Immunohistochemical detection of PAX-FOXO1 fusion proteins in alveolar rhabdomyosarcoma using breakpoint specific monoclonal antibodies.

Alveolar Rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with about 80% of cases characterized by either a t(1;13)(p36;q14) or t(2;13)(q35;q14), which results in the formation of the fusion oncogenes PAX7-FOXO1 and PAX3-FOXO1, respectively. Since patients with fusion-positive ARMS (FP-RMS) have a poor prognosis and are treated with an aggressive therapeutic regimen, correct classification is of clinical importance. Detection of the translocation by different molecular methods is used for diagnostics, including fluorescence in situ hybridization and RT-PCR or NGS based approaches. Since these methods are complex and time consuming, we developed specific monoclonal antibodies (mAbs) directed to the junction region on the PAX3-FOXO1 fusion protein. Two mAbs, PFM.1 and PFM.2, were developed and able to immunoprecipitate in vitro-translated PAX3-FOXO1 and cellular PAX3-FOXO1 from FP-RMS cells. Furthermore, the mAbs recognized a 105 kDa band in PAX3-FOXO1-transfected cells and in FP-RMS cell lines. The mAbs did not recognize proteins in fusion-negative embryonal rhabdomyosarcoma cell lines, nor did they recognize PAX3 or FOXO1 alone when compared to anti-PAX3 and anti-FOXO1 antibodies. We next evaluated the ability of mAb PFM.2 to detect the fusion protein by immunohistochemistry. Both PAX3-FOXO1 and PAX7-FOXO1 were detected in HEK293 cells transfected with the corresponding cDNAs. Subsequently, we stained 26 primary tumor sections and a rhabdomyosarcoma tissue array and detected both fusion proteins with a positive predictive value of 100%, negative predictive value of 98%, specificity of 100% and a sensitivity of 91%. While tumors are stained homogenously in PAX3-FOXO1 cases, the staining pattern is heterogenous with scattered positive cells only in tumors expressing PAX7-FOXO1. No staining was observed in stromal cells, embryonal rhabdomyosarcoma, and fusion-negative rhabdomyosarcoma. These results demonstrate that mAbs specific for the chimeric oncoproteins PAX3-FOXO1 and PAX7-FOXO1 can be used efficiently for simple and fast subclassification of rhabdomyosarcoma in routine diagnostics via immunohistochemical detection.

Insulin Modulates Myogenesis and Muscle Atrophy Resulting From Skin Scald Burn in Young Male Rats.

BACKGROUND: Burn injuries (BIs) due to scalding are one of the most common accidents among children. BIs greater than 40% of total body surface area are considered extensive and result in local and systemic response. We sought to assess morphological and myogenic mechanisms through both short- and long-term intensive insulin therapies that affect the skeletal muscle after extensive skin BI in young rats. MATERIALS AND METHODS: Wistar rats aged 21 d were distributed into four groups: control (C), control with insulin (C + I), scald burn injury (SI), and SI with insulin (SI + I). The SI groups were submitted to a 45% total body surface area burn, and the C + I and SI + I groups received insulin (5 UI/Kg/d) for 4 or 14 d. Glucose tolerance and the homeostatic model assessment of insulin resistance index were determined. Gastrocnemius muscles were analyzed for histopathological, morphometric, and immunohistochemical myogenic parameters (Pax7, MyoD, and MyoG); in addition, the expression of genes related to muscle atrophy (MuRF1 and MAFbx) and its regulation (IGF-1) were also assessed. RESULTS: Short-term treatment with insulin favored muscle regeneration by primary myogenesis and decreased muscle atrophy in animals with BIs, whereas the long-term treatment modulated myogenesis by increasing the MyoD protein. Both treatments improved histopathological parameters and secondary myogenesis by increasing the MyoG protein. CONCLUSIONS: Treatment with insulin benefits myogenic parameters during regeneration and modulates MuRF1, an important mediator of muscle atrophy.

Clonality analysis of combined Brenner and mucinous tumours of the ovary reveals their monoclonal origin.

The derivation of ovarian intestinal-type mucinous tumours is not well established. Some are derived from teratomas but the origin of the majority is not clear. It has been recently proposed that the non-germ cell group may be derived from Brenner tumours, as the association of a mucinous tumour with a Brenner tumour is frequently observed. In order to explore the histogenesis of these neoplasms, we undertook a clonality analysis of the two components of ten combined Brenner and mucinous tumours using a human androgen receptor gene (HUMARA) assay. All eight informative cases of ten showed a concordant X-chromosome inactivation pattern between the two tumour components, indicative of a shared clonal origin (p = 0.0039). Microsatellite genotyping in five of the combined tumours displayed an identical heterozygous pattern with paired Fallopian tube tissue, indicative of a somatic cell origin. In addition, paired box protein 8, a highly sensitive Müllerian epithelial marker, was not detected by immunohistochemistry in either tumour component in any of the ten tumours, suggesting that this subset of mucinous tumours does not originate from Müllerian-derived epithelium. In conclusion, this study demonstrates that in combined mucinous and Brenner tumours, there is a shared clonal relationship between the two different tumour components and suggests that some pure mucinous tumours may develop from a Brenner tumour in which the Brenner tumour component becomes compressed and obliterated by an expanding mucinous neoplasm.

Pax6 controls centriole maturation in cortical progenitors through Odf2.

Cortical glutamatergic neurons are generated by radial glial cells (RGCs), specified by the expression of transcription factor (TF) Pax6, in the germinative zones of the dorsal telencephalon. Here, we demonstrate that Pax6 regulates the structural assembly of the interphase centrosomes. In the cortex of the Pax6-deficient Small eye (Sey/Sey) mutant, we find a defect of the appendages of the mother centrioles, indicating incomplete centrosome maturation. Consequently, RGCs fail to generate primary cilia, and instead of staying in the germinative zone for renewal, RGCs detach from the ventricular surface thus affecting the interkinetic nuclear migration and they exit prematurely from mitosis. Mechanistically, we show that TF Pax6 directly regulates the activity of the Odf2 gene encoding for the appendage-specific protein Odf2 with a role for the assembly of mother centriole. Our findings demonstrate a molecular mechanism that explains important characteristics of the centrosome disassembly and malfunctioning in developing cortex lacking Pax6.

Simultaneous carcinomas of the breast and ovary: utility of Pax-8, WT-1, and GATA3 for distinguishing independent primary tumors from metastases.

Breast carcinomas rarely metastasize to the ovary and are even more rarely present clinically as primary ovarian tumors. However, patients with breast cancer not infrequently develop independent primary ovarian carcinomas. In these cases, distinction between independent primaries and metastatic tumors is crucial. Several comparative immunohistochemical studies have been reported, but few included significant clinicopathologic data and none investigated cases of ovarian and breast carcinomas from the same patients. In this study, we compared 18 cases of patients with bona fide independent breast and ovarian carcinomas (15 high-grade serous and 3 clear cell carcinomas), with 9 cases of patients with known mammary carcinomas (7 lobular and 2 ductal carcinomas) metastatic to the ovary. Immunohistochemical stains for Pax-8, WT-1, and GATA3 were carried out on tissue microarrays (TMA). Most primary ovarian carcinomas were larger than the metastatic tumors (P=0.001) and were diagnosed at an advanced stage. All primary ovarian tumors showed marked nuclear pleomorphism, whereas only 2 metastatic breast carcinomas had Grade 3 nuclei (P=0.000). The vast majority of ovarian metastases (7/9) showed the typical pattern of lobular breast carcinoma. Pax-8 and WT-1 expression were found in 16 of 18 (88%) and 13 of 18 (72%) primary ovarian carcinomas, respectively. In contrast, all primary ovarian carcinomas were negative for GATA3. The 2 Pax-8-negative ovarian carcinomas were also negative for WT-1. With the exception of 3 triple-negative carcinomas, all primary breast carcinomas were positive for GATA3. All metastatic breast carcinomas were positive for GATA3 and negative for Pax-8. WT-1 expression was seen in only 1 of 9 metastatic breast carcinomas (11%). Patients with ovarian metastases had worse prognosis than patients with independent breast and ovarian carcinomas (P=0.000). Pax-8, WT-1, and GATA3 immunoreactions are useful in the distinction between independent primaries and metastatic mammary carcinomas to the ovary in the light of clinicopathologic findings.

Immunohistochemical analyses point to epidermal origin of human Merkel cells.

Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, ß1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. ß1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Circulating tumor cells in peripheral blood or disseminated to bone marrow, a concept commonly referred to as minimal residual disease (MRD), are thought to be key to the prediction of metastasis and treatment efficacy. To date, two MRD markers, MYOD and MYOGENIN, have been tested; however, MRD detection continues to be challenging mainly owing to the closeness of the detection limit and the discordance of both markers in some samples. Therefore, the addition of a third marker could be useful for more accurate MRD assessment. The PAX3 gene is expressed during embryo development in all myogenic precursor cells in the dermomyotome. As RMS cells are thought to originate from these muscle precursor cells, they are expected to be positive for PAX3. In this study, PAX3 expression was characterized in cancer cell lines and tumors, showing wide expression in RMS. Detection sensitivities by quantitative polymerase chain reaction (qPCR) of the previously proposed markers, MYOD and MYOGENIN, were similar to that of PAX3, thereby indicating the feasibility of its detection. Interestingly, the flow cytometry experiments supported the usefulness of this technique in the quantification of MRD in RMS using PAX3 as a marker. These results indicate that flow cytometry, albeit in some cases slightly less sensitive, can be considered a good approach for MRD assessment in RMS and more consistent than qPCR, especially owing to its greater specificity. Furthermore, fluorescence-activated cell sorting permits the recovery of cells, thereby providing material for further characterization of circulating or disseminated cancer cells.

Utility of PAX8 mouse monoclonal antibody in the diagnosis of thyroid, thymic, pleural and lung tumours: a comparison with polyclonal PAX8 antibody.

AIMS: The purpose of this study was to compare the immunohistochemical staining profiles of PAX8-polyclonal, PAX8-monoclonal, PAX5-monoclonal and PAX6-monoclonal antibodies in several histological types of primary thoracic and thyroid tumours. In addition, we analysed PAX8 mRNA expression by using in-situ hybridization. METHODS AND RESULTS: We compared polyclonal PAX8 and monoclonal PAX8, PAX5 and PAX6 antibodies in 962 samples (687 lung carcinomas, 40 malignant pleural mesotheliomas, 138 thymic tumours and 97 thyroid tumours) using the tissue microarray technique. Among thyroid tumours, the monoclonal and polyclonal PAX8 antibodies showed a high positive rate (98.0%). Of 167 polyclonal PAX8 antibody-positive tumours, except for thyroid tumours, 54 cases tested positive for PAX5 and/or PAX6 (31 lung carcinomas and 23 thymic tumours). No PAX8 mRNA expression was detected using RNAscope (in-situ hybridization technique) other than in thyroid tumours. A portion of polyclonal PAX8 antibody-positive tumours showed cross-reactivity for PAX5 or PAX6 protein. CONCLUSIONS: Monoclonal PAX8 antibody showed high specificity to thyroid tumours and was superior to the polyclonal antibody.

Laryngotracheal presentation of anaplastic thyroid carcinoma with squamous differentiation: seven cases demonstrating an under-recognized diagnostic pitfall.

AIMS: To describe a series of anaplastic thyroid carcinomas that mimicked primary head and neck squamous cell carcinoma (HNSCC) by virtue of both morphology and clinical presentation. METHODS AND RESULTS: Seven cases were identified in a 15-year period where a biopsy of an airway lesion that appeared to be squamous cell carcinoma was, in fact, anaplastic thyroid carcinoma. The tumours had squamous and/or spindle cell morphology, with only the squamous component being apparent in the airway biopsy. Some tumours arose within metaplastic (n = 3) or atypical (n = 3) epithelium, supporting the diagnosis of a primary mucosal tumour. Positive PAX8 (n = 5) and TTF-1 (n = 4) staining was identified. CONCLUSIONS: An endotracheal presentation of anaplastic thyroid carcinoma with squamous morphology may be misdiagnosed as a primary head and neck squamous cell carcinoma. PAX8 and TTF-1 expression are helpful in making the distinction, but the problem lies in suspecting a thyroid carcinoma in what appears to be a straightforward diagnosis of HNSCC.

Differential patterns of PAX8, p16, and ER immunostains in mesonephric lesions and adenocarcinomas of the cervix.

Mesonephric remnants, usually located deep in the lateral cervical wall, may become hyperplastic resulting in a florid proliferation. These can be misinterpreted as malignant and confused with endocervical adenocarcinomas. Recent data have shown that PAX2 is diffusely expressed in mesonephric remnants and hyperplasias. PAX8 is a related transcription protein that is expressed in tissues of müllerian and wolffian origin. In this study, we have investigated the utility of an immunohistochemical panel comprising of PAX8, estrogen receptor (ER), and p16 in the differential diagnosis between mesonephric proliferations and cervical adenocarcinomas. A database search was conducted for cases of mesonephric remnants/hyperplasia/carcinoma of cervix and invasive cervical adenocarcinomas. Immunohistochemical stains for PAX8, ER, and p16 were performed using the avidin-biotin peroxidase technique on the most representative tissue. The search yielded 28 cases of mesonephric proliferations of cervix (15 mesonephric remnants, 12 mesonephric hyperplasias, and 1 mesonephric adenocarcinoma) and 16 cases of cervical adenocarcinomas (15 usual type and 1 adenoma malignum). Immunohistochemically, all the mesonephric proliferations, regardless of being benign or malignant, displayed a consistent staining pattern-diffusely and strongly positive for PAX8, negative for ER, and patchy cytoplasmic staining for p16. The usual type cervical adenocarcinomas exhibited a variable staining pattern with PAX8 and ER but all were strongly and diffusely positive for p16. The case of adenoma malignum was PAX8 positive, ER negative, and showed weak and patchy staining with p16. Our study suggests that a panel of immunohistochemical stains composed of PAX8, p16, and ER is useful in the distinction between mesonephric proliferations and cervical adenocarcinomas.

Mesonephric carcinosarcoma involving uterine cervix and vagina: report of 2 cases with immunohistochemical positivity For PAX2, PAX8, and GATA-3.

Mesonephric carcinomas are rare tumors predominantly arising in the uterine cervix from mesonephric remnants. Although the tumor has classic morphologic features, some cases can mimic Müllerian adenocarcinoma and be misdiagnosed, especially those with significant ductal pattern. Moreover, there is an overlap in immunohistochemical results with endometrial and endocervical carcinomas. In this study, we report 2 cases of mesonephric carcinosarcoma, originally diagnosed as Müllerian carcinomas, 1 presenting in the vagina; review immunohistochemical results including positivity for GATA-3, not previously reported and comment on the proposed panel of PAX8, p16, and estrogen receptors as discriminators of Müllerian adenocarcinoma (endocervical or endometrial) versus mesonephric carcinoma.

PAX-8 expression in cutaneous ciliated cysts: evidence for Müllerian origin.

Cutaneous ciliated cysts (CCC) are exquisitely rare, benign cystic lesions demonstrating simple, ciliated epithelial linings reminiscent of fallopian tube epithelium. Most commonly, CCC show a predilection for the lower extremities of young reproductive age women and demonstrate immunohistochemical positivity for estrogen and progesterone receptors, supporting the theory that they are derived from ectopic Müllerian rests. PAX-8 is a paired box gene, important in the development of Müllerian and thyroid organs and has utility in the identification of tumors of Müllerian, renal, and thyroid origin. Prompted by the precedent studies on PAX-8 immunohistochemical expression in tumors of Müllerian origin, this article aimed to explore the utility of this antibody in defining the histogenesis of 2 bona fide cases of CCC, both occurring in young reproductive age women. Herein, 2 prototypic index cases of CCC with strong nuclear positivity for estrogen and progesterone receptors are shown to also have positive nuclear staining for PAX-8, further supporting their likely Müllerian origin. These data support the designation of these lesions as cutaneous Müllerian cysts, distinct from potential ciliated cysts of eccrine origin.

PAX2 and PAX8: useful markers for metastatic effusions.

OBJECTIVE: It was the aim of this study to determine the utility of PAX2 and PAX8 in cytology effusions with metastatic tumor. STUDY DESIGN: PAX2 and PAX8 immunohistochemical staining was performed on cell blocks of 89 pleural, pericardial and peritoneal effusions with benign diagnoses (18 cases), or secondary to renal cell carcinoma (RCC; 9 cases), müllerian carcinoma (21 cases) or non-müllerian carcinoma (41 cases). RESULTS: PAX2 stained 0% (0/18) of controls, 100% (8/8) of RCCs, 35% (7/20) of müllerian carcinomas, and 2% (1/41) of non-müllerian carcinomas. PAX8 stained 6% (1/18) of control cases, 100% (9/9) of RCC cases, 100% (20/20) of müllerian carcinomas, and 5% (2/41) of non-müllerian carcinomas. PAX2 was 35% sensitive and 95% specific for müllerian carcinoma and 100% sensitive and 95% specific for RCC. PAX8 was 100% sensitive and 95% specific for müllerian carcinoma and 100% sensitive and 95% specific for RCC. CONCLUSIONS: PAX8 is more sensitive than PAX2 for metastatic effusions from müllerian carcinomas (100 vs. 35%), while also having a higher intensity of staining than PAX2. However, PAX2 and PAX8 are both highly sensitive and specific for RCCs. PAX2 and PAX8 are valuable diagnostic markers for metastatic müllerian carcinomas and RCCs in effusion cytology. PAX8 is superior for carcinomas of müllerian origin.

PAX immunoreactivity patterns in anaplastic wilms tumors.

This immunohistochemistry study explores particular differential immuno expression patterns of PAX genes in unfavorable histology Wilms tumor (UH) and favorable histology Wilms tumor (FH). Some unique features in unfavorable histology Wilms tumor were discovered and they may provide insightful clues of this unique and both clinically and biologically important phenomenon.

Value of PAX8, PAX2, claudin-4, and h-caldesmon immunostaining in distinguishing peritoneal epithelioid mesotheliomas from serous carcinomas.

Distinguishing between peritoneal epithelioid mesotheliomas and papillary serous carcinomas involving the peritoneum can be difficult on routine histological preparations, but this differential diagnosis can be facilitated by the use of immunohistochemistry. Recent investigations have indicated that PAX8, PAX2, claudin-4, and h-caldesmon are immunohistochemical markers that can assist in distinguishing between these two malignancies; however, much of the information published on the value of these markers is either insufficient or contradictory. The purpose of this study is to resolve some of the existing controversies and to fully determine the practical value of these markers for assisting in the differential diagnosis between peritoneal mesotheliomas and serous carcinomas. In order to do so, a total of 40 peritoneal epithelioid mesotheliomas and 45 serous carcinomas (15 primary, 30 metastatic to the peritoneum) were investigated. PAX8 and PAX2 nuclear positivity was demonstrated in 42 (93%) and 25 (56%) of the serous carcinomas, respectively, whereas none of the mesotheliomas expressed either marker. Forty-four (98%) of the serous carcinomas exhibited claudin-4 reactivity along the cell membrane, whereas none of the mesotheliomas were positive for this marker. All of the serous carcinomas and mesotheliomas were negative for h-caldesmon. Based on these results, it is concluded that PAX8 and claudin-4 have a higher sensitivity and specificity for assisting in discriminating between peritoneal epithelioid mesotheliomas and serous carcinomas when compared with all of the other positive carcinoma markers that are, at present, recommended to be included in the immunohistochemical panels used in this differential diagnosis. Even though it is highly specific, PAX2 has little practical value in the diagnosis of peritoneal epithelioid mesotheliomas as its sensitivity is low. The h-caldesmon is not useful.

Flat pattern of nephrogenic adenoma: previously unrecognized pattern unveiled using PAX2 and PAX8 immunohistochemistry.

Nephrogenic adenoma is a benign lesion of the urinary tract, particularly the urinary bladder. It is a gross and microscopic mimicker of urothelial neoplasm or metastatic carcinoma. Several histological patterns (tubular, tubulocystic, polypoid, papillary, fibromyxoid) have been recognized, but a flat pattern has not been described. Histologically, nephrogenic adenoma consists of tubules, cysts or papillae lined by flat to polygonal cells with frequent hobnail appearance. The stroma is often edematous or has a granulation tissue-like appearance with acute or chronic inflammation. By immunohistochemistry, nephrogenic adenomas are positive for renal epithelial markers CK7, CD10 and alpha-methylacyl-coenzyme A racemase, and negative for bladder urothelium or prostate markers. Recent studies have shown that nephrogenic adenomas are positive for PAX2 and PAX8. We encountered an interesting case of tubular nephrogenic adenoma with adjacent areas suspicious of flat urothelial atypia. Immunohistochemistry for PAX2 and PAX8 were positive in these areas, unveiling a flat pattern of nephrogenic adenoma. This case prompted us to study 15 cases of nephrogenic adenoma to determine additional instances of flat pattern and to assess the value of PAX2 and PAX8 immunoreactivity to diagnose nephrogenic adenoma. PAX2 and PAX8 immunostaining was positive in 14/15 and 15/15 cases, respectively. The flat pattern was present at least focally adjacent to tubular, polypoid and papillary areas, in 8/15 cases of nephrogenic adenoma. In conclusion, the flat pattern is a common finding in nephrogenic adenomas, but easily under recognized by morphologic examination and may be confused with flat urothelial lesions with atypia. Immunostains for PAX2 and PAX8 are useful in the detection of nephrogenic adenomas and particularly unveil those nephrogenic adenomas with flat pattern.

Comparative immunohistochemical analysis of pulmonary and thymic neuroendocrine carcinomas using PAX8 and TTF-1.

PAX8 is expressed in thymic epithelial neoplasms and a subset of neuroendocrine carcinomas of gastrointestinal origin but not pulmonary neuroendocrine carcinomas. Thyroid transcription factor 1 (TTF-1) is known to be positive in pulmonary neuroendocrine carcinomas, but studies investigating its expression in thymic neuroendocrine carcinomas are lacking. To date, there are no comprehensive studies focusing on the comparative expression of PAX8 or TTF-1 in pulmonary and thymic neuroendocrine carcinoma. Twenty-five cases of low and intermediate grade neuroendocrine carcinomas of pulmonary and thymic origin, respectively, were selected for immunohistochemical studies using antibodies directed against PAX8 and TTF-1. The percentage of positive tumor cells as well as the intensity of staining were evaluated and scored. Twenty-one of the pulmonary neuroendocrine carcinomas were classified as low grade (typical carcinoid) and 4 as intermediate grade (atypical carcinoid) tumors; the thymic tumors consisted of 8 low grade and 17 intermediate grade neuroendocrine carcinomas. Only 2 (8%) of the pulmonary tumors showed nuclear expression of PAX8 while 19 (76%) expressed TTF-1. Of the thymic tumors, 8 (32%) were positive for PAX8 and 2 (8%) showed TTF-1 positivity. Primary neuroendocrine carcinomas of the thymus are rare neoplasms that display a more aggressive clinical course than pulmonary neuroendocrine carcinomas, highlighting the importance of the separation of these tumors. To date, there are no specific immunomarkers to distinguish between neuroendocrine carcinomas of pulmonary and thymic origin. The differential expression of PAX8 and TTF-1 may prove useful in this context as a PAX8+/TTF-1- immunophenotype appears to be more common in thymic neuroendocrine carcinomas, whereas the reverse (PAX8-/TTF-1+) is true for most pulmonary neuroendocrine carcinomas.

PAX8 and PAX5 are differentially expressed in B-cell and T-cell lymphomas.

AIMS: The purpose of this study was to evaluate the expression patterns of B-cell specific activator protein (BSAP)/PAX5 and PAX8 in a wide variety of B-cell and T-cell neoplasms. METHODS AND RESULTS: A wide range of B-cell and T-cell neoplasms were subjected to immunohistochemical staining with antibodies against BSAP/PAX5 and PAX8 (polyclonal, pPAX8; monoclonal, mPAX8). Ten non-neoplastic lymph node specimens were examined with the same panel. All of the tested neoplastic and non-neoplastic B-cells reacted with the BSAP/PAX5 and pPAX8 antibodies, but did not show reactivity with the mPAX8 antibody. All tested T-cell neoplasms were negative using the BSAP/PAX5, pPAX8 and mPAX8 antibodies. CONCLUSIONS: This is the first study to show the absence of reactivity to an mPAX8 antibody in an expanded panel of B-cell lymphomas as well as in a variety of T-cell neoplasms. In contrast to the mPAX8 antibody, the pPAX8 antibody shows nuclear positivity in non-neoplastic B cells and mature B-cell neoplasms; however, this expression is probably a result of cross-reactivity with PAX5. Given that many laboratories use the pPAX8 antibody, a clear understanding of the differential staining patterns is necessary. The differential diagnosis of a B-cell lymphoma should be entertained when a pPAX8-positive, epithelial marker-negative neoplasm of uncertain primary origin is encountered.

The utility of PAX8 and IMP3 immunohistochemical stains in the differential diagnosis of benign, premalignant, and malignant endocervical glandular lesions.

UNLABELLED: Glandular lesions of the endocervix can be diagnostically challenging and occasionally the differential diagnosis includes endocervical adenocarcinoma in situ (EC AIS) and well-differentiated endocervical adenocarcinoma (ECA). PAX8 and IMP3 are two markers which have not been well studied in the endocervix. Our aim was to evaluate their immunohistochemical (IHC) expression in benign and malignant endocervical glandular lesions as well as to compare them to the traditionally used panel (Ki-67, p16, CEA). DESIGN: We searched our surgical pathology files for a cohort of benign endocervical glandular lesions as well as premalignant and malignant groups including EC AIS and ECA. An IHC panel consisting of PAX8, IMP3, Ki-67, p16, and CEA was performed on all cases. Immunoreactivity was scored on a degree of positivity (S0=no immunoreactivity, S1=up to 10% cells, S2=between 10 and 50% cells, S3=>50% cells) and intensity (Int0 - absent, Int1 - mild/faint, Int2 - moderate, Int3 - strong). RESULTS: PAX8 showed diffuse positivity (S3) with at least a moderate intensity of staining (Int2) in the benign group. PAX8 was focal (S1) in ECA and faint (Int1), compared to EC AIS, which was moderate (S2) and faint (Int1). IMP3 expression was focal in the benign group (S1), moderate (S2) in EC AIS and moderate-to-diffuse (S2-3) in ECA. IMP3 intensity was faint (Int1) in benign lesions, moderate (Int2) in EC AIS, and strong (Int3) in ECA. Significant Ki-67, p16, and CEA expression was noted in the premalignant/malignant cohort. CONCLUSION: PAX8 and IMP3 can be helpful in the differential diagnosis of benign vs. malignant endocervical glandular lesions. Our study, however, shows that there is some degree of overlap of staining in both the benign and malignant group. As such, PAX8 and IMP3 should always be interpreted with caution and in combination with the histomorphology.

Quantification of ovarian cancer markers with integrated microfluidic concentration gradient and imaging nanohole surface plasmon resonance.

Nanohole array-based biosensors integrated with a microfluidic concentration gradient generator were used for imaging detection and quantification of ovarian cancer markers. Calibration curves based on controlled concentrations of the analyte were created using a microfluidic stepped diffusive mixing scheme. Quantification of samples with unknown concentration of analyte was achieved by image-intensity comparison with the calibration curves. The biosensors were first used to detect the immobilization of ovarian cancer marker antibodies, and subsequently applied for the quantification of the ovarian cancer marker r-PAX8 (with a limit of detection of about 5 nM and a dynamic range from 0.25 to 9.0 µg.mL(-1)). The proposed biosensor demonstrated the ability of self-generating calibration curves on-chip in an integrated microfluidic platform, representing a further step towards the development of comprehensive lab-on-chip biomedical diagnostics based on nanohole array technology.