Cancer Awareness – Cervical Cancer Facts

Approximately 13,000 women in the United States are diagnosed with cervical cancer annually, leading to an annual estimated 4,000 deaths.1 Cervical cancers are comparatively rare in the United States, with about 0.8% of new cancer cases classified as cervical, contributing approximately 0.7% of cancer deaths yearly.1 Worldwide, cervical cancer is the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death, with an annual estimated 527,600 cases and 265,700 deaths.2

Nearly all cervical cancers are caused by high-risk (oncogenic) human papilloma virus (HPV).3,4 HPV subtypes are broadly classified as high-risk (leading to cervical malignant transformation) or low-risk (leading to benign lesion development).5 The high-risk HPV subtypes, HPV 16 and HPV 18, are the most common subtypes found in cervical tumors. Together, HPV 16 and HPV 18 are the causative agents for greater than 70% of invasive cervical cancers worldwide.5,6 It has been estimated that approximately 10% of the worldwide population have a cervical HPV infection at any given time.7 In spite of this high infection rate, nearly 90% of such infections are naturally cleared by the body within a year or two, and only women with persistent high-risk HPV subtype infections are at risk of progression to cervical cancer.8,9 The slow progression of the disease combined with the availability of screening and HPV vaccination is why many experts consider cervical cancer to be nearly completely preventable.10 Invasive cervical cancer can often be successfully treated if detected at an early stage.

As part of our promise of “Fighting Cancer, One Slide at a Time,” Biocare Medical is proud to offer key high-quality immunohistochemistry (IHC) and in situ hybridization (ISH) reagents that aid in early and accurate classification of cervical tumors and presence of HPV.

Key Antibodies for Cervical Cancer

Product Name Clone Catalog Number
HPV-16* CAMVIR-1 CM 186
HPV Cocktail Broad Spectrum* BPV-1/1H8 + CAMVIR-11 CM 177; PM 177
CA 125 OC125 CM 101; PM 101
Carcinoembryonic Antigen (CEA {M}) COL-1 CM 058; PM 058
p63 4A4 CM 163; PM 163; IP 163; OAI 163; VP 163
CD117/c-kit EP10 CME 296; PME 296; IP 296; OAI 296
MCM2 + TOP2A OT18A11 + UMAB146 API 3181
Topoisomerase II alpha O31 ACI 3045; API 3045
VEGF* EP1176Y CME 356

*Research Use Only (RUO)

Key probes for detecting HPV

Product Name HPV Target Subtype (Classification) Catalog Number
HPV Type 16 Probe (Digoxigenin)** HPV 16 (High-Risk) BRA 4047
HPV Type 18 Probe (Digoxigenin)** HPV 18 (High-Risk) BRA 4048
HPV Type 31 Probe (Digoxigenin)** HPV 31 (High-Risk) BRA 4051
HPV Type 51 Probe (Digoxigenin)** HPV 51 (High-Risk) BRA 4052
HPV Type 6 Probe (Digoxigenin)** HPV 6 (High-Risk) BRA 4045
HPV Type 11 Probe (Digoxigenin) HPV 11 (High-Risk)** BRA 4046

**Analyte Specific Reagents (ASR) for clinical Laboratories to use in developing laboratory developed tests.

Clinical References:

1. Siegel RL, Miller KD, Jemal A. CA Cancer J Clin. 2017 Jan;67(1):7-30.
2. Torre LA, et al. Cancer Epidemiol Biomarkers Prev. 2017 Apr;26(4):444-457.
3. http://www.nccc-online.org/wp-content/uploads/2019/01/10ThingsHPV_CCAM-1.pdf
4. https://www.cdc.gov/cancer/knowledge/publications/fact_sheets.htm
5. de Sanjose S, et al. Lancet Oncol. 2010 Nov;11(11):1048-56.
6. Li N, et al.. Int J Cancer 2011;128:927–35.
7. de Sanjose S, et al. Lancet Infect Dis 2007;7:453–9.
8. Shvetsov YB, et al. Clin Infect Dis 2009;48:536–46.
9. Moscicki AB, et al. Vaccine 2012;30Suppl 5:F24–33.
10. Committee on Adolescent Health Care Immunization Expert Work Group. Obstet Gynecol 2015;126:e38–43.

Heat-Induced Epitope Retrieval

Introduction

Formalin has been used since 1893 as the standard fixative for tissue processing in histopathology.1 Due to formalin’s superior preservation of morphological detail, criteria for pathological diagnoses have been established through the observation of formalin-fixed, paraffin-embedded (FFPE) tissue sections stained with hematoxylin and eosin. Although many other fixatives exist, none have supplanted formalin in general use. Formalin is a cross-linking type of fixative, forming methylene bridges between proteins within the tissue.2,3 This cross-linking reaction adversely alters the structure of tissue proteins, resulting in loss of antigenicity.4 One way to restore antigenicity is through the use of heat-induced epitope retrieval (HIER) prior to immunohistochemistry (IHC).

The complexity of the IHC protocol demands a properly-trained, highly-skilled staff to achieve the most accurate, consistent diagnoses. As the enhanced diagnostic utility of IHC has been realized, an increased demand has been placed on the histology laboratory. Automation, and the concomitant standardization and reduction of variability, allows laboratories to achieve the quality, reproducibility, and speed necessary to meet the increased IHC demand.5

HIER

HIER, also known as Antigen Retrieval (AR), is based upon biochemical studies suggesting that the cross-linking reaction between protein and formalin may be reversed by high-temperature heating or strong alkaline hydrolysis.2,3,6 The routine use of HIER prior to IHC has been shown to minimize inconsistency and standardize staining.7-8 The most critical aspects that influence the quality of HIER results: 1) Heating temperature; 2) Heating time; and 3) The pH of the retrieval solution.

HIER is the process of heating the slide-mounted specimen material in an antigen retrieval solution, followed by a cooling-off period. There is no universal antigen retrieval solution available for restoring all antigens.9 The most commonly used antigen retrieval solutions are citrate-based, tris-based, or EDTA-based solutions.9-10 The pH of the HIER solution influences the immunohistochemistry staining intensity significantly and is critical to the effectiveness of HIER for the specific antigen tested.11 HIER at the appropriate pH allows the antigen to regain its natural shape. Some antigens retrieve better in a lower pH solution, such as a citrate buffer, while others will retrieve better in a higher pH solution, such as a tris buffer.9,11 The composition and the pH of retrieval buffers are critical for optimal retrieval and subsequent staining.

One mechanism to explain how HIER works is by hydrolysis of the methylene bridge cross-links.9,11,12,13,14 Breaking the formalin-induced methylene cross-links in the presence of the appropriate pH solution allows the fixed protein to undergo a series of conformational changes to restore, or partially restore, its native structure. The conformational change allows for better access for the antibody to bind to the antigen. The entire process is being driven by thermal energy from the heat source.11 It is hypothesized that the application of heat accelerates breakage of the methylene cross-links. Subsequently, the methylene cross-links get hydrolyzed when that energy threshold has been reached. The amount of energy added during HIER is determined by heating temperature as well as heating time. There is an inverse relationship between heating temperature and heating time: the higher the temperature, the shorter the heating time needed to attain optimal results. Heating at a higher temperature for a shorter duration yields better staining results than heating at a lower temperature for a longer time.9

The source of heat can be from any number of laboratory equipment, including: pressure cookers, steamers, water baths, microwave ovens, and fully automated IHC stainers. Using a laboratory pressure cooker eliminates the irregular heating and temperature variation/hot spots inherent to steamers and microwave ovens.9,10,13 Ultimately, the introduction of automated IHC staining platforms and online heat retrieval techniques solved much of the inconsistency problems encountered with the majority of other heat retrieval devices. Online HIER processes are more reproducible, less damaging to tissue sections, and save a great deal of time. One major attribute of automation is standardization of protocol and reducing variability from user to user with manual HIER and IHC staining processes.

Clinical References:

1. Fox CH, et al. J Histochem Cytochem. 1985 Aug;33(8):845-53.
2. Fraenkel-Conrat H, Olcott HS. J Biol Chem. 1948 Jul;174(3):827-43.
3. Fraenkel-Conrat H, Olcott HS. J Am Chem Soc. 1948 Aug;70(8):2673-84.
4. Montero C. J Histochem Cytochem. 1991 Jun;39(6):741-8.
5. Prichard JW. Arch Pathol Lab Med. 2014 Dec;138(12):1578-82.
6. Shi S-R, Key ME, Kalra KL. J Histochem Cytochem. 2003 Jan;51(1):1-4.
7. Shi S-R, Cote RJ, Taylor CR. J Histotechnol. 1999 Sep;22(3):177-92.
8. Taylor CR, et al. Biotech Histochem. 1996 Jan;71(5):263-70.
9. Ramos-Vara JA, Miller MA. Vet Pathol. 2014 Jan;51(1):42-87.
10. Tacha D, Teixeira M. J Histotechnol. 2002 Dec;25(4):237-42.
11. Shi S-R, Cote RJ, Taylor CR. J Histochem Cytochem. 2001 Aug;49(8):931-7.
12. Guo T, et al. J Histochem Cytochem. 2007 Jul;55(7):763-72.
13. Van Hecke D. J Histotechnol. 2002 Mar;25(1):45-54.
14. Gown AM. Am J Clin Pathol. 2004 Feb;121(2):172-4.

Cancer Awareness – Early Detection and Tumors of Unknown Origin

Biocare Medical takes great pride in our promise of “Fighting Cancer, One Slide at a Time.” Our focus in the fight against cancer is realized through our high-quality immunohistochemistry (IHC) and in situ hybridization (ISH) reagents and instrumentation. Specifically, our sensitive and specific antibodies, molecular probes, and detection chemistries support early and accurate classification of many cancer types, including tumors of unknown origin. Early detection of cancer significantly increases the chances for successful treatment and can effectively reduce the associated mortality, while accurate diagnosis of tumors of unknown origin allows for determination of the best treatment option.

Early Diagnosis

Early diagnosis for the nine most common cancers (bladder, colon, breast, cervical, ovarian, malignant melanoma, lung, prostate, and testicular cancers), significantly increased survival rates1. When the cancer was diagnosed at stage one or two, 80% of patients survived for at least 10 years.

The appropriate immunohistochemical panels, as well as ISH and other molecular testing, are a critical component in the systematic algorithm of specific, accurate diagnosis2. In the past decade, the clinical diagnostic precision of organ- and tumor-specific immunomarkers, along with the clinical validation of effective IHC panels, has improved significantly.

Tumor of Unknown Origin

When the lineage of a tumor can be identified through standard morphological means, organ- or tumor-specific immunomarkers would be utilized to determine an accurate diagnosis. If the tumor lineage cannot be easily identified via morphological analysis, IHC plays a critical role in the determination of the site of primary origin2,3. Often, the first diagnostic IHC panel will include antibodies directed against lymphoid antigens (Leukocyte Common Antigen [LCA] Cocktail), epithelial antigens (Pan Cytokeratin [AE1/AE3]), melanocytic antigens (S100 protein, SOX10), and sarcoma antigens (Muscle Specific Actin).

Mainline Screening Immunomarkers for Tumors of Unknown Origin

Lymphoma Carcinoma Melanoma Sarcoma
Leukocyte Common Antigen (LCA) Cocktail Pan Cytokeratin [AE1/AE3] S100 Protein Cocktail/SOX10 Muscle Specific Actin

For each of the mainline screening immunomarkers in the table above, once a positive categorical origin is confirmed (lymphoma, carcinoma, melanoma, or sarcoma), additional markers will be tested to determine organ-specific origin of the primary tumor2-4.

Secondary Screening Panels for Tumors of Unknown Origin

Lymphoma Carcinoma Melanoma Sarcoma
CD20 [L26] Cytokeratin LMW (8/18) HMB45 Smooth Muscle Actin
PAX-5 p63 + CK5 / Desmoglein 3 + p40 (M) Tyrosinase Desmin
CD3 [LN10] Cytokeratin 7 / Cytokeratin 19 MART-1 Cocktail Myogenin
CD15 Cocktail / CD30 (Ki-1) CDX2 (M) + CDH17 (RM) / Cytokeratin 20 Pan Melanoma Cocktail-2 CD31
ALK [5A4] TTF-1 + Napsin A (RM) Microphthalmia Transcription Factor (MiTF) CD34
Kappa (M) + Lambda (P) Estrogen Receptor (ER) [SP1]* / Progesterone Receptor (PR) [16]* N/A CD99
CD68 / CD163 c-erbB-2/HER2 N/A ERG
CD10 Prostein + PSA + NKX3.1 / ERG N/A N/A
Cyclin D1 Synaptophysin N/A N/A
CD4 + CD8 PAX8 (M) N/A N/A
Bcl-2 / Bcl-6 [LN22] SALL4 N/A N/A
CD7 CD56 / MASH1 N/A N/A
Terminal Deoxynucleotidyl Transferase Hepatocyte Specific Antigen / Arginase-1 N/A N/A
MUM-1 / CD138 Glypican-3 (GPC3) N/A N/A
N/A GATA-3 + Uroplakin II N/A N/A

*These products are Research Use Only, not FDA cleared for clinical diagnosis.

Clinical References:

1. Wise J. BMJ. 2016 Jun 12; 353:i3277.
2. Selves J, et al. Cancers. 2018; 10(4):108.
3. Kandukuri SR, et al. Arch Pathol Lab Med. 2017 Aug; 141(8):1014-1032.
4. Chu P, Wu E, Weiss LM. Mod Pathol. 2000 Sep;13(9):962-72.

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HPV Probes Now Available from Biocare Medical

Biocare Medical, LLC is pleased to announce the availability of a comprehensive line of HPV probes for in situ hybridization. Each digoxigenin-labeled individual probe is designed to detect HPV DNA viral subtypes 6, 11, 16, 18, 31 or 51.

Almost 80 million Americans are infected with Human Papilloma Virus (HPV), with about 14 million becoming newly infected each year. HPV is considered to be the most common sexually transmitted infection. Although relatively rare, cervical cancer resulting from HPV infection with so-called “high risk” viral subtypes afflicts over 11,000 women every year in the United States. Low risk HPV subtypes are responsible for 360,000 annual cases of genital warts and a smaller number of recurrent respiratory papillomatosis cases.

“With the introduction of our line of HPV probes, Biocare Medical strives to improve the molecular diagnostic tools available to pathologists, as well as advance diagnostic confidence. This is especially critical in cases of discriminating HPV-positive vs. HPV-negative oropharyngeal squamous cell carcinoma, where a correct diagnosis reduces therapy-related morbidity,” says Dr. David Tacha, Chief Scientific Officer of Biocare Medical.

About Biocare Medical
Biocare Medical, LLC is an innovator in developing and supplying world class automated immunohistochemistry (IHC) instrumentation and the full range of reagents for IHC and ISH lab testing. Biocare Medical is the market leader in simultaneous Multiplex IHC and antibody development, which aids in the assessment of clinical cases and accelerates turnaround time. The company’s customers include clinical histology laboratories, pharmaceutical companies, CROs, and biotechnology companies as well as academic, government, military, and other non-profit laboratories. Biocare Medical offers an expanding portfolio of integrated products to address the rapidly growing cancer and infectious disease diagnostic and research markets using tissue immunostaining and in situ hybridization methods. Biocare Medical is headquartered and has manufacturing facilities in Concord, California, and has a global distribution network. For more information, please visit www.biocare.net.