Zyto-Facts 1-2018

News for Pathology and Immunohistochemistry

3. Histologica, Oberhausen

15–16 June 2018 Congress Centrum Oberhausen Oberhausen, Germany

Maastricht Pathology 2018 19–22 June 2018 Maastricht Exibition and Congress Center Maastricht, Netherlands

30e Congrès de l’Association Française d’Histotechnologie 2018 21–22 June 2018 Ecole d‘Ingénieur de Purpan Toulouse, France

30th European Congress of Pathology 8–12 September 2018 Euskalduna Conference Centre 4 Bilbao, Spain

Herbsttagung 2018 der ÖGPath/ IAP Austria 27–28 September 2018 Medizinische Universität Graz Austria

6. Deutsche Pathologietage 26–28 Oktober 2018 Maritim Hotel pro Arte Berlin, Germany

EditorialAccurate diagnoses as well as reliable prognostic and predictive information are the basis for making

confident decision concerning the appropriate individual treatment of cancer patients. Tissue-based

diagnostics like immunohistochemistry are becoming an increasingly important part of this process.

High quality diagnostic reagents along with an in-depth knowledge of the characteristics and quality of

different antibodies and clones as well as of protocols for tissue processing and staining are of essential

importance. This issue includes:

a comparison of the different specificities of “pan” or “broad-spectrum” cytokeratin antibodies

a discussion of the advantages and disadvantages of two TTF-1 antibodies clones

a description of how to process calcified tissue for optimal staining results

Enjoy reading this newsletter!Karl-Georg Lintermann

Dr. Karl-Georg Lintermann PhD, Export manager

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MEDICA 2018 12–15 November 2018 Messe Düsseldorf, Düsseldorf, Germany

Carrefour Pathologie 2018 22–23 November 2018 Palais des congrès Paris, France

Confident in differentiating mucinous ovarian and colorectal adenocarcinoma? SATB2 is the marker of choice

Bibliography

[1] Magnusson K et al. SATB2 in combination with cytokeratin 20 identifies over 95 % of all colorectal carcinomas. Am J Surg Pathol 35:937-948, 2011

[2] Lin F et al. Cadherin-17 and SATB2 are sensitive and specific immunomarkers for medullary carcinoma of the large intestine. Arch Pathol Lab Med 138:1015-1026, 2014

[3] Moh M et al. SATB2 Expression Distinguishes Ovarian Metastases of Colorectal and Appendiceal Origin From Primary Ovarian Tumors of Mucinous or Endometrioid Type. Am J Surg Pathol 40:419-432, 2016

[4] Pérez Montiel D et al. The value of SATB2 in the differential diagnosis of intestinal-type mucinous tumors of the ovary: primary vs metastatic. Ann Diagn Pathol 19:249-252, 2016

[5] Logani S et al. Use of novel immunohistochemical markers expressed in colonic adenocarcinoma to distinguish primary ovarian tumors from metastatic colorectal carcinoma. Mod Pathol 18:19-25, 2005

[6] Vang R et al. Immunohistochemical expression of CDX2 in primary ovarian mucinous tumors and metastatic mucinous carcinomas involving the ovary: comparison with CK20 and correlation with coordinate expression of CK7. Mod Pathol 19:1421-1428, 2006

[7] Yang C et al. Diagnostic Utility of SATB2 in Metastatic Krukenberg Tumors of the Ovary: An Immunohistochemical Study of 70 Cases With Comparison to CDX2, CK7, CK20, Chromogranin, and Synaptophysin. Am J Sur Path 42:160-171, 2018

SATB2 on colon

SATB2 on colon carcinoma

SATB2 on colon carcinoma

SATB2 is a transcription factor involved in the reg-ulation of osteoblastic and neuronal differentiation. Immunohistochemical studies have shown its specif-ic expression in normal colorectal epithelium as well as in colorectal adenocarcinomas. Antibodies against SATB2 are recommended for inclusion in a panel of CK7, CK20 and CDX2 in order to identify the primary origin of poorly differentated adenocarcinoma [1, 2].Two recent studies address the value of SATB2 in the differential diagnosis of primary ovarian muci-nous adenocarcinoma and metastasis of colorectal/appendical adenocarcinoma [3, 4]. Determination of the primary can be challenging because metas-tasis of extraovarian origin may mimic histological and immunohistochemical features of some prima-ry ovarian mucinous tumors. In particular, several studies have shown a significant portion of primary ovarian adenocarcinomas to be positive with the colorectal marker CDX2 [5, 6]. In contrast, SATB2 immunoreactivity was not de-tected in ovarian adenocarcinomas lacking mature teratoma. SATB2 expression in ovarian metastasis could, however, be detected in 75% of colorectal

adenocarcinomas and 86 % of high and low grade appendiceal adenocarcinomas. No SATB2 expression was found in ovarian metastasis of pancreatic, gas-tric, gallbladder, or endocervical origin [3]. In another study, all 80 primary ovarian tumors not associated with cystic teratoma were SATB2 nega-tive and all 20 metastatic ovarian tumors of the co-lon and appendix were positive for SATB2 [4].In both studies, SATB2 was highly specific for colorec-tal and appendiceal metastases versus primary ovar-ian mucinous tumors with the exception of SATB2 expression found in teratomas of ovarian primaries. A new study investigated the diagnostic utility of SATB2 in metastatic Krukenberg tumors (MKTs), which is the third most frequent metastasis in the ovary. Immunohistochemical staining with SATB2 antibodies on 70 MKTs of various origins were per-formed. They describe SATB2 as a useful marker for determining the primary sites of MKTs of the ovary. In particular, SATB2 was the best marker for distinguishing MKT-metastatic adenocarcinomas ex goblet cell carcinoids [AdexGCC] from MKTs-stom-ach and MKTs-colorectum [7].

Description Status Format Dilution Amount Cat. No.

SATB2 Clone: SATBA4B10 Host: Mouse

RUO Concentrate 1:50 - 1:100 0.5 ml MSK101-05

Product information

Cytokeratins (CK) are intermediate filaments that constitute the cytoskeletal structure of virtually all epithelial but also of some non-epithelial cells. They have been classified into basic (CK1 to 8) and acid (CK9 to 20) subfamilies and may also be distin-guished by their molecular weights and their tissue distribution. Immunohistochemistry using antibodies against cytokeratins helps to classify undifferentiated neoplasia and metastases of unknown origin. Varia-tions exist regarding sensitivity and specificity of the various broad spectrum or „pan“ cytokeratin antibod-ies. The well-known broad spectrum CK antibody AE1 & AE3 is a mixture of two different clones of anti-cy-tokeratin monoclonal antibodies, AE1 and AE3. Clone AE1 detects the high molecular weight cytokeratins 10, 14, 15, and 16, as well as the low molecular weight cytokeratin 19. Clone AE3 detects the high molecular weight (HMW) cytokeratins 1, 2, 3, 4, 5, and 6, as well as the low molecular weight (LMW) cytokeratins 7 and 8. It is notewothy that hepatocellular carcinomas are characteristically negative or only focally weakly positive with anti-cytokeratin AE1 & AE3. This is due

to the fact that AE1 & AE3 does not recognize cytoker-atin 18, which is expressed in most ductal and glan-dular epithelia. In addition, renal cell carcinomas and sometimes adrenal, prostate, and neuroendocrine carcinomas can be negative with AE1 & AE3 [1 – 4].In order to overcome the low sensitivity of AE1 & AE3 in certain carcinomas, Zytomed Systems created the antibody Cytokeratin Pan Plus by adding the antibody clone 5D3 to the cocktail of clones AE1 and AE3. 5D3 detects CK8 and 18.Two other „Pan“ clones were launched recently by Zy-tomed Systems, Cytokeratin Pan OSCAR and Cytoker-atin Pan, clone KL1. A recent publication showed the superiority of clone OSCAR in detecting metaplastic breast carcinoma (MBC), especially spindle cell carci-nomas [5]. Clone KL1 displays the broadest reactivity detecting cytokeratins 1, 2, 5, 6, 7, 8, 10, 11, 14, 16, 17, 18, and 19*.[6]Cytokeratin Pan Plus, KL1 and OSCAR react with a broad range of cytokeratins and therefore detect most liver and kidney carcinomas and are likely to detect more micrometastases in lymph nodes.

Bibliography

[1] Moll R et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11-24, 1982

[2] Ordóñez NG. Broad-spectrum immunohistochemical epithelial markers: a review. Hum Pathol 44:1195-1215, 2013

[3] Chu PG and Weiss LM. Keratin expression in human tissues and neoplasms. Histopathol 40:403-439, 2002

[4] Wells CA et al. The immunocytochemical detection of axillary micrometastases in breast cancer. Brit J Cancer 50:193-197, 1984

[5] Galera P et al. Diagnosis of Metaplastic Breast Carcinoma: Keratin OSCAR Versus Other Cytokeratins. Appl Immunohistochem Mol Morphol 24:622-626, 2016

[6] Viac J et al. Reactivity pattern of a monoclonal antikeratin antibody (KL1). J Invest Dermatol 81:351-354, 1983

Cytokeratin (Pan), clone KL1, on renal cell carcinoma

* Not available in all countries. Please contact your local distributor. | Reactivity according to Ordóñez (2013): 1, 2, 5, 6, 7, 8, 11, 14, 16, 17, 18

Reactivity with Cytokeratins

basic (type II) acidic (typeI)

Description Dilution Volume Cat. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Cytokeratin (Pan) Clone: AE1 + AE3Host: Mouse

-6 ml MSG019

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓16 ml BMS006

1:100 – 1:2000.5 ml MSG019-051 ml MSK019

Cytokeratin (Pan Plus) Clone: AE1 + AE3 + 5D3Host: Mouse

- 6 ml MSG098✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

1:50 – 1:100 0.5 ml MSG098-05

Cytokeratin (Pan)*Clone: KL1Host: Mouse

- 6 ml MSG113

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓1:100

0.5 ml MSK113-05

1 ml MSK113

Cytokeratin (OSCAR)Clone: OSCARHost: Mouse

- 6 ml Z-465-26-Y✓ ✓ ✓ ✓

1:40 1 ml Z-465-01-Y

Zytomed Systems‘ antibodies against Cytokeratins (selection)

„Broad spectrum“ or „Pan“ CK antibodies – Clones in comparison

Focus: lab workDecalcification of tissue specimensIn routine histopathology, decalcification of bone and other calcified tissue is a prerequisite for the suitability of a specimen for the embedding proce-dure, for cutting, and for subsequent diagnostic tech-niques. Decalcification is usually carried out between fixation and routine processing to paraffin.Three main types of decalcifying agents are alter-natively based on strong mineral acids, weaker or-ganic acids or those composed of chelating agents like EDTA (e. g. Titriplex).EDTA works by capturing the calcium ions from mineralized tissue. The process is slow but very gentle and should be the method of choice for im-munohistochemistry, in situ-hybridization or PCR.Decalcification is a standard procedure in many histopathology labs and is always a compromise between speed and optimal morphology of the tis-sue. Aspects of the process that are important for achieving high quality results are detailed in the following paragraphs.EDTA is depleting in the decalcification process by binding with calcium: Instead of increasing the EDTA concentration of the decalcification buffer (which would affect tissue morphology), we rec-ommend using a large volume and renewing it sev-eral times during the process.

Increasing temperature during the process will speed up the procedure at the expense of tissue quality. According to our experiences, during incu-bation the temperature should not exceed 37 °C. Decalcification works better at high pH (up to pH 10) but tissue preservation will be not as good as with the standard pH 7.0. Gentle agitation may in-crease the decalcification rate slightly.Determination of the end-point of decalcification is an important step. Incompletely decalcified tis-sue may be difficult to section (with damage to a microtome possibly occurring), whereas over-de-calcification compromises morphology and impairs subsequent staining.X-ray and chemical testing using ammonium oxalate solutions are accurate but not routinely used in labs to prove complete decalcification. A physical test like bending the tissue or inserting a pin or razor blade into the specimen is not very precise and there is a risk of damaging the specimen, especially when done by inexperienced personnel. However, it’s quick, con-venient, and sufficient in most cases.When proceeding with standard embedding steps keep in mind that decalcified tissue could contain denser areas and thus requires a protocol that uses longer rather than shorter incubation times.

Suggested reading for the topic of SATB2 as a marker of colorectal origin

Kyra B. Berg, MD; David F. Schaeffer, MD, FRCPC

SATB2 as an Immunohistochemical Marker for Colorectal Adenocarcinoma: A Concise Review of Benefits and PitfallsFrom the Department of Pathology, University of British Columbia, Vancouver, Canada (Dr Berg) and the Department of Pathology, Vancouver General Hospital, Vancouver, British Columbia, Canada (Dr Schaeffer).

Berg and Schaeffer provide an overview of the current literature and summarize their practical experiences with SATB2. They describe SATB2 as a robust colorectal and appendiceal marker if used as part of a panel approach and as being more specific (but less sensitive) than CDX2 for an adenocarcinoma of unknown origin.

Open access to the full text under the following link: https://doi.org/10.5858/arpa.2016-0243-RS

Our lab recommends the following protocol for iliac crest punctures

Decalcification solution: •Prepare 10 % Formalin by mixing 200 ml of 37% Formalin + 940 ml Aqua dest. •Stir for 24h at 60 °C •Adjust pH to 7.0 with 40 % NaOH, store in the dark •Dissolve 135 g EDTA (Titriplex III) in 900 ml 10 % Formalin •Incubate the specimen for about 8 h at 37 °C in decalcification solution.

CD138 on iliac crest (plasmocytoma)

CD20 on iliac crest

Pax5 on iliac crest

Focus: lab work

Product information

Thyroid Transcription factor 1 (TTF-1) a member of the NKX2 family of transcription factors is a tissue specific transcription factor, expressed in the forebrain, thy-roid and lung. TTF-1 plays a key role in morphogenesis of the lungs. Antibodies to TTF-1 are widely used in pathology diagnostics to identify lung and thyroid or-igin, not least because TTF-1 retains its high specificy and rather good sensitivity in metastatic and poorly differentiated tumors.As a result of new therapies available for lung cancer, differentiation of pulmonary adenocarcinoma (ADC) vs squamous cell carcinomas of the lung (SqCC) has become more and more important in the clinical man-agement of lung cancer patients. TTF-1 is the classical marker for ADC in this setting as part of a panel that includes p40, Cytokeratin 5 & 6 and Napsin A.In addition to the well established clone 8G7G3/1, Zytomed Systems recently added clone SPT24 to

their portfolio. Several studies have shown that both clones have different sensitivities for ADC and SqCC. In general, SPT24 is regarded as more sensitive for ADC whereas 8G7G3/1 is more specific [1, 2]. The higher sensitivity of clone SPT24 is reflected in the results of the external quality scheme NordicQC. In run 46, 2016, clone SPT24 is described as a „very robust and sensi-tive“ marker for the demonstration of TTF-1 with 90% of all participants achieving good or optimal results. The number of false positive staining of SPT24 in SqCC can be significantly reduced by optimizing the titer and by using appropriate cut-offs [3]. In contrast to SPT24, clone 8G7G3/1 shows additional reactivity with hepatocytes and hepatocellular car-cinomas (HCC) and therefore can be useful in distin-guishing HCC from histological mimics. Immunohisto-chemistry using 8G7G3/1 in HCC results in a granular cytoplasmic stain pattern.

TTF-1 antibodies – A comparison of clones 8G7G3/1 and SPT24

Bibliography

Masai K et al. Expression of squamous cell carcinoma markers and adenocarcinoma markers in prima-ry pulmonary neuroendocrine carcinomas. Appl Immunohistochem Mol Morphol 21:292-295, 2013

Matoso A et al. Comparison of thyroid transcription factor-1 expression by 2 monoclonal antibodies in pulmonary and nonpulmonary primary tumors. Appl Immunohistochem Mol Morphol 18:142-149, 2010

Tacha D and Zhou D TTF-1 [SPT24] Staining Specificity in Lung Adenocarcinoma vs. Lung Squamous Cell Carcinoma is Markedly Improved with Titer Optimization and Cut-Off Values. USCAP 2015, Biocare Medical, Concord, www.biocare.net

TTF-1 staining on pulmonary adenocarcinoma

TTF-1 staining on thyroid gland

Cytoplasmatic staining of TTF-1 (8G7G3/1) on hepatocytes

Description Status Format Dilution Volume Cat. No.

TTF-1Clone: SPT24Host: Mouse

CE/IVDReady-to-use - 6 ml MSG111

Concentrate 1:100 – 1: 200 0.5 ml MSK111-05

TTF-1Clone: 8G7G3/1Host: Mouse

CE/IVDReady-to-use -

6 ml MSG00416 ml BMS016

Concentrate 1:200 - 1:5000.5 ml MSK004-051 ml MSK004

p40Clone: BC28Host: Mouse

CE/IVDReady-to-use -

6 ml MSG09716 ml BMS050

Concentrate 1:200 - 1:5000.5 ml MSK097-051 ml MSK097

Napsin AClone: BC15Host: Mouse

CE/IVDReady-to-use - 6 ml RBG059

Concentrate 1:100 – 1: 200 0.5 ml RBK059-05

Cytokeratin 5 & 6Clone: D5/16B4Host: Mouse

CE/IVDReady-to-use -

6 ml MSG03416 ml BMS017

Concentrate 1:50 - 1:1000.5 ml MSK034-051 ml MSK034

NL_E_1_2018

Your local contact:

contact

ZYTOMED SYSTEMS GmbH Anhaltinerstraße 16 14163 Berlin | Germany Fon +49 30 804 984 990 Fax +49 30 804 984 999international@zytomed-systems.de www.zytomed-systems.com

Bibliography

[1] World Health Organization (WHO). Her-pes simplex virus. http://www.who.int/mediacentre/factsheets/fs400/en Accessed May 13, 2016

[2] Adams RL et al. The immunocyto-chemical detection of herpes simplex virus in cervical smears – a valuable technique for routine use. J Pathol 143:241, 1984

[3] Tomita T et al. Identification of herpes simplex virus infection by immu-noperoxidase and in situ hybridization methods. Virchows Arch A Pathol Anat Histopathol 419:99-105, 1991

Herpes simplex virus type I (HSV I, which mostly induces “cold sores”) and Herpes simplex virus type II (HSV II, which mostly causes genital herpes) are two highly contagious members of the herpesvirus family that infect humans. About 3.7 billion people are infected with HSV I and 417 million are infected with HSV II under the age of 50 [1].HSV infection commonly affects mucoepithelia or skin. HSV can infect a large variety of organs includ-ing brain, lung, liver, GI-tract and adrenal glands and lead to life-threatening conditions especially in cases involving immunosuppressed patients. A fast and re-liable diagnosis increases a patient´s odds of survival.

Zytomed Systems’ new antibody cocktail reacts with HSV type I and II specific antigens and with antigens common to HSV type I and II virus. It reacts with all the major glycoproteins present in the viral envelope as well as with at least one core protein [2, 3].The Cell Control Array Virus (MB-CC VIR) containing cell lines infected with CMV, HSV type I and type II, EBV, and Polyomavirus/SV40 is perfectly suited as a positive control for immunohistochemistry us-ing the HSV Cocktail. The small size of the control block sections allows patient material sections and control block sections to be mounted on the same slide.

HSV I & II – An Antibody Cocktail against Herpes simplex

Description Status Format Dilution Amount Cat. No.

HSV Cocktail Clone: polyclonal + DBM15.69Host: Rabbit + Mouse

CE/IVD Ready to use - 6 ml COG006

Description Status Amount Cat. No.

Cell Control Array Virus Research use only 1 Block MB-CC VIR

Product description

EBV

Muscle HSV I HSV II SV40

CMV

Mai-2018