Zyto-Facts 1-2020News for Pathology and Immunohistochemistry

104. Jahrestagung der Deutschen

Gesellschaft für Pathologie 04 – 06 June 2020 Hotel Estrel Berlin, Germany

Carrefour Pathology 2020 02 – 05 November 2020 Palais des Congrès Paris, Paris, France

EditorialI am pleased to present the current issue of our newsletter Zytofacts, in which we again cover a broad area of anatomical pathology. An often overlooked parameter of the immunohistochem-ical staining process is the use of the optimal diluent. Examples of the most efficient antibody/diluent pairings are shown in the article on page 2.

So called personalized medicine will continue to be a major topic in the coming years. Two articles address issues related to the screening of patient tissue with PD-L1 and ROS1 antibod-ies. We provide an IHC protocol for the establishment of PD-L1 clone CAL10 for the screening of patients that could potentially benefit from the therapy with the immune checkpoint inhibitor Pembrolizumab. Our optimal results in the NordiQC quality scheme show clearly that lab devel-oped tests (LDT) can match expensive companion diagnostic tests. Our launch of the ROS1 cell control array provides a tool for the quality management of ROS1 immunohistochemistry.

The long awaited p16INK4a is available on the market again as a CE/IVD classified antibody. On page 5 we outline background information about the signalling pathways involved.

An additional article discusses the use of our new Pan Trk antibody for the screening of different tumor tissue for NTRK-fusions.

Enjoy the reading, Karl-Georg Lintermann

Dr. Karl-Georg Lintermann Export manager

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Suggested reading for the topic of colorectal markers in mucinous tumors

Brettfeld S M et al. Archives of Pathology & Laboratory Medicine 143:1119-1125, 2019

SATB2 versus CDX2: A Battle Royale for Diagnostic Supremacy in Mucinous Tumors.

Brettfeld and colleagues describe the pros and cons of these two important markers for colorectal origin. SATB2 and CDX2 immunohistochemical stains were performed on whole sections from 44 mucinous colorectal carcinomas and 175 non-colorectal mucinous tumors. Both antibodies were regarded as accurate markers for colorectal origin. They especially highlight the importance of different cut offs for each antibody in the diagnostic routine. Open access to the full text under the following link: https://www.archivesofpathology.org/doi/full/10.5858/arpa.2018-0337-OA

Description Cat. No.

MSH2 (FE11) MSK031-05

MSH6 (SP93) 513-3934

PAX-8 (BC12) MSK107-05

GATA3 (L50-823) MSK100-05

Optimized formulations of antibody diluents can improve the antibody performance. The per-fectly matched antibody diluent often provides greater dilution, higher specificity, and en-hanced stability for storage. A variety of differ-ent antibodies showed enhanced staining and

reduced background in immunohistochemistry when diluted with our new Antibody Diluent C. In our test series we found significant enhance-ment of the signal in comparison to our stan-dard Antibody Diluent (ZUC025) for the following antibodies:

We continue to recommend our Antibody Diluent B when working with MLH1 (MSK045-05).

Description Status Format Volume Cat.No.

Antibody Diluent CE/IVD Ready-to-use100 ml ZUC025-100

500 ml ZUC025-500

Antibody Diluent B CE/IVD Ready-to-use25 ml ZUC051-025

100 ml ZUC051-100

Antibody Diluent C RUO Ready-to-use25 ml ZUC103-025

100 ml ZUC103-100

Do you use the right diluent?Influence of the antibody diluent on immunostainings

PAX-8 (MSK107-05) diluted 1:50 with Antibody Diluent C (ZUC103) on renal cell carcinoma

PAX-8 (MSK107-05) diluted 1:50 with Antibody Diluent (ZUC025) on renal cell carcinoma

MSH6 (513-3934) diluted 1:100 with Antibody Diluent (ZUC025) on colon carcinoma

MSH6 (513-3934) diluted 1:100 with Antibody Diluent C (ZUC103) on colon carcinoma

Description Cat. No.

CD10 (56C6) MSK070

PD-L1 (CAL10) RBK063-05

Napsin A (BC15) RBK059-05

TTF-1 (SPT24) MSK111-05

Product information

The NordiQC Companion module (C5) focused on the accuracy of the PD-L1 IHC assays in order to identify patients with non-small cell lung carcinoma (NSCLC) or urothelial carcinomas that benefit from immune therapy with pembrolizumab (Keytruda®).PD-L1 expression levels in the circulated material used for the assessment were primarily characterized by the CE/IVD approved companion diagnostic IHC as-say, 22C3 pharmDx, SK006 Dako/Agilent based on the tumour proportion score (TPS; first line treatment TPS ≥ 50 %, second line treatment TPS ≥ 1 %) for NSCLC

and on a combined positive score (CPS) indicating a CPS of ≥10 as cut-off level for being “positive” and CPS <10 being “negative” for urothelial carcinomas. Both slides were stained in our lab using the following protocol: Pre-treatment at 110° (DC-Module, Biocare Medical) for 7 min in citrate buffer (ZUC028), antibody PD-L1, clone CAL10 (RBK063-05), dilution 1:100, incuba-tion for 45 min at room temperature, two step poly-mer detection kit (POLHRP100) and DAB High contrast (DAB5000PLUS). The incubation steps were performed according to the datasheet. We recommend using our

new Antibody Diluent C (ZUC103) for dilution. Other protocols that were assessed as optimal in run C5 used EDTA buffer for pre-treatment. Both PD-L1 (lung) and PD-L1 (uro) slides provided optimal assessments.These results clearly show that concentrated antibod-ies like the clone CAL10 used in optimized laboratory developed assays show comparable results to ap-proved companion diagnostic assays.You can download our individual results of the last NordiQC runs from our webpage: www.zytomed-sys-tems.com/downloads/certificates/round-robin-trial).

NordicQC results for PD-L1 Clone CAL10 in external quality scheme

Description Status Format Dilution Volume Cat. No.

PD-L1 (CD274)Clone: CAL10Host: Rabbit

CE/IVDReady-to-use - 6 ml RBG063

Concentrate 1:100 – 1:200 0.5 ml RBK063-05

Tissue PD-L1 reaction pattern*1 Placenta Positive control2 + 3 Tonsil Positive control4 NSCLC No; <1 %5 NSCLC No; <1 %6 NSCLC Low; ≥1-49 %7 NSCLC Low; ≥1-49 % / High ≥50 %**8 NSCLC Low; ≥1-49 %9 NSCLC Excluded (too few tumour cells)10 NSCLC High; ≥50 %11 NSCLC Low; ≥1-49 % / High ≥50 %**12 NSCLC Low; ≥1-49 % / High ≥50 %**13 NSCLC High; ≥50 %

Tissue PD-L1 reaction pattern*1 Placenta Positive control2 + 3 Tonsil Positive control4 Urothelial carcinoma ≥10 (Immune cells)5 Urothelial carcinoma <106 Urothelial carcinoma <107 Urothelial carcinoma ≥10 (Immune cells + tumour cells)8 Urothelial carcinoma ≥10 (Tumour cells)9 Urothelial carcinoma ≥10 (Tumour cells)10 Urothelial carcinoma ≥10 (Immune cells + tumour cells)

Scans of stained TMAs used for the NordiQC PD-L1 C5 assessment

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4

7

2

5

8

3

6

9 10

1

4

7

2

5

8

3

6 9

10 11 12 13

* Tumor proportion score (TPS) determined by PD-L1 IHC 22C3, SK006 Dako performed in NordiQC reference lab.

** These tumor cores displayed tumor heterogeneity. Depending on the blocks and reference PD-L1 levels from which the sectioned slides were circulated, the level of PD-L1 was categorized into one of the percentage ranges (TPS) highlighted in the table.

* Combined positivity score (CPS) determined by PD-L1 IHC 22C3, SK006 Dako performed in NordiQC reference lab.

NordicQC results for PD-L1 Clone CAL10 in external quality scheme

Bibliography

[1] Uguen A, de Brakeleer M. ROS1 fusions in cancer: a review. Future Oncol 12:1911-1928, 2016

[2] Mazières J et al. Crizotinib therapy for advanced lung adenocarcinoma and a ROS1 rearrangement: results from the EUROS1 cohort. J Clin Oncol 33:992-999, 2015

[3] Shaw AT et al. Crizotinib in ROS1-rear-ranged non-small-cell lung cancer. N Engl J Med 371:1963-1971, 2014

[4] Bubendorf L et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 469:489-503, 2016

[5] Selinger CI et al. Screening for ROS1 gene rearrangements in non-small-cell lung cancers using immunohis-tochemistry with FISH confirmation is an effective method to identify this rare target. Histopathology 70:402– 411, 2017

[6] Luk P et al. Biomarkers for ALK and ROS1 in Lung Cancer. Arch Pathol Lab 142: 922-928, 2018

ROS1 is a receptor tyrosine kinase (TK) with struc-tural similarity to the anaplastic lymphoma kinase (ALK) protein. ROS1 translocations define a subset of patients with non-small cell lung carcinoma (NS-CLC). About 1 – 2 % of NSCLC cases have a ROS1 rear-rangement leading to overexpression of a chimeric ROS1 protein with constitutional TK activity, result-ing in unregulated cell growth [1]. Clinical studies and case reports show that treatment with tyrosine kinase inhibitors such as crizotinib and ceritinib rep-resents an effective therapeutic strategy in patients with activating ROS1 rearrangements [2,3]. Due to the low incidence of ROS1 translocations, a screen with anti ROS1 antibodies on NSCLC tissue sections is often performed, followed by confirma-tory FISH analysis of ROS1 positive tissue. A number of studies have shown a good correla-tion between ROS1 FISH and ROS1 IHC with report-ed sensitivities of 94 % to 100 % and specificities of 76 % – 100 %. The different results obviously de-pend, at least in part, on the cutoffs used to deter-mine positivity [4-6]. ROS1 IHC tests have several pitfalls which could lead to false positive results. EGFR-mutant tumors can show strong staining as well as mucinous adenocarcinomas which show a more granular staining pattern. Positive staining

can occur on reactive pneumocytes, alveolar mac-rophages and giant cells. Due to the subjective interpretation of staining inten-sities and inter-observer variability of ROS1 IHC, an ad-ditional FISH or a molecular analysis is recommended in weak or focal cases, especially in medical facilities with less experienced physicians [6].The establishment of a ROS1 immunohistochemistry as well as its routine use in diagnostics requires a validated process using appropriate controls [4]. The Cell Control Array ROS1 (IHC) block serves as a posi-tive control for the detection of ROS1 protein in lung and other tissues. It is a homogenous paraffin block including two ROS1 positive cell lines, one ROS1 neg-ative cell line, and one core of heart muscle tissue. The two positive ROS1 cell lines differ in their lev-el of ROS1 expression, showing weak and medium expression, respectively. In addition, the Cell Control Array ROS1 can be used for the detection of mRNA of a CD74-ROS1 fusion using real-time PCR.The small size of the control block sections allows for simultaneous mounting of patient material sections and control block sections on the same slide. Thus, you will have an on-slide control array staining (OSCAR) proving a regular stain even after years of storage.

Description Form Volume Cat. No.

Cell Control Array ROS1 (IHC) 1 Block2 ROS1 positive cell lines, 1 ROS1 negative cell line,1 core of heart muscle tissue

MB-CC ROS1

Product information

Cell Control Block ROS1 (IHC) for the establishment, validation and quality control of ROS1 immunohistochemistry

Protocol

• Pre-treatment in EDTA-Buffer (ZUC029-500), 35 min, steamer

• ROS-1, clone D4D6 (Cell Signaling Technology)

• dilution 1:100 for 60 minutes at RT• Polymer detection Kit POLHRP-100

(25 min / 30 min)• DAB High Contrast (DAB500PLUS)

10 min

negative

Heart muscle ROS1 medium ROS1 low

Description Status Format Dilution Volume Cat. No.

p16INK4a

Clone: JC2Host: Mouse

CE/IVDReady-to-use - 6 ml MSG123

Concentrate 1:50 – 1:100 0.5 ml MSK123-05

Bibliography

[1] Serrano M et al. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366:704-707, 1993

[2] Sherr CJ et al. D-type cyclins and their cyclin-dependent kinases: G1 phase integrators of the mitogenic response. Cold Spring Harb Symp Quant Biol 59:11-19, 1994

[3] Mirzayans R et al. Role of p16INK4a

in Replicative Senescence and DNA Damage-Induced Premature Senes-cence in p53-Deficient Human Cells. Biochem Res Int 2012:951574, 2012

[4] Rocco JW & Sidransky D. p16 (MTS-1/CDKN2/INK4a) in cancer progression. Experimental Cell Research 264:42-55, 2001

[5] Li J et al. The Regulatory Mechanisms of Tumor Suppressor p16INK4a and Relevance to Cancer. Biochemistry 50:5566-5582, 2011

[6] Lukas J et al. Retinoblastoma-pro-tein-dependent cell-cycle inhibition by the tumour suppressor p16. Nature 375:503-506, 1995

[7] Shain AF et al. Test Characteristics of Specific p16 Clones in the Detection of High-grade Squamous Intraepithe-lial Lesions (HSIL). Int J Gynecol Pathol 37:82-87, 2018.

The p16INK4a protein, also known as Cyclin Depen-dent Kinase Inhibitor 2A, was discovered in the early 1990s and has been studied intensively since then because of to its ability to influence the pro-gression of the cell cycle at the transition from the G1 to the S phase [1]. The 16 kDa protein was de-scribed as a specific inhibitor of cyclin-dependent kinase 4 (Cyclin Dependent Kinase 4, CDK4) - INK4 stands for Inhibitor of Cyclin Dependent Kinase 4.Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) regulate the cell cycle at the end of the G1 phase. During the G1 phase, the cyclin D level in the cell rises, cyclin D binds to CDK4 and CDK6, and the resulting cyclin/CDK protein complexes phos-phorylate the retinoblastoma protein (Rb). The phosphorylation inactivates the Rb protein and transcription factors of the E2F family are released and mediate the expression of a large number of proteins such as Cyclin E, Cyclin A and the thymi-dine kinase that are essential for the progression of the cell cycle and for DNA synthesis [1, 2].p16INK4a binds to CDK4 and/or CDK6 and inhibits the catalytic activity of the cyclin/CDK complexes. Rb remains inactive and binds and inhibits E2F, which prevents the cells from entering the S phase of the cell cycle and thereby prevents cell proliferation. Numerous other functions have been described for p16INK4a over time: e.g., it conveys the MDM2-de-pendent degradation of p53, suppresses the ki-nase activity of c-Jun-N-terminal kinases (JNK), is involved in the regulation of AKT/ Survivin signaling and represses the transcription of numerous genes, such as RB, TP53, VEGF (Vascular Endothelial Growth Factor), MMP-2 (Matrix Metalloproteinase 2) or NF-κB [3]. It is therefore not surprising that p16INK4a plays a critical role in central processes such as cell cycle, senescence and apoptosis.

p16INK4a is encoded by the CDKN2A gene, one of the most studied tumor suppressor genes. Muta-tions, DNA methylation and homozygous or het-erozygous gene loss can influence the expression of the CDKN2A gene. Post-translational modifica-tions modulate the functionality and activity of the protein. A reduction in activity up to the complete inactivation of the tumor suppressor p16INK4a in-volved in the regulation of the G1 phase is a com-mon and early event in the pathogenesis of solid tumors [4, 5].While in these tumors the p16INK4a protein is missing, or the expression level can be very diverse, over-expression of the protein can be found in tumor cells of HPV-induced carcinomas [6]. The already described Rb and transcription factor E2F complex also inhibits, among others, the transcription of the CDKN2A gene and thus the expression of p16INK4a. If the viral oncoprotein E7 is expressed as a result of an HPV infection, it binds and inactivates the Rb protein. E2F transcription factors are thus released, p16INK4a is expressed and accumulated in the cell, and yet the cell continues to go through the cell cycle unhindered [5, 6].Zytomed Systems’ antibody against p16INK4a, clas-sified as CE/IVD, is used to localize the p16INK4a

protein in tissue sections of formalin-fixed and paraffin-embedded tissue. The antibody was intensively tested in our own as well as in in-dependent pathology laboratories using dif-ferent manual and automatized methods and protocols. Clone JC2, also known as 16P04/JC2 or 16P04, outperformed the commonly used clone E6H4 in a meta-analysis of 32 papers addressing the specificity, sensitivity, positive predictive value, and negative predictive value of different p16 clones [7].

Product information

Clone JC2 a CE/IVD classified antibody for the detection of p16INK4a

p16INK4a, clone JC2, on Cervical Intraepithelial Neoplasia (CIN3), pre-treatment T-EDTA pH 9.0, dilution 1:50

Protocol

• Pre-treatment in EDTA-Buffer (ZUC029-500), 35 min, steamer

• ROS-1, clone D4D6 (Cell Signaling Technology)

• dilution 1:100 for 60 minutes at RT• Polymer detection Kit POLHRP-100

(25 min / 30 min)• DAB High Contrast (DAB500PLUS)

10 min

NL_E_1_2020

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

Pan TrkEfficient and reliable screening for NTRK fusions by immunohistochemistry

The family of neurotrophic tyrosine kinase (NTRK1/2/3) genes encodes the TrkA, TrkB and TrkC protein kinases. The three family members are ac-tivated by different neurotrophins like NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-4 (neurotrophin-4), and NT-3 (neurotro-phin-3). Neurotrophin signalling activates cellular pathways involved in the development and matu-ration of the central and peripheral nervous systems through regulation of proliferation, differentiation, and survival of sympathetic and nervous neurons. Fusions of the receptor tyrosine kinases genes NTRK1, NTRK2 and NTRK3, which result in overex-pressed and constitutively active Trk fusion pro-teins, are present in a variety of different malig-nancies. NTRK fusions with various partners are highly recurrent in certain rare malignancies, such as secretory carcinoma of the breast or salivary gland and infantile fibrosarcoma, but they are also infrequently seen in some common cancers, such

as melanoma, glioma and carcinomas of the thy-roid, lung and colon. Expression of the wild-type Trk protein is seen in some neuroendocrine tumors but is absent or extremely low in most solid tumors. With the approval of specific Trk inhibitors like larotrectinib, the assessment of NTRK fusion be-came a routine method for patients with locally advanced or metastatic cancers [1]. Several studies using Pan Trk antibodies on different tumor tissues showed a high sensitivity and specificity for NTKR fusions ranging from 79 % to 100 % respectively [1-3]. The European Society for Medical Oncology (ESMO) therefore recommends either front-line sequencing (preferentially RNA-sequencing) or immunohistochemistry as methods for screening, especially in tumors where NTRK1/2/3 fusions are uncommon [4].We now offer the rabbit monoclonal primary anti-body EPR17341 which is directed against a common part of the C-terminal region of the Trk proteins.

March-2020

Product information

Prevalence of NTKR fusions in various tumors

Bibliography

[1] Solomon JP et al. NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls. Mod Pathol. 33:38-46, 2020

[2] Hechtman J et al. Pan-Trk Immunohis-tochemistry Is an Efficient and Reliable Screen for the Detection of NTRK Fusions. Am J Surg Pathol. 41:1547–1551, 2017

[3] Rudzinski ER et al. Pan-Trk immunohis-tochemistry identifies NTRK rearrange-ments in pediatric mesenchymal tu-mors. Am J Surg Pathol 2018, 42:927-935

[4] Marchiò C et al. ESMO recommen-dations on the standard methods to detect NTRK fusions in daily practice and clinical research. Ann Oncol. 301417-1427, 2019

Description Status Format Dilution Volume Cat. No.

Pan TrkClone: EPR17341Host: Rabbit

RUO Concentrate 1:50 – 1:1000.1 ml RBK069-01

0.5 ml RBK069-05

Tumor Percentage

Inflammatory myofibroblastic tumor 17.7 %

Salivary gland carcinoma 5.08 %Thyroid carcinoma 2.28 %Sarcoma 0.68 %Glioma/neuroepithelial tumor 0.55 %Appendiceal adenocarcinoma 0.48 %

(modified from Solomon et al. Mod Pathol. 33:38-46, 2020)

Tumor Percentage

Melanoma 0.36 %Pancreatic adenocarcinoma 0.34 %Neuroendocrine tumor 0.31 %Colorectal carcinoma 0.31 %Cholangiocarcinoma 0.25 %Lung adenocarcinoma 0.23 %Breast carcinoma 0.13 %