Correction to: Comparative analysis of confocal microscopy on fresh breast core needle biopsies and conventional histology.

Following publication of the original article [1], the authors reported an added data on Table 1 in their paper. The original article [1] has been updated.

Comparative analysis of confocal microscopy on fresh breast core needle biopsies and conventional histology.

BACKGROUND: Evaluation of core needle biopsies (CNB) is a standard procedure for the diagnosis of breast cancer. However, tissue processing and image preparation is a time- consuming procedure and instant on-site availability of high-quality images could substantially improve the efficacy of the diagnostic procedure. Conventional microscopic methods, such as frozen section analysis (FSA) for detection of malignant cells still have clear disadvantages. In the present study, we tested a confocal microscopy scanner on fresh tissue from CNB with intention to develop an alternative device to FSA in clinical practice. PATIENTS AND METHODS: In 24 patients with suspicious breast lesions standard of care image-guided biopsies were performed. Confocal images have been obtained using the Histolog™ Scanner and evaluated by two independent pathologists. Hematoxylin-Eosin (H&E) histological sections of the biopsies were routinely processed in a blinded fashion with respect to the confocal images. RESULTS: In total 42 confocal images were generated from 24 biopsy specimens, and available for analysis within a few minutes of taking the biopsy. This resulted in 2 × 42 = 84 pathologic evaluations. In four cases, a pathologic diagnosis was not possible with confocal microscopy. An exact correlation based on the B-classification was reached in 41 out of 80 examinations and in another 35 cases in a broader sense of correspondence definition (i.e. malignant vs. benign). CONCLUSIONS: As a reliable on-site method, the Histolog™ Scanner provides a visualization of cellular details equivalent to the H&E standards, permitting rapid and accurate diagnosis of malignant and benign breast lesions. Furthermore, this device offers great potential for immediate margin analysis of specimen in breast conserving therapy.

Clinical use of biomarkers in breast cancer: Updated guidelines from the European Group on Tumor Markers (EGTM).

Biomarkers play an essential role in the management of patients with invasive breast cancer. For selecting patients likely to respond to endocrine therapy, both oestrogen receptors (ERs) and progesterone receptors (PRs) should be measured on all newly diagnosed invasive breast cancers. On the other hand, for selecting likely response to all forms of anti-HER2 therapy (trastuzumab, pertuzumab, lapatinib or ado-trastuzumab emtansine), determination of HER2 expression or gene copy number is mandatory. Where feasible, measurement of ER, PR and HER2 should be performed on recurrent lesions and the primary invasive tumour. Although methodological problems exist in the determination of Ki67, because of its clearly established clinical value, wide availability and low costs relative to the available multianalyte signatures, Ki67 may be used for determining prognosis, especially if values are low or high. In oestrogen receptor (ER)-positive, HER2-negative, lymph node-negative patients, multianalyte tests such as urokinase plasminogen activator (uPA)-PAI-1, Oncotype DX, MammaPrint, EndoPredict, Breast Cancer Index (BCI) and Prosigna (PAM50) may be used to predict outcome and aid adjunct therapy decision-making. Oncotype DX, MammaPrint, EndoPredict and Prosigna may be similarly used in patients with 1-3 metastatic lymph nodes. All laboratories measuring biomarkers for patient management should use analytically and clinically validated assays, participate in external quality assurance programs, have established assay acceptance and rejection criteria, perform regular audits and be accredited by an appropriate organisation.

Comparison of two neoadjuvant chemoradiotherapy regimens in patients with potentially curable esophageal carcinoma.

The implementation of neoadjuvant chemoradiotherapy (CRT) in esophageal cancer (EC) patients has led to improved survival rates. Worldwide, different CRT regimens are applied. It is unknown how these regimens relate to each other regarding efficacy. Therefore, the aim of this study was to determine the preferred regimen regarding toxicity of, response to CRT, and long-term survival after esophagectomy in EC patients. EC patients in two centers who underwent CRT with different regimens prior to surgery were included in this study. CRT consisted of 50.4Gy combined with two cycles of cisplatin and 5-FU(center A), or 41.4Gy combined with five cycles of carboplatin and paclitaxel (center B). Toxicity, response to therapy and long-term survival were compared between groups. One hundred sisty-five patients were included. Forty-one percent of patients in center A developed ≥1 toxicity ≥ grade 3 versus 25% in center B (P = 0.025). CRT with a cisplatin-based regimen was an independent predictor for development of toxicity ≥ grade 3 (P = 0.043). There were no differences in response between both regimens (P = 0.904). Three-year survival was 61% (A) versus 57% (B) (P = 0.725). The carboplatin/paclitaxel/41.4Gy regimen causes less toxicity compared to the cisplatin/5-FU/50.4Gy regimen with nonsignificant differences in response rates and long-term survival; therefore our results support this regimen to be the preferred regimen for EC patients.

Reliability of receptor assessment on core needle biopsy in breast cancer patients.

We compared the breast core needle biopsy and the resection specimen with respect to estrogen (ER), progesterone (PR) and human epidermal growth factor receptor 2 (HER2) status to identify predictors for discordant findings. We retrospectively collected data from 526 newly diagnosed breast cancer patients. ER, PR and HER2 status had been assessed in both the core needle biopsy and resection specimen. The assessment of ER by immunohistochemistry (IHC) in core needle biopsy was false negative in 26.5% and false positive in 6.8% of patients. For the PR status the false negative and false positive results of core needle biopsy were 29.6% and 10.3%, respectively. The results of the HER2 status, as determined by IHC and silver in situ hybridization (SISH), were false negative in 5.4% and false positive in 50.0%. We need to be aware of the problem of false negative and false positive test results in ER, PR and HER2 assessment in core needle biopsy and the potential impact on adjuvant systemic treatment. With current techniques, we recommend using the resection specimen to measure these receptors in patients without neoadjuvant treatment. A better alternative might be the use of tissue microarray, combining both core needle biopsy and resection specimen.

Activated macrophages containing tumor marker in colon carcinoma: immunohistochemical proof of a concept.

The presence of carcinoembryonic antigen (CEA)-containing activated macrophages has been demonstrated in peripheral blood from patients with colorectal carcinoma. Macrophages migrate from the circulation into the tissue, phagocytose debris, and return to the bloodstream. Hence it seems likely that activated macrophages containing tumor debris, i.e., tumor marker, are present in the stroma of colorectal carcinoma. After phagocytosis, they could follow a hematogenic or lymphogenic route to the peripheral blood. The aim of this study is to assess the presence of tumor marker-containing activated macrophages in the stroma of colon carcinoma and in regional lymph nodes. From 10 cases of colon carcinoma, samples of tumor tissue and metastasis-free lymph nodes were cut in serial sections and stained for CD68 to identify macrophages and for CEA, cytokeratin, or M30 presence. Slides were digitalised and visually inspected using two monitors, comparing the CD68 stain to the tumor marker stain to evaluate the presence of tumor marker-positive macrophages. Macrophages containing tumor marker could be identified in tumor stroma and in metastasis-free regional lymph nodes. The distribution varied for the different markers, CEA-positive macrophages being most abundant. The presence of macrophages containing tumor marker in the tumor stroma and lymph nodes from patients with colon carcinoma could be confirmed in this series using serial immunohistochemistry. This finding supports the concept of activated macrophages, after phagocytosing cell debris, being transported or migrating through the lymphatic system. These results support the potential of tumor marker-containing macrophages to serve as a marker for diagnosis and follow-up of colon cancer patients.

The effect of a phasic oral contraceptive containing Desogestrel on seborrhea and acne.

OBJECTIVE: To investigate the effect of an oral contraceptive (Novial) on facial seborrhea and moderate acne. METHODS: In this nonrandomized, group-comparative trial, women aged between 18 and 30 with seborrhea were enrolled. Women used either a phasic oral contraceptive containing ethinylestradiol (35/30/30 microg) and Desogestrel (50/100/150 microg (DSG-OC)) (n = 177) or no hormonal treatment (control group; n = 77) for four cycles. RESULTS: Overall, 161 subjects (90.9%) in the DSG-OC group and 73 subjects (94.4%) in the control group completed the study. In the DSG-OC group, the total score for sebum excretion decreased by 0.71 nL/cm(2) (95% CI 0.36-1.05). In the control group, sebum excretion increased by 0.05 nL/cm(2) (-0.55 to 0.46). The difference between groups (0.78 nL/cm(2) (0.19-1.36)) was statistically significant (P = 0.010). The number of active follicles/cm(2) decreased by 0.86 (0.44-1.28) and 0.08 (-0.53 to 0.69), respectively (P = 0.029). The difference in qualitative scores between the groups (0.93 (0.08-1.78)) was statistically significant ( P = 0.032). Overall, 19.3% of the subjects reported an AE (18.1% in the DSG-OC group, 22.1% in the control group). CONCLUSION: Novial effectively reduces seborrhea after only four cycles of treatment and may be a suitable oral contraceptive for women wishing to improve their facial skin condition.

Determination of threshold values for determining the size of the fraction of steroid hormone receptor-positive tumor cells in paraffin-embedded breast carcinomas.

BACKGROUND: For 4 years we used a multiparameter DNA flow cytometric (MP-FCM) technique to assess steroid hormone receptor expression in the diagnostic workup of routinely processed formalin-fixed, paraffin-embedded breast carcinomas as an alternative to immunohistochemistry (IHC) for the quantification of hormone receptor-positive cells. In all cases a positive fraction of hormone receptor-expressing epithelial cells was detected. This observation raised the question of what the cutoff value might be to distinguish receptor-negative from receptor-positive tumors. METHODS: In our search for a possible threshold value of positivity for estrogen receptor (ER) and progesterone receptor (PR) in MP-FCM, we developed four steps. First, we compared IHC results in our own laboratory with the results obtained by MP-FCM on a small series of breast tumors (n = 42). Second, after collecting our first 843 tumors, we made a comparison with the literature of the distribution of receptor positivity according to age classes. Third, using the most likely threshold that resulted from this comparison, we compared a subset of 340 node-negative tumors for their combined ER/PR profiles with the data from a similar group of node-negative tumor cases from the National Cancer Institute's Surveillance, Epidemiology and End-Result (SEER) study. Fourth, with the results of these comparisons, we prospectively collected IHC data and MP-FCM results of the same tumor samples for a period of 1 year. In this way, we collected data for an additional 180 tumors. RESULTS: The first step in this process resulted in an previous publication where 20% of steroid hormone receptor-positive cells seemed to be an acceptable cutoff point for positivity. However, the second step provided the best correlation at approximately 35% of ER reactive cells in the cytokeratin-positive cell population. With this cutoff, the distribution of combined ER/PR profiles in our patient population of node-negative breast cancers also showed a distribution similar to the data from the SEER study. The fourth step, using the 35% threshold value, resulted in a good correlation (r = 0.85, P < 0.0001) for ER and PR between IHC and MP-FCM in the 180 tumors investigated. CONCLUSION: By comparing in-house data with those from large external data collections in the literature, a threshold percentage can be defined that distinguishes steroid hormone receptor-negative from hormone receptor-positive tumors. As a result, information about DNA content and cell cycle distribution can be obtained. This observational study provides additional support to our opinion that MP-FCM is an alternative for IHC determination of ER and PR positivity. It is more objective and quantification can be done more appropriately. The additional value of this approach is that we generate continuous variables of ER/PR content instead of categorical classes, which can be used at different threshold levels for evaluation of clinical relevance.

Multiparameter flow cytometry in the diagnosis of a gynaecologic double tumor: a case report.

PURPOSE: An uncommon clinical presentation of metastatic tumor will often lead to additional diagnostic examinations. The patient of the present study was known to have endometrial cancer which was thought to be limited to the endometrium. Three months postoperatively, she developed ascites due to spread of the tumor, which is rarely seen in low-stage endometrial cancer. METHOD: Multiparameter flow cytometry using both cell phenotype information and DNA ploidy was performed. RESULTS: Retrospectively, the patient was diagnosed as having a DNA-diploid epithelial tumor of the endometrium as well as a DNA-aneuploid epithelial tumor in the left fallopian tube. It was shown that 3 months after primary surgery she developed ascites caused by metastatic tumor from the primary fallopian tube cancer. CONCLUSION: The complete diagnosis was made using multiparameter flow cytometry which, at present, is not routinely applied in gynecologic pathology.

An immunohistochemical study of the clearance of apoptotic cellular fragments.

We investigated the distribution and fate of apoptotic bodies during human development and in the adult, using an antibody (M30) that recognizes a neo-epitope formed early in the apoptotic cascade by caspase cleavage of cytokeratin 18. In the fetus, we found extensive accumulation of M30-positive, non-phagocytosed fragments in the red pulp of the spleen, subcutaneous and submucosal vessels, the interstitium of the lung, and the glomerular mesangium of the kidneys. In the liver, M30-immunoreactive fragments were found inside macrophages in the sinusoids. The number of these fragments and the intensity of the immunostaining increased with the gestational age of the fetus. In the adult, M30-positive fragments were barely detectable in normal tissues. However, many pathological situations, including both chronic degenerative processes and metastatic cancer, were associated with accumulation of M30-positive fragments in the red pulp of the spleen. In the liver and kidney, no fragments could be detected. Remarkably, 13 of the 16 patients with metastasized cancer showed pronounced accumulation of M30-positive fragments containing hematoxylin-reactive material in the red pulp of the spleen. In the non-cancerous cases, such DNA-containing fragments were only seen in 9 of 94 cases. The results show that when apoptotic activity is high, as during development in the fetus or during metastasis and other pathological processes in the adult, the phagocytic clearance of apoptotic bodies can be overloaded. These apoptotic fragments then accumulate in the spleen. The visual detection of apoptotic fragments is concluded to reflect increased cell turnover.

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer.

AIM: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. METHODS: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 microm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100,000 cells were analysed on the flow cytometer. RESULTS: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple non-SLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. CONCLUSION: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.

Steroid receptor heterogeneity in relation to DNA index in breast cancer: a multiparameter flow cytometric approach on paraffin-embedded tumor samples.

Steroid hormone (estrogen and progesterone) receptor (ER and PR) status at the time of breast carcinoma surgery is used as a marker for hormone dependency to guide adjuvant therapy. In a significant number of cases a discrepancy exists between the detected number of hormone receptors and the response to hormonal therapy. One of the explanations for this could be intratumoral heterogeneity. Our objective was to investigate the heterogeneity of steroid hormone receptor expression in breast cancer by using multiparameter flow cytometry (MP-FCM) on routinely processed formalin-fixed, paraffin-embedded tumors. A series of 232 routinely processed breast carcinomas were analyzed using a recently developed technique for the isolation of single cells from paraffin-embedded material. After dewaxing and rehydrating, 50-microm thick sections were heated for 2 hours at 80 degrees C in a citrate solution. Single-cell suspensions were prepared by a short pepsin digestion. The obtained single-cell suspensions were immunostained simultaneously for cytokeratin and ER or PR. Finally, DNA was stained using propidium iodide, after which the samples were analyzed on a flow cytometer. The fractions of ER- and PR-positive cells were determined in the total, as well as the G0 /G1 fraction of the diploid, and in case of nondiploid tumors, also in the G0 /G1 fraction of the aneuploid cell population. Of 232 cases, 88 (38%) were diploid, 38 (16%) were tetraploid, and 106 (46%) were aneuploid. In the diploid tumors the mean fraction of ER- and PR-positive cells was 81% and 76%, respectively. The ER- and PR-positive fractions in the total cytokeratin-positive fraction decreased significantly in the tetraploid (56% and 55%, respectively) and aneuploid tumors (both 47%, p < 0.0001). When analyzing the ER- and PR-positive fractions separately in the diploid and aneuploid cell populations of the nondiploid tumors, it became apparent that the ER and PR status in the diploid fraction of the tumor was significantly higher than in the aneuploid fraction (p < 0.0001). For the tetraploid tumors the mean ER- and PR-positive fractions were 79% and 76%, respectively, in the diploid fraction, and this decreased to 45% in the aneuploid cell subpopulation. In the aneuploid tumors this decrease was even more drastic: in the diploid cell population the ER- and PR-positive fractions were 66% and 62%, while this was 38% and 39% in the aneuploid population. These findings illustrate clearly the existence of a heterogeneous distribution of ER/PR expression in breast cancer, related to the loss of a diploid DNA index. Because of its objective quantification of subfractions within the same tumor, MP-FCM can be regarded as a superior method compared to more conventional techniques such as immunohistochemistry and biochemistry.

Flow cytometric DNA and phenotype analysis in pathology. A meeting report of a symposium at the annual conference of the German Society of Pathology, Kiel, Germany, 6-9 June 2000.

This meeting report summarizes the presentations of three different groups that are active in the field of flow cytometry (FCM) in relation to diagnosing and classification of proliferative disorders. The report starts with the contribution from Regensburg about the developments in DNA FCM, the progression to dual parameter determinations, and combination of immunophenotyping in combination with DNA. In the second part, the use of FCM for the detection of isolated tumor cells in the peripheral blood from patients with prostate or breast cancer is discussed in a contribution from Münster. In the third part, from Heerlen, the use of multi-parameter FCM on formalin-fixed paraffin-embedded tissues from solid tumors is discussed as a new development and application in routine surgical pathology.

Cancer incidence in relatives of patients with testicular cancer in the eastern part of The Netherlands.

OBJECTIVES: To investigate the incidence of malignant tumors in first-degree relatives of patients with testicular cancer. METHODS: Information about the occurrence of cancer in relatives of patients treated for testicular germ cell cancer (TC) at the Department of Urology of the University Medical Centre Nijmegen from 1986 to 1997 was collected using postal questionnaires from 379 (72%) of 524 patients. The expected numbers of cancers in relatives were computed from age- and sex-specific incidence data in the Netherlands Cancer Registry. The observed/expected (O/E) ratios with 95% confidence intervals (CIs) were calculated using Byar's approximation of the exact Poisson test. RESULTS: The O/E ratio for developing cancer in the families of patients with TC was 1.2 (95% CI 1.0 to 1.3). Among first-degree relatives of patients with TC, more TC was observed than expected (O/E 3.3; 95% CI 1.4 to 6.9). The risk for brothers of patients with TC increased 5.9-fold (95% CI 2.2 to 12.8). Both the risk of developing lung cancer (O/E 1.5) and malignancy of the female genital tract in sisters (O/E 2.8) was slightly increased. In contrast, the risk of urinary tract malignancies (O/E 0.3) and other and unknown primary tumors (O/E 0.2) had a lower incidence among relatives. However, both the increased and decreased risk of nontesticular cancer for first-degree relatives may have been caused by misclassification. CONCLUSIONS: TC clusters in families were more pronounced among brothers than among fathers and sons. This study supports previous reports that families of patients with TC do not seem to be prone to nontesticular cancer. Additional investigations in families with TC are recommended to map candidate genes for TC.

Immunohistochemical profiles of 30 monoclonal antibodies against cytokeratins 8, 18 and 19. Second report of the TD5 workshop.

In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 microg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340-365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311-335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.

A novel flow cytometric steroid hormone receptor assay for paraffin-embedded breast carcinomas: an objective quantification of the steroid hormone receptors and direct correlation to ploidy status and proliferative capacity in a single-tube assay.

Semiquantitative estimation of steroid hormone receptors by immunohistochemistry applied to paraffin sections is common practice in surgical pathology. Flow cytometric (FCM) analysis of estrogen receptor (ER) and progesterone receptor (PR) levels provides a faster and more objective quantitative assay. However, a major problem in such FCM analyses of solid tumor samples is the admixture of tumor cells with normal epithelial, stromal, and inflammatory cells. The aim of the underlying study was to investigate the applicability of a recently developed multiparameter flow cytometric methodology for the accurate estimation of the fraction of steroid hormone receptor-positive tumor cells and to explore whether this multiparameter approach allows the detection of specific, clinically relevant subsets of tumors, based on a combination of ploidy level, steroid hormone receptor status, and cell cycle characteristics. For this purpose, samples of 42 breast cancer patients, from which routine immunohistochemistry for ER and PR also was available, were analyzed. From each case, a cell suspension was prepared from the paraffin block by applying a heating and short pepsin digestion step to 50-microm-thick sections. These cell suspensions were double-immunostained for cytokeratin to identify the epithelial cells, and ER or PR, whereas DNA was quantitatively stained with propidium iodide using an optimized protocol. In the entire group of breast tumors, the percentages of ER- and PR-positive cells were registered in the epithelial subfraction, in combination with DNA ploidy and S phase fraction (SPF). A significant correlation was found between the fraction of hormone receptor-positive cells as found by the immunohistochemical and FCM procedures. For ER, a correlation coefficient of r = .87 was found, and for PR r = .62, both P < .0001. It became clear that all the diploid breast tumors had more than 30% tumor cells positive for ER with a SPF lower than 10%, whereas aneuploid tumors contained on average a smaller percentage of steroid hormone receptor-positive cells, and simultaneously an SPF greater than 10%. Our results show that this multiparameter FCM analysis allows an objective and reproducible quantification of the fraction of steroid hormone receptor-positive cells in the relevant epithelial cell compartment in relation to DNA ploidy status and proliferative capacity in a single-tube assay.

Clonality assessment of lymphoproliferative disorders by multiparameter flow cytometry of paraffin-embedded tissue: an additional diagnostic tool in surgical pathology.

A major drawback of immunohistochemical detection of monoclonality in B-cell lymphoproliferative disorders is the lack of contrast between surface-immunoglobulin staining and extracellular immunoglobulin staining. To bypass this drawback, immunophenotyping of single-cell suspensions by flow cytometry is commonly used. Although the expression of immunoglobulin light chain subtype can be quantified rapidly and reliably, the technique is hampered by the requirement of fresh unfixed material. We applied a recently developed technique for the isolation of single cells from formalin-fixed, paraffin-embedded material to measure clonality in B-cell lymphoproliferative disorders (lymphoid tissue (n = 10) and non-Hodgkin's B-cell lymphoma (n = 10). Immunocytochemistry indicated that common cell surface markers as well as the immunoglobulin light chains could be detected in the cell suspensions derived from archival material. In addition, the technique also allowed combined high-resolution DNA flow cytometric analysis. To investigate the effect of formalin fixation on cross-linking of extracellular immunoglobulins to lymphocytes, a double-immunostaining experiment for both light chain immunoglobulins (kappa and lambda) was performed. This experiment showed that this cross-linking was minimal (less than 2%). All cases of reactive lymphoid hyperplasia were DNA diploid and showed a polyclonal expression of immunoglobulin light chains. In contrast, in 9 of 10 non-Hodgkin's B-cell lymphomas, monoclonality was established on the basis of light chain expression, whereas only 6 of 9 cases were conclusive by immunohistochemistry. Four of the 9 cases were DNA aneuploid. One case did not show light chain expression at all by both techniques. However, this case could be classified as malignant by flow cytometric analysis because of the DNA-aneuploid nature of the B-cell subpopulation. The average S-phase fraction (SPF) of the B cells in the reactive lymphoid tissues was 3.5%. The mean SPF values for B cells in DNA-diploid cases of lymphomas was 3.0%, whereas the mean SPF of B cells in DNA-aneuploid cases was 6.1%. The presented technique is superior to immunohistochemistry for the detection of monoclonality in B-cell lymphoproliferative disorders and therefore provides a powerful tool to support the diagnosis of malignant lymphoma in routinely processed archival samples of lymphoid tissues.