge initiale d'un prélèvement en anatomie et cytologie pathologiques en cancérologie (rapport intégral).

CONTEXT: The Standards, Options and Recommendations (SOR) collaborative project was initiated in 1993 by the Federation of the French Cancer Centres (FNCLCC), with the 20 French Regional Cancer Centres, several French public university and general hospitals, as well as private clinics and medical speciality societies. Its main objective is the development of serviceable clinical practice guidelines in order to improve the quality of health care and the outcome of cancer patients. The methodology is based on a literature review, followed by critical appraisal by a multidisciplinary group of experts. Draft guidelines are produced, then validated by specialists in cancer care delivery. OBJECTIVES: Produce clinical practice guidelines for the management and shipment of histological and cytopathological cancer specimens using the methodology developed by the Standards, Options and Recommendations project. METHODS: The FNCLCC designated the group of experts. Available data were collected by a search of Medline and lists selected by experts in the group. A first draft of the guidelines was written, then validated by independent reviewers. RESULTS: The main recommendations are: 1/ High-quality transmission of information between professionals is essential to the management of cancer specimens in order to assure high-quality diagnosis and evaluation of prognostic factors; 2/ Written procedures concerning sample shipment, handling, storage, registration, tracking and fixation exist; these procedures, as well as the necessary shipping material, will be sent to all clinical services involved; 3/ When possible, fresh, unfractionated, oriented surgical samples will be submitted to the same histological and cytopathological laboratory; 4/ Samples collected for extemporaneous examination, freezing or cell culture must be shipped immediately under appropriate storage conditions; 5/ Once frozen, samples can be stored in a deep freezer at temperatures of -80 degrees C or below, or kept in liquid nitrogen; 6/ Fixing tissues shortly after sample collection is essential to prevent cell lysis; 7/ Computerised systems will be used to assure correct specimen registration and tracking in histological and cytopathological laboratories.

[Standards, options and recommendations for the composition of anatomic and surgical pathology reports or cytopathology reports in oncology]. / Standards, Options et Recommandations pour la rédaction d'un compte rendu d'anatomie et cytologie pathologiques en cancérologie.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the Federation of the French Cancer Centres (FNCLCC), the 20 French Cancer Centres and specialists from French Public Universities, General Hospitals and Private Clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and outcome for cancer patients. The methodology is based on literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. OBJECTIVES: To develop practice guidelines according to the definitions of the Standards, Options and Recommendations project for the content of the anatomic and surgical pathology or cytopathology reports in field of oncology. METHODS: Data were identified either by searching on Medline or via members of the expert groups personal references lists. When the guidelines were defined, the document was submitted to 49 independent reviewers, and to the medical committees of the 20 French Cancer Centres. RESULTS: The main recommendations for the composition of the anatomic and surgical pathology or cytopathology reports in oncology are 1/ The reports must contain the identification of the pathologist, of the patient and of the specimen, a gross description for the surgical specimen, eventually a microscopic description, the diagnosis, all the elements essential for establishing the prognosis and for the clinical care, and a conclusion. 2/ The reports could contain some comments. 3/ The reports must be brief, precise, clear, homogeneous and ideally standardised, in order to be comprehensible for all the clinicians and the pathologists.

[Immunohistochemical determination of hormonal receptors on cell-blocks from fine-needle cytopunctures of breast carcinoma]. / Détermination des récepteurs hormonaux par immunohistochimie sur vytoblocs de cytoponctions des cancers du sein.

We assessed the reliability of hormonal receptors (HR) by means of immunohistochemisty (IHC) on cell blocks obtained from diagnostic fine-needle cytopunctures in a group of 142 primary breast carcinoma. The results were compared to biochemical assessment (EIA) on their corresponding tissue samples (118 surgical specimens and 24 core needle biopsies). Percentage of stained nuclei and a score incorporating the proportion and the intensity of positive nuclei were evaluated. A two-group classification (cutoff 10% of stained nuclei) was used to define HR status. Highly positive tumors (>= 50% of stained nuclei) were also individualized. Regarding HR status, concordance rate between immunostaining and biochemical assessment was 86.6% for ER and 76.8% for PR. Major discrepancies were found in 6.3% and 15.5% of cases for ER and PR, respectively. A good correlation was also observed between quantitative values obtained by the two methods (r = 0.69 for ER and 0.60 for PR). Discrepancies were mainly related to weak positive staining, values close to the respective cutoffs and when biochemical evaluation was performed on core needle biopsies. We conclude that IHC on cell blocks prepared from fine-needle cytopuncture specimens of breast carcinomas is useful as a routine procedure for hormonal receptor determination especially when planning neoadjuvant treatment.

CCND1 mRNA overexpression is highly related to estrogen receptor positivity but not to proliferative markers in primary breast cancer.

To elucidate the role of CCND1 alterations in sporadic breast cancer we investigated the possible link between CCND1 mRNA levels versus estrogen-receptor (ER) status and a proliferation marker, S-phase fraction (SPF), measured by flow cytometry. CCND1 expression was quantified by means of real-time quantitative RT-PCR in a well-characterized series of 33 primary breast cancer patients. Eighteen tumors (54.5%) showed CCND1 overexpression ranging from 3.3 to 29.5 times the level observed in normal breast tissue. Seventeen (94.4%) of the 18 cases with CCND1 overexpression were ER-positive compared to seven (46.7%) of the 15 cases with normal CCND1 expression (p=0.0074). CCND1 overexpression was independent of SPF and DNA-ploidy status. These data suggest that the CCND1 gene does not act as an oncogene responsible for more rapid cell proliferation in breast cancer, but could be involved in the regulation of hormone sensitivity associated with ER.

Immunohistochemistry on cell blocks from fine-needle cytopunctures of primary breast carcinomas and lymph node metastases.

We assessed the reliability of prognostic biologic markers by means of immunohistochemistry on cell blocks obtained from diagnostic fine-needle cytopunctures of breast carcinomas and their lymph node metastases. Immunohistochemical studies of MIB-1 (Ki-67), estrogen receptors (ER), progesterone receptors (PR), p53, and c-erb-B-2 were performed in 55 cases of primary breast carcinoma on cell blocks (cytoblock technique) and on their corresponding tissue samples (46 mastectomy specimens and 9 Trucut biopsies) and in 38 cases on cell blocks from fine-needle cytopunctures of both the primary breast tumors and their concurrent lymph node metastases. Interobserver reproducibility ranged from 87 to 100%, depending on the marker. A good correlation was observed between immunostaining assessment on cell blocks and on the corresponding tumor tissues as follows: Ki-67 (85%), ER (96%), PR (82%), p53 (76%), and c-erb-B-2 (84%). An excellent correlation was observed between cell-block results for primary tumors and node metastases; however, a far higher percentage of Ki-67-positive nuclei was observed in the nodes than in the corresponding tumors in seven cases. All nodes corresponding to ER- or PR-negative tumors were also negative, whereas the nodes corresponding to two ER-positive and one PR-positive tumor were negative. Marked discrepancies were also noted with p53 in two cases and with c-erb-B-2 in two cases. Most discrepancies occurred with Trucut biopsies and with breast tumors that contained a large intraductal component. We conclude that cell blocks prepared from fine-needle cytopuncture specimens of breast carcinomas and their node metastases are useful when planning neoadjuvant treatment.

[Standards, Options and Recommendations (SOR) for drafting of anatomic and surgical pathology reports or cytopathology reports in oncology]. / Standards, Options et Recommandations (SOR) pour la rédaction d'un compte rendu d'anatomie et de cytologie pathologiques en cancérologie.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the Federation of the French Cancer Centres (FNCLCC), the 20 French Cancer Centres and specialists from French Public Universities, General Hospitals and Private Clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and outcome for cancer patients. The methodology is based on literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. OBJECTIVES: To develop practice guidelines according to the definitions of the Standards, Options and Recommendations project for the content of the anatomic and surgical pathology or cytopathology reports in field of oncology. METHODS: Data were identified either by searching on Medline or via members of the expert groups personal references lists. When the guidelines were defined, the document was submitted to 49 independent reviewers, and to the medical committees of the 20 French Cancer Centres. RESULTS: The main recommendations for the drafting of the anatomic and surgical pathology or cytopathology reports in oncology are: 1) The reports must contain the identification of the pathologist, of the patient and of the specimen, a gross description for the surgical specimen, eventually a microscopic description, the diagnosis, all the elements essential for establishing the prognosis and for the clinical care, and a conclusion. 2) The reports could contain some comments. 3) The reports must be brief, precise, clear, homogeneous and ideally standardised, in order to be comprehensible for all the clinicians and the pathologists.

[Intraoperative cytology of breast lesions]. / La cytologie extemporanée en pathologie mammaire.

Evaluation of intraoperative cytology and frozen sections for breast lesions is essential to single-stage and cost effective management. The aim of this study is to evaluate the diagnostic accuracy and the potential role of intraoperative cytology. The results of the study undertaken at the René Huguenin Center and the data of the literature suggest that intraoperative cytology may be helpful in some cases, especially as an adjunct to frozen sections. If frozen sections could be avoided any time clear features of benignity or malignancy are offered by both clinical data and macroscopic and cytologic examination, this approach does have limitations. Intraoperative cytology should not be used as an alternative to frozen sections except, in a few cases, when technical conditions required for them are not available or suitable. It can also constitute a good way for continuous education of our cytotechnologists.

[Breast cytopathology. Diagnostic difficulties and limits]. / Cytopathologie mammaire. Difficultés et limites diagnostiques.

Diagnostic difficulties can arise during the cytologic diagnosis of almost all types of breast lesions. "True" difficulties, which are discussed herein, should be differentiated from difficulties due to faulty technique. The frequency of diagnostic difficulties varies across lesions: the proportion of "suspicious" specimens is 4% in adenofibroma, 5 to 7% in cancer, and 17% in epithelial duct hyperplasia, and the false-negative rate in cancer is 3 to 5%. Many difficulties can be overcome by a good knowledge of breast cytopathology. Others are insuperable and should remain so to avoid diagnostic mistakes. In these cases, which should be considered "suspicious", the clearly written documented report should request a histological study. The distinction between duct carcinoma and lobular carcinoma remains difficult, and that between invasive carcinoma and intraductal carcinoma requires a histologic study.

Location of several putative genes possibly involved in human breast cancer progression.

Cancer is a genetic disease resulting from an accumulation of genetic abnormalities in various regulatory genes. Most studies on genetic alterations in human breast cancer have involved primary tumors. The possible involvement of specific tumor suppressor genes in the later stages of cancer progression is poorly documented. We investigated allelic losses associated with breast cancer progression by analyzing 55 polymorphic markers on 11 autosomal chromosomes in a series of 49 relapses (23 local recurrences and 26 distant metastases). All of the loss of heterozygosity (LOH) regions reported in primary breast tumors were frequent in both series of relapses. These results suggest that the allelic losses that are common to the different series of samples occur very early during tumor progression. This study points to candidate metastasis-related genes targeted by LOH on chromosome arms 3p21.3, 16q22.2-23.2, and, possibly, 7q31 but provides no clear evidence of LOH affecting previously described metastasis-related genes such as NME1, MTS1, and TSG101.

Low frequency of TSG101/CC2 gene alterations in invasive human breast cancers.

Large intragenic deletions of the TSG101/CC2 gene were recently reported in seven of 15 primary metastatic breast cancers. Although the number of samples was small, this observation suggested that TSG101/CC2 alterations were a major event in breast carcinogenesis. To study the frequency of these deletions in invasive breast cancers we analysed 189 primary invasive breast tumours and 59 breast cancer metastases. We detected intragenic rearrangements in only three samples (two primary tumours and one metastasis). Northern blot analysis of 43 tumours without rearrangements failed to detect any abnormalities. Furthermore, we studied TSG101/CC2 in 11 human breast adenocarcinoma cell lines by Southern blot, RT-PCR and sequencing of the entire coding region of the gene, and detected no abnormalities. These results show that genetic alteration of TSG101/CC2 is a rare event in breast cancer.

Loss of heterozygosity on chromosome arm 16q in breast cancer metastases.

One of the main genetic abnormalities associated with breast carcinogenesis is the loss of genetic material from chromosome arm 16q. Different groups have identified two regions (16q22.1 and 16q24-ter) that are frequently deleted in primary tumors, suggesting the presence of tumor suppressor genes in these regions. Little is known about the late stages of tumor progression in this respect, and we, therefore, analyzed biopsy specimens of breast cancer metastases for deletions in these critical regions of 16q. We examined fine needle cytopunctures from 24 metastases, each with lymphocyte DNA, for allelic imbalance on 16q by means of polymerase chain reaction (PCR) with 15 highly polymorphic markers. All the metastatic samples showed deletion of at least one informative locus on 16q. The loss of heterozygosity (LOH) pattern often indicated the loss of a complete long arm of chromosome 16 (13 cases); nevertheless, in the remaining 11 samples, partial LOH patterns were observed. A small region of overlap (SR02) in 16q22.1 was frequently involved, whereas another (SR01) in 16q24-ter was affected in only two cases. A third region of LOH in 16q22.2-q23.2 was found in 6/11 samples. These results suggest that at least three different regions are involved in allelic imbalance on chromosome arm 16q in breast cancer. Loss of material from the third region could be a major event in the genesis of metastases.

DNA ploidy and S-phase fraction by image and flow cytometry in breast cancer fine-needle cytopunctures.

The aim of this study was to achieve a better definition of the respective indications of flow cytometry (FCM) and image cytometry (ICM) in clinical practice by comparing their efficiency in a series of 104 primary breast carcinomas that were sampled by means of fine-needle cytopuncture and analyzed by both methods. The comparison involved the DNA content and the estimation of S-phase fraction (SPF). For this purpose, a manual rectangular model was used for ICM, and Modfit software was used for FCM. With respect to DNA ploidy, the concordance rate between the two methods was 87% (DNA diploid vs. DNA nondiploid) and 81% when subclasses of DNA ploidy were used (hypodiploid, diploid, hyperdiploid, tetraploid, or multiploid). True discordance was observed in 19 cases (18%). Eleven of these discordances were the result of an underestimation of multiploidy by FCM. In the 91 cases evaluable by ICM, the SPF median value was 3.5 for DNA-diploid tumors and 8 for DNA-nondiploid tumors. In the 67 cases evaluable by FCM, the SPF median value was 2.31 for DNA-diploid tumors and 5.25 for DNA-nondiploid tumors. The concordance between ICM and FCM in the 46 uniploid tumors was 0.90. Our results led us to obtain samples for the two techniques systematically, to perform FCM routinely because it is more rapid and adequate in a large number of cases, to confirm by ICM any DNA-diploid or DNA near-diploid peak revealed by FCM, and to use ICM when FCM is inadequate, i.e., in case of low cellularity (fewer than 5000 cells for analysis of the cell cycle), suspected tetraploidy, samples not exclusively composed of malignant cells, the identification of several morphologically different malignant populations, or populations with large coefficients of variation and high background.

Cytologic nuclear grading of fine needle cytopunctures of breast carcinoma. Comparison with histologic nuclear grading and image cytometric data.

OBJECTIVE: To evaluate nuclear grading on fine needle cytopunctures of breast carcinoma, which is of special interest when neoadjuvant chemotherapy is planned. STUDY DESIGN: In a prospective study, we compared cytologic grading, based on nuclear parameters (pleomorphism and mitosis), to modified Scarff-Bloom-Richardson histologic grading in 105 primary operable breast carcinomas. The results of these two nuclear grading systems were compared to Feulgen image analysis data from the corresponding cytologic samples. RESULTS: The concordance rate between the two grading systems was 76%. Concordance between cytologic and histologic grading was observed more frequently in purely invasive carcinomas (85%) than in cases combining invasive and in situ components (56%). A highly significant relationship was observed between the two grading systems and indices of proliferative activity (S-phase fraction, proliferation index, 5c exceeding rate and endoreduplication rate), particularly in concordant grading. Furthermore, nuclear area correlated with the results of the two grading systems. CONCLUSION: Cytologic nuclear grading appeared to be a reliable tool for a large proportion of breast tumors. Despite difficulties related to tumor heterogeneity, which could be detected by careful cytologic examination, it is a useful alternative to histologic grading.

[Cytopathology of the breast]. / La cytopathologie mammaire.

Cytologic diagnosis is a useful tool for diagnosis of breast lesions. It is also useful in the follow-up of non operated lesions and overall in the follow-up of breast carcinomas. Technical improvements (immunocytochemistry, flowcytometry, image analysis) are applicable to cytologic material and permits to obtain prognostic factors in breast cancer. Quality of samples, smears, stainings and of cytologic interpretation are prerequisite for the reliability of this method.

[Predictive value of cytology for the efficacy of chemotherapy in breast cancers]. / Valeur prédictive de la cytologie pour l'efficacité de la chimiothérapie dans les cancers du sein.

For the past decade, primary chemotherapy has been extensively used in breast cancer treatment. However, valuable indicators of tumor response are needed. Fine-needle cytopuncture allows the study of malignant cells by cytologic examination, image analysis and flow cytometry. Some parameters evaluated before treatment (cytologic grade, nuclear area and S phase) or at the beginning of treatment (cytomorphologic and cell kinetic changes) have been shown to be correlated with tumor response. The methods, their value and limitations, and results of the literature are discussed.

Sequential cytopunctures during preoperative chemotherapy for primary breast carcinoma. II. DNA flow cytometry changes during chemotherapy, tumor regression, and short-term follow-up.

Between May 1986 and May 1987, 35 primary noninflammatory breast carcinomas (T3N0-N1M0) were studied by means of DNA flow cytometry (FCM-DNA) before and after each of three courses of preoperative chemotherapy (doxorubicin, vincristine, cyclophosphamide, methotrexate, and 5-fluorouracil) to assess initial nuclear DNA content, initial S-phase fraction (SPF), and the impact of chemotherapy on these parameters. Correlations were sought with objective regression and short-term follow-up. Four sequential cytopunctures were performed for cytologic examination and FCM-DNA analyses. Ten tumors were diploid and 25 aneuploid. No significant changes in FCM-DNA parameters during chemotherapy were observed in diploid tumors, and no regression was seen in nine of the ten cases. Among the 25 aneuploid tumors, 10 showed changes in DNA content and/or kinetic parameters. A significant correlation was observed between objective regression and initial FCM-DNA content (P = 0.008), initial SPF (P = 0.004), and changes in FCM-DNA patterns observed during chemotherapy (P = 0.00005). During the follow-up period (range, 27 to 41 months), 13 patients had relapses. Patients with aneuploid tumors were more likely to have relapses (n = 11) than patients with diploid tumors (n = 2), and patients with high SPF were more likely to have relapses than patients with low SPF, but the differences were not significant. Similarly, changes in FCM-DNA parameters were observed more often in patients who had relapses, but, again, the difference was not significant. In 5 of the 13 patients who had relapses, FCM-DNA analyses were performed on cytopunctures of the recurrences: patterns were identical to those observed before treatment even when the primary tumor regressed or showed changes in FCM-DNA parameters during chemotherapy.

Evaluation of breast carcinoma chemosensitivity by flow cytometric DNA analysis and computer assisted image analysis.

Flow cytometric (FCM) DNA and S-Phase (S%) analyses were compared to computerized image analysis (SAMBA 2005) in 27 breast carcinomas (T3, N0-N1, M0) treated by 3 cycles of preoperative Adriamycin, vincristine, cyclophosphamide, methotrexate, 5-fluorouracil (AVCMF) chemotherapy (CT). Twelve carcinomas had shown objective regression and 15 no regression. Samples studied were obtained by sequential fine-needle cytopunctures. Comparing DNA profiles obtained by both methods before and after the first cycle, it appears that tumors can be divided into 3 groups. In the first group (10 cases), no changes were observed after the first cycle of CT. These tumors before treatment had either single DNA peak without cells in S% and G2M or a major peak with a small S% and G2M peak. The second group (9 cases) showed some changes in DNA profiles with an increased G2M peak but no additional values; these tumors before treatment had a small S% and a G2M peak. In the third group (8 cases), before treatment, all were non-diploid with high S% and high G2M. After the first cycle, all showed obvious changes in DNA profiles with a decrease of the G0/G1 peak and an increased S% and G2M with dispersed additional values along the scale in (G2M) x 2 and (G2M) x 4 regions. When changes were compared to tumor regression in the 1st and 2nd groups, 1/10 and 3/9 cases, respectively, were evaluated as objective regression. In the third group, all had objective regression (p less than 0.001). In most cases, a good correlation was observed with both methods.(ABSTRACT TRUNCATED AT 250 WORDS)

[Puncture of bone lesions under tomodensitometric control]. / Ponction des lésions osseuses sous contrôle tomodensitométrique.

Sixty CT-guided fine needle biopsies were performed on 59 patients. Of the 40 lesions in patients with a previous history of cancer, malignancy was confirmed by cytological examination in 27 cases and benignity in three cases. Of the 20 lesions in patients with no previous history of cancer, malignancy was confirmed in nine cases (45%) and benignity in five cases (25%). This precise and safe procedure shows 78% overall accuracy in this series.