Correlation between CD8+ T cells specific for prostate-specific antigen and level of disease in patients with prostate cancer

Modest work has been performed to improve the sensitivity of residual disease detection or investigate the contribution that the immune system makes in controlling metastatic tumor growth, in particular, the frequency and biological actions of peptide-specific CD8+ T lymphocytes in limiting metastatic disease and/or maintaining remission. Fifty-three peripheral blood samples from 32 prostate cancer (PC) patients were investigated for the presence of circulating prostate-specific antigen (PSA)-expressing cells (CPECs) using a highly sensitive and specific assay combining immunomagnetic epithelial cell enrichment with nested RT-PCR of PSA mRNA. Using HLA-A2 tetramer complexes, frequency of CD8+ T cells specific for PSA-derived peptides was determined. Additionally, serum concentrations of PSA and testosterone were measured. CPECs were detected in 26% of peripheral blood samples from PC patients. CD8+ T cells specific for PSA-derived peptides were detected at low frequency in HLA-A2-positive PC patients. The correlation between these PSA-specific CD8+ T cells and residual prostate tumor cells and clinical measures was investigated. Our data suggest that frequency of PSA-specific CD8+ T cells is correlated to CPECs, but not to serum PSA level.     

Pathologic evaluation of sentinel lymph nodes in colorectal carcinoma

BACKGROUND: The identification of lymph node metastases in colorectal resection specimens is necessary for accurate tumor staging. However, routine lymph node dissection by the pathologist yields only a subset of nodes removed surgically and may not include those nodes most directly in the path of lymphatic drainage from the tumor. Intraoperative mapping of such sentinel lymph nodes (SLNs) has been reported in cases of melanoma and breast cancer. We applied a similar method to cases of colorectal carcinoma, with emphasis on the pathology of the SLNs. METHODS: Eighty-three consecutive patients with colorectal carcinoma were evaluated after intraoperative injection of 1 to 2 mL of 1% isosulfan blue dye (Lymphazurin) into the peritumoral subserosa. Blue-stained lymph nodes were suture-tagged by the surgeon within minutes of the injection for identification by the pathologist, and a standard resection was performed. Designated SLNs were sectioned at 10 levels through the block; a cytokeratin immunostain (AE1) was also obtained. To evaluate the possibility that increased detection of metastases in the SLN might be solely due to increased histologic sampling, all initially negative non-SLNs in the first 25 cases were sectioned also at 10 levels. RESULTS: Sentinel lymph nodes were identified intraoperatively in 82 (99%) of 83 patients and accounted for 152 (11.9%) of 1275 lymph nodes recovered, with an average of 1.9 SLNs per patient. A total of 99 positive lymph nodes (38 positive SLNs and 61 positive non-SLNs) were identified in 34 node-positive patients. The SLNs were the only site of metastasis in 17 patients (50%), while 14 patients (41%) had both positive SLNs and non-SLNs. Three patients (9%) had positive non-SLNs with negative SLNs, representing skip metastases. In patients with positive SLNs, 91 (19%) of 474 total lymph nodes and 53 (12%) of 436 non-SLNs were positive for metastasis. In patients with negative SLNs, 8 (1%) of 801 total lymph nodes and 8 (1.2%) of 687 non-SLNs were positive for metastasis. Multilevel sections of 330 initially negative non-SLNs in the first 25 patients yielded only 2 additional positive nodes (0. 6%). All patients with positive SLNs were correctly staged by a combination of 4 representative levels through the SLN(s) together with a single cytokeratin immunostain. CONCLUSIONS: Intraoperative mapping of SLNs in colorectal carcinoma identifies lymph nodes likely to contain metastases. Focused pathologic evaluation of the 1 to 4 SLNs so identified can improve the accuracy of pathologic staging.     

Undesirable cytokeratin immunoreactivity of native nonepithelial cells in sentinel lymph nodes from patients with breast carcinoma

CONTEXT: Use of cytokeratin immunohistochemistry on histologically negative sentinel lymph nodes (SLNs) in patients with breast carcinoma has been shown to be efficient in detecting false-negative nodes. Several recent studies have shown that micrometastases detected by immunohistochemistry constitute an independent predictor of disease-free period and overall survival for breast cancer patients. It has been demonstrated that the fibroblastic type of reticulum cells in lymph nodes also express cytokeratin, and unawareness of such cytokeratin reactivity in SLNs could result in difficulty in the interpretation of the results of immunohistochemistry. OBJECTIVES: To study the incidence of undesirable cytokeratin reactivity in reticulum cells and other native nonepithelial cells of SLNs and to compare the immunoreactivity of 3 commonly used cytokeratin antibodies (AE1/AE3, pancytokeratin [pan-CK], and CAM5.2). DESIGN: Immunohistochemistry with pan-CK, AE1/AE3, and CAM5.2 antibodies was performed on paraffin sections of SLNs from patients with breast cancer. Correlation of undesirable cytokeratin reactivity with size and metastatic status of the SLNs was also analyzed. PATIENT MATERIAL: Paraffin sections of 84 SLNs from 38 consecutive patients with breast cancer in our tertiary-care, teaching hospital. RESULTS: Cytokeratin reactivity was found in reticulum cells and plasma cells in 29 (35%) and 9 (10%) of the 84 SLNs, respectively, with pan-CK and CAM5.2 but not with AE1/AE3 (P <.001). The presence of cytokeratin-positive reticulum cells did not correlate with the size and metastatic involvement of the SLNs. CONCLUSIONS: The incidence of undesirable keratin reactivity in SLNs from breast cancer patients could be limited by using an AE1/AE3 antibody cocktail. The AE1/AE3 antibody cocktail is a sensitive epithelial marker and appears to be more specific in recognizing epithelial cells in SLNs.     

Prostate cancer invasion is influenced more by expression of a CD44 isoform including variant 9 than by Muc18

The standard form of cell adhesion glycoprotein CD44 is a metastasis suppressor in prostate cancer. However, we previously showed by RT-PCR and Western blotting that cancer overexpresses unique CD44 variant v7-v10 isoforms. Muc18 is another cell adhesion marker reportedly overexpressed by prostate cancer. Matched frozen section-confirmed tumor and benign tissues were harvested from 10 prostatectomy specimens and tumor was microdissected from two lymph node metastases. Tissues were homogenized for RNA preparations, and RT-PCR was performed for the CD44v7-v10 sequence. In cultured prostate cancer cells, we caused RNA interference against CD44v9 and/or Muc18. We used PC3M cells and a derivative cell line called G(s)alpha, that constitutively expresses this G-protein and is more invasive. Lipofection was performed for a green fluorescent protein plasmid and for two 22-mer DNA fragments, cloned into a plasmid expression vector to generate hairpin, interfering dsRNA. Assays for invasion into Matrigel, a basement membrane matrix, were performed in 4-5 experiments. RT-PCR demonstrated expression of a 608 bp band representing CD44v7-v10 or a 638 bp band of CD44v6-v10 in prostate cancer tissues and metastases but not benign tissue. Cultured G(s)alpha cells overexpressed CD44v9 by comparison with PC3M cells. At 90 h after 6-hour lipofection, protein silencing was evident by Western blots. Silencing the CD44v9 expression reduced invasiveness into Matrigel to 21.6+/-7.0% in PC3M cells (P<0.001) and 31.2+/-18.3% in G(s)alpha cells (P=0.001), compared to cells exposed to transfection vehicle alone. Silencing Muc18 expression reduced invasiveness to 76.9+/-13.5% of the control value in PC3M cells (P<0.05) and 84.8+/-29.9% in G(s)alpha cells (P=0.18). Prostate cancer invasion is facilitated more by its overexpression of CD44 variant 9 than by Muc18. Its relative overexpression by G(s)alpha cells is a novel finding, suggesting a link between signal transduction and cell adhesion marker expression.     

乳腺癌前哨淋巴结术中分子诊断的临床实用性初探

目的 探讨GeneSearchTM乳腺淋巴结检测试剂盒(以下简称GeneSearch)在乳腺癌前哨淋巴结(SLN)术中诊断的临床实用性.方法 对复旦大学附属肿瘤医院2009年2月至6月诊治的88例乳腺癌患者行SLN活检.首先垂直长轴将所得淋巴结切成数块厚约2 mm的组织块,对各切面进行术中细胞印片后,奇数号组织块用于术后连续切片检查,偶数号组织块采用GeneSearch进行检测,应用即时荧光定量逆转录聚合酶链反应检测SLN中CK19和乳腺球蛋白表达的Ct值.将GeneSearch以术后连续切片的诊断为准,与术中细胞印片、术后连续切片的病理结果分别进行比较.结果 88例共获得225枚SLN,其中宏转移淋巴结27枚,微转移淋巴结9枚,阴性淋巴结189枚(其中5枚为孤立肿瘤细胞).从切割淋巴结开始到最终形成报告,GeneSearch耗时范围为35～45 min(平均40 min).基于淋巴结数目,GeneSearch与术后连续切片的总体符合率为95.6%(215/225),其检测敏感度为86.1%(31/36),均高于术中细胞印片[分别为94.7%(213/225)和72.2%(26/36)].SLN转移灶大小与CKl9和乳腺球蛋白的Ct值存在统计学相关性(P＜0.01).结论 GeneSearch用于SLN术中诊断时,其检测敏感度高于术中细胞印片,达到比较满意的效果,但在应用中仍存在一些问题.     

Evaluation of axillary sentinel lymph node biopsy by immunohistochemistry and multilevel sectioning in patients with breast carcinoma

BACKGROUND: Axillary lymph node dissection for evaluation of the presence or absence of metastatic disease is the single most important prognostic factor for patients with newly diagnosed primary breast cancer. Recently, sentinel lymph node (SLN) biopsy is being investigated as an alternative to the evaluation of the entire axilla. We evaluated whether the application of multilevel sectioning and immunohistochemistry in SLNs will increase the accuracy of detection of metastatic deposits. METHODS: Between October 1998 and July 1999, 38 patients with breast carcinoma (25 ductal, 5 lobular, 4 tubular, and 4 mixed ductal and lobular) underwent successful SLN biopsy followed by complete axillary node dissection. Sentinel lymph nodes were localized with a combination of isosulfan blue dye and radionuclide colloid injection. Frozen sections and permanent sections of SLNs were examined. All negative SLNs were examined for micrometastases by 3 additional hematoxylin-eosin (H&E)-stained sections and immunohistochemistry with the cytokeratins AE1/AE3. RESULTS: Sentinel lymph nodes were successfully identified surgically in 38 (93%) of 41 patients. There was a 97% correlation between the results of the frozen sections and the permanent H&E-stained sections. Twelve (32%) of 38 patients showed evidence of metastatic disease in their SLN by routine H&E staining. In 7 (58%) of 12 patients with positive nodes, the sentinel node was the only positive node. The 26 patients with negative SLN examination by H&E were further analyzed for micrometastases; 5 (19%) were found to have metastatic deposits by immunohistochemistry. Of these patients, 2 were also converted to node positive by detection of micrometastatic disease by examination of the additional H&E levels. CONCLUSIONS: Sentinel lymph nodes can be accurately identified in the axilla of breast cancer patients. Evaluation of SLNs provides reliable information representative of the status of the axilla in these patients. Immunohistochemistry and, to a lesser degree, detailed multilevel sectioning are able to further improve our ability to detect micrometastatic disease in SLNs of breast cancer patients.     

Analysis of sentinel node biopsy - a single-institution experience supporting the use of serial sectioning and immunohistochemistry for detection of micrometastases by comparing four different histopathological laboratory protocols

Grabau D, Ryden L, Fernö M & Ingvar C
(2011) Histopathology 59 , 129–138 Analysis of sentinel node biopsy – a single‐institution experience supporting the use of serial sectioning and immunohistochemistry for detection of micrometastases by comparing four different histopathological laboratory protocols Aims: Detecting micrometastases (>0.2 and ≤2 mm/>200 cells) and isolated tumour cells (ITCs; ≤0.2 mm/<200 cells) is important for staging of breast cancer patients. The aim of this study was to systematically compare several laboratory protocols used to detect metastases after initial intraoperative frozen section examination. Methods and results: Four different protocols for the work‐up of sentinel lymph nodes (SLNs) after frozen sectioning were applied in the routine diagnostic process from 2001 to 2009. In addition, team‐work with a limited number of laboratory technicians and pathologists handling SLNs was introduced in 2008. The present study shows that there were, overall, significantly more node‐positive patients in the period when team‐work and intensive step sections including immunohistochemistry (IHC) were used (P = 0.01). This resulted in 13% more patients being found to have ITCs and micrometastases than in a time period when only step sections were performed. No increase in the number of false‐negative frozen sections was seen. Conclusions: Future guidelines for pathological work‐up of sentinel nodes in women with breast cancer might include team‐work and IHC if frozen sections are used intraoperatively.     

Implementation of step sectioning in the examination of sentinel lymph nodes to improve the detection of micrometastases in breast cancer patients

The object of this study was to examine whether a new protocol for examination of sentinel lymph nodes (SLNs) would lead to the detection of more metastases. Sections of 1 mm would identify most SLN macrometastases, and step sections at intervals of 200-250 μm would identify most micrometastases. A total of 111 breast cancer patients who underwent the SLN procedure at St. Olavs University Hospital in Trondheim, Norway in 2008 were included in the study group. Their SLNs were processed according to a new standardized protocol with sections of 2-3 mm being step sectioned at intervals of 200-250 μm. A total of 109 breast cancer patients undergoing the SLN procedure in 2007 were used as a reference group. Metastases were found in 29% of the cases, compared with 26% in the reference group. Step sectioning of SLNs revealed metastases in five cases initially found to be negative. The metastases of the study group were smaller, with a median value of 1.25 mm compared with 4.25 mm in the reference group. Step sectioning led to the detection of metastases in SLNs initially found to be negative. The median size of the metastases was considerably smaller in the study group than in the reference group.     

Iatrogenically false positive sentinel lymph nodes in breast cancer: Methods of recognition and evaluation

With the introduction of sentinel lymph node (SLN) biopsy as a standard procedure for staging clinically node negative breast cancer patients, meticulous pathologic evaluation of SLNs by serial sections and/or immunohistochemistry for cytokeratins has become commonplace in order to detect small volume metastases (isolated tumor cells and micrometastases). This practice has also brought to the fore the concept of iatrogenically false positive sentinel nodes secondary to epithelial displacement produced largely by preoperative needling procedures. While this concept is well described in the clinical and pathologic literature, it is, in our experience, still under-recognized, with such lymph nodes frequently incorrectly diagnosed as harboring true metastases, possibly resulting in unwarranted further surgery and/or chemotherapy. This review discusses the concept of displaced epithelium in the histologic evaluation of breast surgical specimens and provides a stepwise approach to the correct identification of iatrogenically transported displaced epithelial cells in sentinel lymph nodes.     

Mammaglobin expression in gynecologic malignancies and malignant effusions detected by nested reverse transcriptase-polymerase chain reaction

The detection of micrometastatic disease remains a challenge for the diagnosis and monitoring of malignant disease. RT-PCR for human mammaglobin (hMAM) was recently shown to provide a sensitive method for assessing circulating breast cancer cells in peripheral blood. This study was aimed at investigating hMAM expression in normal and malignant tissue from the female genital tract and the prostate as well as in malignant effusions derived from gynecologic malignancies. hMAM expression was analyzed with nested RT-PCR in 152 samples of normal (n = 73) and malignant epithelial tissues (n = 79) and in 33 specimens of various normal mesenchymal tissue types. We found hMAM expression was not restricted to the normal mammary gland and breast carcinoma but was also detectable in most specimens of benign and malignant epithelial tissue from the ovary (97% versus 95%), uterus (both 100%), and cervix (91% versus 90%). Notably, hMAM expression was also found in benign prostatic hyperplasia (45%) and in prostate cancer (55%). A much lower expression rate was found in various normal and benign mesenchymal tissues (12%). In keeping with our previous data, hMAM expression was absent in all control samples (n = 124) of peripheral blood and bone marrow from healthy volunteers and patients with hematologic malignancies. In pleural or peritoneal effusions (n = 42) from patients with carcinomas of the breast, endometrium, or ovary, hMAM positivity was noticed in the majority of cases (74%), whereas only 52% of the specimens were cytologically positive for tumor cells. In conclusion, hMAM expression assessed by nested RT-PCR is a sensitive molecular marker for detecting micrometastatic tumor spread into pleural effusions and ascites from patients with breast cancer and various other gynecologic neoplasms.     

Sperm-associated antigen 9 is a novel biomarker for colorectal cancer and is involved in tumor growth and tumorigenicity

Colorectal cancer (CRC) is the second most common tumor in developed countries. The present study was undertaken to determine the expression of the sperm-associated antigen 9 gene (SPAG9) as a possible biomarker in CRC, to investigate its correlation with humoral immune response and different stages and grades in CRC patients, and to explore its possible role in colon tumorigenesis in vitro and in an in vivo mouse model. SPAG9 expression was determined by RT-PCR, in situ RNA hybridization, and immunohistochemistry. Humoral response against SPAG9 was detected by enzyme-linked immunosorbent assay and Western blotting. SPAG9 gene silencing was performed using plasmid-based small interfering RNA to study various malignant properties of colon cancer cells in vitro and in vivo. The majority of CRC patients showed SPAG9 expression and generated humoral response. There was a close relationship between SPAG9 protein expression and humoral immune response in the majority of early-stage CRC patients, indicating that anti-SPAG9 antibodies could be a novel serum biomarker for early diagnosis. The down-regulation of SPAG9 (mediated by small interfering RNA) inhibited malignant properties in in vitro and significantly suppressed tumor growth in vivo. These findings collectively suggest that SPAG9 may have a role in tumor development and early spread and thus could serve as a novel target for early detection and for cancer immunotherapy.