Anwendung der PCR-Methode im oncgnostics-Labor

The method of the PCR stands for the polymerase chain reaction. The underlying process has fundamentally changed medical and biotechnological research. It is now considered the most important laboratory method for investigating the molecular structure of our genetic material (= DNA). This is partly due to the fact that the PCR analysis is versatile. It is used in all oncgnostics projects and is an elementary part of our GynTect test for cervical cancer. However, the PCR test recently gained publicity through its use in the current worldwide testing of Corona.

What is PCR used for?

Laboratories use the methodology for amplifying DNA in the shortest possible time. Quick runs, so-called Fast-PCRs, take place within 10 minutes. Others last between one and two hours.

Take our cervical cancer test GynTect: The aim of the PCR is to find out whether certain, epigenetically modified gene sequences that only occur in cancer cells, are present. These are known as methylation of DNA.

In order to clarify this question via PCR based diagnosis, you need the following:

  • DNA from the sample to be examined. The sample is taken at the gynecological practice where the affected woman is cared for.
  • Nucleotides: These are the basic chemical building blocks of DNA which are needed to make copies of the given DNA strand.
  • Polymerases: This is a class of enzymes that is essential for the replication of DNA.
  • Specific primers: The artificially generated, short oligonucleotide chains serve as starting molecules for the polymerase. They dock on the DNA strand, marking the point at which the polymerase begins its work. Depending on which regions (= biomarkers) of the DNA are to be detected, other primers serve as keys for the PCR method.
  • Finally, a buffer solution is needed in which the reaction runs and which keeps the pH during the reaction stable.

Polymerase chain reaction: Process in the thermal cycler

The replication of the DNA usually takes place in three steps in a so-called thermal cycler. This laboratory device regulates the exact temperatures during the biochemical reaction.

  1. The first step is denaturation. The sample is heated to 94-96 °C. This causes the two strands of the double-stranded DNA to separate from one another.
  2. For the second phase of primer hybridization, the sample is rapidly cooled down to 50-65 °C. The respective temperature depends on the primers used, which serve as specific keys as described above. The primers now dock at the suitable points on the two single strands of the DNA.
  3. In the final step of elongation, also called extension, the single strands of the DNA become double strands again. The temperature is increased again to 68-72 °C. The polymerase attaches to the small areas of double-stranded DNA that were created by the primers. With the help of the loose nucleotides, it completes the double strand.

In the last phase, around 500 base pairs are formed within 30 seconds. The length of the third step depends on the length of the DNA sequence to be examined. Once the third step is completed, a new PCR cycle starts. As a result, the DNA amount in the areas of the markers to be detected increases exponentially. More than one billion copies can be available after just 30 cycles. The entire process usually takes one hour.

Important: The DNA sample is only duplicated if it contains the gene sequence you are looking for. In the case of GynTect or Corona, the DNA sample would be positive in this case.

PCR for diagnostics: What happens when using GynTect?

When performing GynTect, we use the described procedure. If there is a methylation of DNA for the corresponding gene sequence, which occurs exclusively in cancer cells and cancer precursor cells, the DNA can be replicated using specific GynTect primers. The result is positive. In this case, the sample lights up.

A specific fluorescent dye is applied for the optical signal. In successive cycles, this is repeatedly stored in double-stranded pieces of DNA that the PCR generates. The more duplicated DNA there is, the stronger the light signal. The cycle from which the resulting luminous signal significantly exceeds the background light is called the Cycle threshold, also known as Ct. For the GynTect test, a certain amount of DNA methylation data is at least necessary for a sample to be recognized as positive. However, the proportion is less than one percent. For Corona PCR tests, some few viruses in a sample are required to yield a positive result. The lower the Ct value, the more viruses were present in the original sample.

If the sample is negative, no methylation of DNA is detected. In this case, the PCR does not generate any DNA fragments and the dye is not stored anywhere. Accordingly, the sample does not emit a light signal.

You can see the preparation of the PCR for GynTect in this video.

Advantage and history of the PCR analysis

The advantage clearly lies in its ease of use. The tool proves specific genetic information in an uncomplicated and targeted manner. Here it is key that the primers specifically recognize the target sequence. In the example of GynTect, we apply primers that only fit certain, in this case six different, gene regions, and the primers only fit if the regions were previously methylated.

Research into the diagnostic method dates back to the 1950s. In its present sense, it was invented in 1983 and only 10 years later, in 1993, it was awarded the Nobel Prize for Chemistry. Today, many different PCR thermal cyclers are used in most laboratories.

2020 was a challenging year – also for our company. Nevertheless, we look back on successful achievements.

GynTect® convinces in test comparison

In November, the journal Clinical Epigenetics published a comparative study on cervical cancer diagnostics tests. Two tests based on epigenetic markers, oncgnostics’ GynTect® and QIAGEN’s QIAsure, were tested on a selection of patient samples. Both tests may be used to identify clinically relevant, HPV-induced cervical disease that may develop into cancer. The aim of the study was to determine not only the sensitivity but also the specificity of the tests, which means how frequently a test delivers a false-positive result. Both tests demonstrated a very good sensitivity for high-grade lesions, especially for cancer cases. However, for GynTect®, the specificity was significantly higher, which means that the rate of false-positive results among healthy HPV-positive women was much lower. Thus, GynTect® may be preferable, due to its higher specificity for CIN2+ or CIN3+.

Achievements: active in research despite Corona

In August 2020, a study on psychological stress in connection to abnormalities found in cervical cancer screening was published, with our contribution. More than 3700 women participated in this scientifically supported online survey. Women with abnormal Pap smear findings or with an HPV infection stated, among other concerns, that they are worried about developing cancer. Even though neither an abnormal Pap smear finding nor an HPV infection does provide a reliable indication of cancer. Nearly half of those affected even expressed fears of dying from cervical cancer. The results were published in the journal Archives of Gynecology and Obstetrics.

Furthermore, we have made significant progress in the diagnostics of Head-and-Neck tumors: Since 2019, we have developed a test based on proprietary DNA methylation markers that may be used in Head-and-Neck cancer diagnostics. In 2020, the corresponding study “OncSaliva” started in the University Hospital Jena. Four more centers will be added by the end of the first quarter of 2021. A total of 150 patients provide saliva and blood samples at the time of their cancer surgery. In addition, we obtain tissue samples from the tumor for analysis. The samples are tested for the methylation markers. 150 healthy subjects are also included, from whom tissue and saliva samples are collected. During the subsequent post-surgical follow-up care, the patients enrolled in the study regularly provide saliva samples for recurrence detection for up to two years. Our scientists analyze the methylation markers in the saliva. This non-invasive method may enable a timely detection of recurrences in the follow-up. The study will run until the end of 2023.

Outlook for 2021: New cooperation and appearance at Eurogin

The results of the GynTect®-PRO study will be available in May. Our company started a three-year follow-up study with the GynTect® test procedure in 2017. For this trial, patients from ten study centers in Germany were enrolled. It is intended to show that young patients with a negative GynTect® result do not develop cervical cancer despite conspicuous changes observed at the cervix uteri. Instead, the cell changes heal by themselves.

In 2021, we will reach another milestone in our international sales activities. Our company recently negotiated a partnership with the international diagnostics group EUROIMMUN, a PerkinElmer company.

Save the Date: We will participate at the international, multidisciplinary HPV congress Eurogin from May 30th to June 1st 2021.

 

Picture: Pharmaceutical biotechnologist Theresa Erler in the oncgnostics laboratory / © Eberhard Schorr

The World Health Organization (WHO) sees an increasing HPV vaccination worldwide as a great opportunity in the fight against various types of cancer. At the organization’s annual meeting, WHO director Tedros Adhanom Ghebreyesus declared that he wanted to use the HPV vaccine more widely. The aim is to act against the globally increasing cases of cervical cancer. With additional treatments and new tests, five million deaths could be avoided by 2050 and many lives could be saved, said Ghebreyesus[1].

What is the strategy of WHO? In the following, we will take a closer look at the dangers coming from a human papillomavirus infection (HPV). We will also discuss the opportunities that vaccination offers against HPV.

What happens with an HPV infection?

HPV infection is primarily a sexually transmitted infection. In fact, most sexually active people become infected with the virus at least once in their lives[2]. However, most HPV infections have no apparent symptoms. They heal on their own. They can no longer be detected after just one or two years.

Regardless of the inconspicuous course, a distinction is made between low-risk HPV types and high-risk HPV types. The former is, for instance, responsible for HPV genital warts that require medical treatment. If an infection with high-risk HPV does not heal by itself, it can develop into cancer through various preliminary stages. This lasts on average 10 to 15 years[3].

According to the Center for Cancer Registry Data, around 6,250 women and 1,600 men develop HPV-related cancers in Germany every year[4].

Types of cancer caused by HPV include:

  • Cervical cancer
  • Carcinomas in the genital area: vagina, vulva, penis and in the anus
  • Head-and-Neck tumors in the area of ​​the throat, tonsils and the base of the tongue

What diseases does HPV vaccination protect against?

Vaccines against the main high-risk types of HPV have been around for several years[5]. The human papillomavirus vaccine has been used in Europe and the USA since 2006. In 2020, a first study from Sweden demonstrated the success of vaccinating against cervical cancer. According to the study results, women who were vaccinated against HPV up to the age of 17 had an 88 percent lower risk of cervical cancer than unvaccinated women[6].

Furthermore, the vaccine is used as a preventive measure for Head-and-Neck malignancies. Anna-Bawany Hums works as a molecular biologist in the research and development department of oncgnostics GmbH: “Head-and-Neck tumors that are traced back to papillomavirus develop over even longer periods of time than cervical cancer. Therefore, there are no current studies that show the effects of HPV vaccinations on these cancers[6]. This makes it all the more important to improve the diagnosis for this disease at the same time. We at oncgnostics are researching how to detect Head-and-Neck tumors in their early stages using non-invasive diagnostic methods. In this way we want to increase the chance of a therapeutic success. The earlier cancer is detected, the better the prospects of a cure are for those who are affected”.

Who should be vaccinated?

The German Standing Committee on Vaccination (STIKO) states that the most effective HPV vaccine age, both for boys and girls, should be between 9 and 14. Child vaccinations should be executed due to several reasons:

  • Protection against individual HPV types can no longer be established if there is a permanent infection or even a precancerous stage. The HPV vaccination is therefore ideally carried out before the first sexual contact is made. Missed vaccinations should be made up as soon as possible. This can be done up to the age of 17 years.
  • Studies show that younger girls have a better immune response to the HPV vaccine than older girls. Correspondingly, vaccination for kids only requires two doses of vaccine.

Whether you are vaccinated or not: HPV Prevention is central

HPV-induced cancers develop over many years and precursors. Even after the HPV vaccination, a residual risk remains. Regular preventive examinations are therefore of crucial importance. In Germany, for instance, a new program for early detection of cervical cancer has been in place since 2020. The program includes co-testings with Pap smear and HPV test for women aged 35 and over.

At its annual meeting, the WHO urged the 194 member countries that at least 70 percent of women should be tested for cervical cancer by the age of 35. In addition, by 2030 at least 90 percent of girls should be fully vaccinated against HPV before they turn 15 years old.

There are still no standardized rules worldwide on systematic preventive examinations for Head-and-Neck tumors. Therefore, it is important to consult a doctor soon in order to clarify complaints in the mouth and throat area at an early stage.

 

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[1] WHO stellt Strategie zur Bekämpfung von Gebärmutterhalskrebs vor. In: Ärzteblatt, 17. November 2020. Online:  www.aerzteblatt.de/nachrichten/118411/WHO-stellt-Strategie-zur-Bekaempfung-von-Gebaermutterhalskrebs-vor

[2] Robert Koch Institut (2020): HPV (Humane Papillomviren): Antworten auf häufig gestellte Fragen (FAQ) zu Erreger und Impfung. Online:  www.rki.de/SharedDocs/FAQ/Impfen/HPV/FAQ-Liste_HPV_Impfen.html;jsessionid=3F10B057E8CD109E0C8EF04167FB32E6.internet072?nn=2375548

[3] Bundesgesundheitsministerium (2020): Verbesserte Früherkennung von Gebärmutterhalskrebs seit Januar 2020. Online: www.bundesgesundheitsministerium.de/themen/praevention/frueherkennung-vorsorge/frueherkennung-von-gebaermutterhalskrebs.html

[4] Robert Koch Institut (2018): RKI-Ratgeber. Humane Papillomviren. Online: www.rki.de/DE/Content/Infekt/EpidBull/Merkblaetter/Ratgeber_HPV.html

[5] Krebsinformationsdienst: Humane Papillomviren und Krebs. Online: www.krebsinformationsdienst.de/vorbeugung/risiken/hpv.php

[6] Robert Koch Institut (2020): HPV (Humane Papillomviren): Antworten auf häufig gestellte Fragen (FAQ) zu Erreger und Impfung. Online: www.rki.de/SharedDocs/FAQ/Impfen/HPV/FAQ-Liste_HPV_Impfen.html;jsessionid=3F10B057E8CD109E0C8EF04167FB32E6.internet072?nn=2375548

 

Almost half of mankind owns this organ and every human life starts in it: the uterus. At the latest, with the first menstruation every woman becomes aware of her own uterus. But how exactly does the female reproductive organ look and what functions does it fulfil?

The uterus is one of the female internal reproductive organs. It consists of muscles whose shape resembles an inverted pear and is located slightly above the pubic bone. In an adult woman, the womb is 7-10 cm and weighs 50-60 g. It is supported by the pelvic floor muscles.

The uterus consists of two parts

Uterus Womb

The upper, thicker part of the uterus is called the body (Corpus Uteri), whilst the lower, narrower section forms the cervix (Cervix Uteri).

In the upper part of the uterine body, the fallopian tubes are located on both sides and transport the mature egg into the uterine cavity, where a fertilised egg may implant itself and develop into an embryo, in other words, a baby. However, this requires a well-built endometrium. Hormonal factors cause the endometrium to regenerate cyclically. If no pregnancy occurs, it is shed and menstruation occurs.

The connection between the uterus and the vagina is the cervix; while the cervical canal opens into the womb, the external orifice of the uterus leads to the vagina.

The uterus during pregnancy

When a baby is developing in the womb, the uterus does amazing things: it expands, stretches and thickens to accommodate the developing baby, the placenta and the amniotic fluid. The womb alone can weigh about one kilo during pregnancy. During birth, the muscles of the womb contract and different contractions eventually help to give birth to the baby.

Uterus diseases

If the pelvic floor is weak, the uterus may sag. Besides, benign tumours, so-called myomas, may form in the musculature of the uterus. A very common gynaecological disorder is endometriosis. The endometrium is mislocated outside the womb, which often is very painful for women and frequently leads to failure to conceive.

Uterine cancer affects the uterine body and is usually caused by hormones, but sometimes also by genetic predisposition. Unusual bleeding may be a symptom of cancer and should be checked.

For example, if a woman becomes infected with HPV (Human Papillomavirus) during sexual intercourse, in most cases, the HPV infection heals on its own. However, in rare cases, the infection persists and changes the tissue around the cervix. These changes (dysplasias) may also regress. However, precancerous stages up to and including cervical cancer may develop. For this reason, it is important to regularly visit the gynaecologist for a cancer screening, to detect cervical cancer early. If possible, in its preliminary stages.

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Cover Foto: GoodStudio/Shutterstock.com
Graphic: Blamb/Shutterstock.com

During the first days of March, Dr. Alfred Hansel took a close look at the healthcare system of Cuba. The Managing Director of oncgnostics GmbH participated in the Cuban-German Healthtech Summit in Havana. Cervical cancer and its early detection is a big issue in Cuba.

Dr. Alfred Hansel, Managing Director of oncgnostics GmbH, attended the event to get a first-hand impression of the Cuban health-care system and the corresponding market. During a delegation trip organised by the German Federal Ministry for Economic Affairs and Energy, representatives of 10 German companies from the medical sector met with representatives of the Cuban health-care industry. The latter gave an insight into their work and the Cuban health care system, while the German companies gave short presentations to the Cuban audience. Opportunities for cooperation were discussed in subsequent bilateral talks. Dr. Hansel had a particularly thorough exchange with representatives of the Hermanos Ameijeiras Clinical-Surgical Hospital, who was very interested in GynTect, the cervical cancer screening test developed by oncgnostics GmbH, and who invited Dr. Hansel for a visit at the hospital’s lab.

Early detection of cervical cancer

The Cuban health-care system is progressive. Pap smears have been used for cervical cancer screening since the late 1960s. Cuban women can undergo the test once every three years. And it was successful! The number of new cases as well as the death rate fell steadily. But since the 1990s the trend has reversed, with more and more women suffering from cervical cancer and even more women dying of it. The reasons are yet unknown. An examination every three years may not be enough. Moreover, only about 70% of women actually go for the regular cervical cancer screening.

Cooperation on a study

The Hermanos Ameijeiras Clinical-Surgical Hospital, the largest hospital in Cuba and a reference centre for research and education, would like to contribute to improving cervical cancer prevention in Cuba by means of a clinical trial in the near future.

‘Representatives of the hospital showed great interest in GynTect, our test for the detection of cervical cancer, and are currently in the starting phase of a study that will demonstrate the advantage of the HPV test over the Pap smear. We have talked intensively about using GynTect to complement the study. In the next few days, we will develop the ideas raised in Cuba in order to possibly implement them soon’, says Dr. Alfred Hansel

Human papillomaviruses are a group of widespread viruses. In most cases, people affected by the virus are unaware of the infection. However, certain types of HPV are responsible for the development of tumours, such as cervical cancer.

Almost everyone gets infected with HPV at some point in their lives. HP viruses are mainly transmitted by skin-to-skin contact, but also by sexual intercourse. The infection often remains undetected because no symptoms are noticed and the infection heals by itself. However, harmless or unpleasant warts, such as genital warts, can occur. In a few cases and with certain types of HPV, chronic HPV infection occurs, which can last for several years. This can lead to malignant cell changes. The consequence may be precancerous lesions or cancer. In particular, HP viruses are responsible for the development of cervical cancer. However, tumours in the mouth, vagina, penis and anal area can also be caused by human papillomaviruses.

High-risk HPV types

Not all HP viruses are causing cancer. To date, more than 200 HPV types have been conclusively identified, according to the Robert Koch Institute. They can be classified into five different groups: Alpha, beta, gamma, mu and nu HPV. Only the alpha-HPV group can infect the skin and mucousal cells in humans.

In general, these types can be subdivided into high- and low-risk types. The low-risk types (“low risk” viruses) can cause warts in the genital area. However, a life-threatening disease can be rarely feared when infected with low-risk HPV types. In contrast, high-risk types (high-risk viruses) can cause malignant cell changes, i.e. cancer.

Protection against HPV infection

Vaccination against HPV is the most effective protection against an HPV infection. Not even condoms provide sufficient protection during intercourse. Children aged nine and older can be vaccinated against certain high-risk HPV types. In general, vaccination should preferably be applied before the first sexual intercourse. This is true for both boys and girls alike, because vaccination not only protects against cervical cancer. There are also other types of cancer associated with HPV, as mentioned above. In addition to vaccination, women should visit their gynaecologist regularly for cancer screening.

From 18 to 21 November we attended MEDICA 2019, in Düsseldorf. We were at the joint booth for the industry association medways e.V. in the world’s largest trade fair for the medical sector.

Record Number of Exhibitors and Visitors

This year the trade fair broke its own record once again with around 5,500 exhibitors and 121,000 visitors. About 170 different countries gathered at the No. 1 platform for international business. Many visitors came by oncgnostics’ booth to learn about our latest developments.

“For us, MEDICA is the most important trade fair of the year. This is where the industry comes together, where we make and maintain important contacts. As exhibitors, we are able to introduce ourselves, and we are very happy to use our own booth for meetings. At the same time, it is always worth taking a tour around the fair to get up to date,” says Dr Alfred Hansel, oncgnostics’ Managing Director, describing his impressions.

Pitch at COMPAMED

Dr. Peter Haug

Simultaneously to MEDICA, COMPAMED, the trade fair for medical suppliers, took place. Our Head of Business Development & Licensing, Dr Peter Haug, accepted the invitation of IVAM, the professional association for microtechnology, and gave a sales pitch at the High-tech for Medical Devices forum.

“Oncgnostics GmbH is not only a manufacturer of reliable and innovative devices for cancer diagnostics: It is also of interest as a development partner to other pharmaceutical and diagnostic companies that would like to develop reliable tests based on epigenetic markers for new indications with us. Moreover, thanks to our strategic partnership with GeneoDx, a subsidiary of the Chinese SINOPHARM Group, we are also able to offer high-volume orders,” summarises Dr Peter Haug.

Oncgnostics GmbH is greatly honoured to have been selected by EURONEXT for the TechShare programme. We will be participating in top-class workshops and coaching sessions for half a year.

High-quality Coaching for Start-ups

As one of eleven German start-ups in the medical and biotech sectors, we will have the opportunity to broaden our horizons over the next six months. We will learn how a potential IPO should be prepared, and for which companies it can be useful. In addition, we can look forward to high-quality coaching directly at our premises in Jena. With the acquired knowledge, we will be able to constantly work on and strengthen our company strategy.

Learning from the Success of Others

Euronext TechShare Campus 2019 Rotterdam | photo: Robert Tjalondo © | www.rockinpictures.com

Dr Peter Haug, Head of Business Development & Licensing at oncgnostics, was present at the Kick-off Campus Erasmus Centre for Entrepreneurship at the University of Rotterdam. The extensive programme was supported by top speakers such as Onno van de Stolpe, CEO and founder of Galapagos NV. The Belgian drug research company was founded in 1999 and successfully went public just six years later: ‘It is very inspiring to hear about the success stories of former start-ups,’ says Dr Peter Haug, describing van de Stolpe’s presentation. ‘In addition, EURONEXT has put together a fascinating selection of high-profile European start-ups. I already knew some of them before, and look forward to networking with the others, all of whom are facing similar next steps and challenges as we are at oncgnostics GmbH.’

After the kick-off in Rotterdam with around 150 international participants, they will continue in different groups. Oncgnostics GmbH will participate in four workshops in Munich, together with six other companies from the Biotech and Medtech sectors. Next spring, all participants will meet in Lisbon for the closing event.

Dr. Alfred Hansel in Spanien

On 12 June, the conference ‘XIII Jornada de Formación en Patología del Tracto Genital inferior y Colposcopia‘ was held in Madrid. Oncgnostics’ managing director Dr Alfred Hansel was one of the speakers at the training event for gynaecologists.

Experts Exchange Knowledge

At the conference, experts updated their knowledge on HPV-related cancers in women’s genital tract. In his lecture, Dr Alfred Hansel described possible cervical cancer diagnostics that go beyond HPV testing. In addition, the participants were able to join various workshops, such as colposcopy courses. Furthermore, the participants had the opportunity to visit the NIMGenetics, distributor of GynTect® in Spain, booth at the industrial exhibition event, and inform themselves about the test.

Investor Days

On 18 June oncgnostics managing director, Dr Alfred Hansel, visited Investor Days Thüringen in Erfurt, and was pleased to receive the ‘bm|t sustainable business award’.

Thuringia’s Sector Network Meeting

As a large sector network meeting, Investor Days bring together the different parties involved: Young companies, investors, decision makers and representatives from economy, science and politics. Selected formats and an entertaining evening programme encourage visitors to get to know each other and exchange knowledge.

Meet the Rising Stars

During the so-called ‘Meet the Rising Stars’, Oncgnostics GmbH presented itself. Managing Director Dr Alfred Hansel presented the current developments of the company to the audience in a 7-minute pitch.

The surprise of the day was the awarding of the ‘bm|t sustainable business award’ to oncgnostics GmbH: ‘We are delighted to receive this award,’ says Dr Alfred Hansel, ‘especially since we did not expect it at all. Thank you very much!’

About Investor Days

The Investor Days Thüringen is an initiative of the Foundation for Technology, Innovation and Research Thuringia (STIFT) and the beteiligungsmanagement thüringen gmbh (bm|t) to bring together innovative start-ups and growth companies with potential cooperation or business partners, as well as investors and business angels.