Gynecological Tumors with NTRK and other Kinase Fusions
About the speaker
Dr. Michael Michal is a practicing surgical pathologist in both private and academic pathology departments in Pilsen, Czech Republic. He is the faculty of Medicine in Charles University. Both Dr. Michaels practice and research focused primarily on mesenchymal tumors and sarcomas. He has long been with Bioptical Laboratory that was involved in screening and testing various tumors for several pharma companies over years. The private pathology lab, Bioptical laboratory, is one of the largest bioptical, cytological and molecular pathology centers in Europe. By now Dr Michal and his team have screened by immunohistochemistry over 10,000 of tumors and they have done molecular testing over 4000 of tumors by mostly NGS testing this year. That's how he got involved in this modern and novel chapter of pathology focused on and NTRK fusions in various tumors.
The studies of NTRK fusion in genealogical tumors are no older than two years. Dr Michal concluded his talk as the first introduction to the current state of knowledge. Keep in mind when we re-visit this topic in 3-5 years from now, it may look significantly different.
The presentation was divided into four parts:
A brief overview of extrauterine soft tissue tumors with fusions involving NTRK and other kinase genes (RET, BRAF, etc.)
NTRK- rearranged uterine tumors
COL1A1-PDGFB-fusion – associated uterine fibrosarcoma
The recent advance of studies in uterine inflammatory myofibroblastic tumor
Extrauterine soft tissue tumors with fusions involving NTRK and other kinase genes
Speaking only about NTRK fusions, they are present in about 0.2-0.7% of all soft tissue tumors. Based on the study we look at this, these rearrangements are much more common in the pediatric population. However, identical tumors both clinically and morphologically can be found in adults as well. The first NTRK-rearranged tumor was discovered in 1998. It was infantile fibrosarcoma, as well as cellular congenital mesoblastic nephroma, which was basically an identical tumor when it occurs in the kidneys. It was called with this different name for various reasons. It had been a complete silence in this soft tissue chapter for the following 16 years. Thanks to widespread use of next generation sequencing technique in the routine pathology practice, it was not until 2014 that other NTRK gene fusions had been gradually detected in mesenchymal tumors with various morphologies such as low to intermediate-grade malignant peripheral nerve sheath tumor (MPNST), low-grade spindle cell sarcoma, myopericytoma, hemangiopericytoma-like tumors. One tumor was called lipofibromatosis-like neural tumor. Also, inflammatory myofibroblastic tumor has shown to contain NTRK genes in a small subset of cases. So did a small part of adult fibrosarcoma. Infantile fibrosarcomas have shown to contain not only ETV6- NTRK3, but also other NTRK fusions. If we look at this list, we can speculate the situation in soft tissue is complicated. Some experts think that we have several different entities that all have NTRK fusions. Others think it's one single entity with wide morphological spectrum. It takes time to figure this out. We focus only on this subset of soft tissue tumors with NTRK fusions for two reasons. The first is that this subset is well defined. The second and most important reason is that identical tumors very likely occur in the uterus as well in the genital tract.
Morphological and molecular features
The classic study was done by a group of Dr Antonescu . These tumors had spindle cell morphology and one subset was low-grade spectrum with low cellularity and mild atypia. They had low mitotic activity. Importantly, they had stromal or perivascular hyalinization, which was a nice morphological clue, which was not 100% specific though for these tumors, but they helped the diagnosis a lot There were also high-grade subsets of the same tumors, they looked basically like fibrosarcoma, or other people would call this like intermediate-grade malignant peripheral nerve sheath tumor (MPNST). While most of high-grade subset lost the vascular hyalinization, the subset retained it and could help the diagnosis. The consistent expression of S100 and CD34 antibodies is diagnostically useful. We had a couple of them in our archive here in Pilsen that showed transformation from low-grade area to high-grade area. It's clear that these tumors were related together. It's basically one tumor. These tumors had a very wide and atomic distribution. They affected both superficial and deep soft tissues, as well as viscera. Few cases have been found in stomach, pancreas, and bones. We see no reason why these tumors cannot affect the uterus as well. The morphological grading might have a prognostic relevance. The low severity low-grade tumors, indicated as LC, all had no evidence of disease or at most local recurrence during the follow-up. Other studies on these tumors basically confirm the indolent behavior of these low-grade tumors. In contrast, the tumors with increased cellularity, indicated by IC and the morphology of fibrosarcoma behaved aggressively and could eventually cause death. It killed the patient in a subset of cases. With regards to the molecular aberrations found in these tumors, half of these were NTRK rearrangements mostly NTRK1 but also NTRK2 while other studies have found NTRK3 rearrangements as well. The other half showed alternative kinase fusion genes such as BRAF or another related kinase gene, RAF1.
Dr Michal published the first study that these tumors can also harbor RET gene fusions . This was confirmed by subsequent studies with more cases . These studies showed and confirmed that tumors with RET gene fusions demonstrated similar clinical and morphological spectrum to tumors with NTRK gene fusions.
Summary of the chapter
The various kinase fusion genes such as NTRK1/2/3, BRAF, RET or RAF1 may give rise to tumors that have identical morphology.
There have been many morphological patterns described
Currently it's unclear whether these tumors with these various morphologies represent separate entities or whether they all should be grouped together under a heading such as mesenchymal tumors with kinase fusions.
However, this is a well-defined subset of the neoplasms with a very wide anatomic distribution which shows CD34 and S100 co-expression and includes both low-grade and high-grade tumors.
Those low-grade tumors seem to have a good prognosis.
On the contrary, those with high-grade features may behave aggressively, and the low-grade tumors can progress into the high-grade tumors.
The low-grade tumors progress into high-grade tumors.
NTRK- rearranged uterine tumors
To date, there is a very limited experience with NTRK- rearranged uterine tumors. Only 16 cases have been reported based on three case series and two case reports . The uterine tumors with NTRK fusions showed strong predilection for uterine cervix. 14 out of 16 reported cases were located in the cervix. They often presented as a cervical polyp, and typically they presented in women in their early 20s till late 40s. The size of the tumors varied between 1.5 to 23 centimeters. The average size was seven centimeters and they usually presented in FIGO stages IA or IB. There were no specific presenting symptoms. The most common symptom was vaginal bleeding while many of them were asymptomatic.
Morphological features- 15 out of 16 reported cases showed a fibrosarcoma -like morphology, basically, identical morphology as we've seen in the soft tissue tumors in the high-grade spectrum. This fibrosarcoma-like morphology included fascicular proliferation of spindle cells with mild to moderate atypia. The miotic activity was highly variable from zero to 50. It could not be much more variable than this. Usually there were microscopically infiltrative borders. Although they looked circumscribed microscopically, they were infiltrated tumors. The necrosis was present in about half of cases. The scattered lymphocytic infiltrates were present in most cases, which was a useful clue.
Of note, no tumor with pleomorphism as seen in uterine leiomyosarcomas have been reported to harbor NTRK fusions so far. The gene fusions are mainly seen in spindle cell sarcoma or fibrosarcoma with mild to moderate atypia.
One study pointed out that this spindle cell proliferation often entrapped the endocervical glands, and then the tumor appeared and resembled to adenosarcoma with stromal overgrowth. Here we can see basically what resembles the benign part of adenosarcoma. This was NTRK1-fusion associated uterine fibrosarcoma. Other features of adenosarcoma were missing, such as the submucosal stromal condensation or periglandular stromal cuffing. These features were not present in the extrauterine tumors.
Dr Michal reported one case with quite different morphologies. It was a patient in her 20s and s we had three years of follow-up for her. This tumor showed a very different morphology that consisted of very bland spindle epithelioid in some parts even plasmacytoid- like cells that had ample deeply eosinophilic cytoplasm. There was virtually no mitotic activity. He tried to look to over 100x high-powered fields and didn't find any clear-cut mitosis. The stroma was predominantly moderately myxoid in these areas, but there were also areas with more prominent myxoid change. Most importantly, especially in these myxoid areas, we could see a very prominent stromal and perivascular hyalinization which resembled very closely counterparts in soft tissue tumors with the low-grade spectrum.
Most of the tumors contained capillaries with more or less pronounced hyalinization and in between these capillaries where these epithelioid cells with rich eosinophilic cytoplasm. This myxoid parts come with the more prominent perivascular hyalinization and here we can see the hyalinization was also present in the less myxoid areas.
All cases showed at least focally S100 positivity. It's definitely a very helpful feature especially when we consider there are very few other tumors in the uterus that could be S100 positive- they can be very rare MPNST or perhaps some melanomas. Mainly metastatic melanomas could be S100 positive. Slightly over half of cases were also CD34 positive. Most of these cases were diffusely positive.  The pan-Trk immunohistochemical stains were positive in all cases, mostly in the cytoplasm. The negative stains include smooth muscle actin (SMA), which was negative in the vast majority and only four out of 16 cases were focally positive. BCOR stains were mostly negative with focally positives in two tumors. Alike, CD10, SOX-10, desmin, keratins, ER, and PR were all negative. stains in these tumors.
It is designed to be expressed in tumors with NTRK1/2/3 fusions. Thinking of low incidence of physiological Trk expression, and a low incidence of NTRK fusions, we regard pan-Trk IHC as a great screening method to discover the rarely present NTRK fusions in organs including colon, lung, thyroid and some others. However, the stains are less reliable in tissues with a high incidence of physiological Trk expression, particularly in neural and muscle tissues. It generally has lower sensitivity for NTRK3 fusions, which was observed by several independent studies. If we speak about soft tissue sarcomas, as a whole, pan-Trk IHC shows 80% sensitivity and o 74% specificity for Trk fusions, respectively. It’s better than nothing in soft tissue sarcomas but it's not that great. However, both of these parameters may be higher in the uterine pathology, as there seems to be a prevalence of NTRK1 fusions and potential differential diagnostic entities are not as broad as in soft tissue. 
There has been no formal study for gynecological tumors that would test all the sarcomas or all differential diagnostic entities. But several studies have tested some of these sarcomas that occur in the uterus. The pan-Trk focal to diffuse expression was found in about 6% of uterine leiomyosarcomas. The consistent, meaning in almost all cases, diffuse expression of pan-Trk was found including in BCOR-rearranged sarcomas as well as in YWHAE-rearranged sarcomas and also in about 100% of solitary fibrous tumors, which are however, quite rare in the uterus. We can see the stromal diffuse staining in these two uterine sarcomas, BCOR-rearranged sarcoma and YWHAE-rearranged sarcoma. 
Molecular genetic features
These tumors harbored NTRK1 gene fusions, most notably fused to TPM3 gene which were found in over half of all NTRK-rearranged cases. only four out of 16 cases, harbored NTRK3 gene fusions. No NTRK2 fusion has been described so far, as far as I know. 
Outcome and treatment- Out of 15 cases with follow up, 10 patients had no evidence of disease. However, four out of 10 patients had less than 12 months of follow-up available. Three patients were alive with disease. The first patient with 30 months of follow-up had a multi-focal tumor spread throughout the pelvis and abdominal cavity including distant metastasis. Second patient with 50 months of follow-up had tumor recurrence in the vagina. And the third patient with 7 months of follow-up had a suspected lung metastasis treated by standard chemotherapy. In spite of that, she later developed a recurrence in the vagina. So far one patient was confirmed to die of disease. She had lung metastasis 12 months after initial presentation then she was treated with standard chemotherapy. In spite of that she developed distant metastases later on. Eventually, she died about six years after initial presentation. 
Most notably, there was one patient with a tumor harboring NTRK3 fusion. She was initially treated with hysterectomy followed by five cycles of chemotherapy and also by pelvic radiation. But still, she developed liver metastasis, which were confirmed by biopsy at 16 months after the operation. After sequencing of this tumor, she was started on larotrectinib. And 15 months after the treatment, there was no radiological evidence of residual tumor, both in the thorax, abdomen or pelvis. Like soft tissue sarcomas with NTRK fusions, it seems that targeted therapy will work in the uterus as well. 
First, in general, the constellation of these following features supports the diagnosis of NTRK- rearranged uterine tumor in all situations. If we see a combination of these features, this differential diagnosis should go higher on our list. It includes location in the uterine cervix. Importantly from the morphological perspective, these tumors show uniform spindle cytomorphology with mild to and most moderate atypia. The typical leiomyosarcomas should not be associated with this fusion as far as we know. The focal S100 protein expression seems a great screening marker. The pan-Trk immunohistochemistry seems to be very useful for the detection. These tumors show fluorescent CD34 expression and this expression should favor this diagnosis. Even if all these features are present, we should always confirm the diagnosis by molecular methods. Since both NTRK1 and NTRK3, and other fusion genes can be involved, methods like FISH or RT-PCR which are aimed at one specific operation are not the most appropriate. We would suggest using NGS assays which are quite widely available today - either we have them at workplace ideally or we just try to submit specimens to somewhere else for confirmation.
The other four subsets of uterine sarcomas should be separated.
Leiomyosarcoma: the typical LMS sarcomas are more pleomorphic, desmin-expressed in most instances, diffuse SMA expression. However, around 6% may express pan-Trk. Be noted the subset of LMS is not pleomorphic. They can be spindle cell monomorphic. So, in this case, IHC will be helpful.
BCOR-rearranged sarcomas: from morphological perspective, the tumor looks somewhat similar. However, we usually can find more myxoid matrix in this tumor. Also, these tumors are consistently CD10 and BCOR positive. These tumors also consistently expressed pan-Trk. As far as I know, they are S100 negative. So that's going to help a lot
Inflammatory myofibroblastic tumors (IMT): these tumors are more often in the uterine corpus. They're usually more myxoid. The muscle markers are usually positive in these tumors. Using immunohistochemistry can be of help. However, a subset of IMTs may harbor NTRK fusions. The relationship between NTRK-rearranged IMTs to those with NTRK- rearranged uterine sarcoma remains unclear.
COL1A1-PDGFB -rearranged uterine sarcoma: it has similar clinicopathological features, no S100 protein expression and no pan-Trk expression.
Reviewing the reported uterine cases, the high-grade tumors in soft tissue are either closely, related, or identical to those we see in the uterus. The similarities are also valid given the fluorescence S100, CD34 co-expression. Dr Michal popped a hypothesis based on only on one single report and he suggested we wait and see more data to validate it. His hypothesis is that we will see more and more cases from the uterus that look like the low-grade soft tissue counterpart with this prominent perivascular hyalinization. So far, there's just one case with this morphology together with S100, CD34 co-expression.
Another feature that possibly support the close relationship of the uterine tumors to the soft tissue counterpart is based on one short report that has been published lately. It described RET-SPECC1L uterine sarcomas with basically the same morphology of fibrosarcoma-like appearance, coherent with CD34, S100 co-expression and surprisingly this tumor retained the perivascular hyalinization even in the high-grade areas.This tumor had a similar clinicopathological features to those tumors we just were talking about and the patient has been disease- free for two years. The molecular spectrum of the uterine tumors will be also broader and will not include only NTRK 1/2/3 rearrangements, but we'll also show maybe rare RET rearrangements and perhaps also BRAF or RAF1 rearrangements as we see in soft tissue.
Summary of the chapter
There is very limited experience.
These tumors however seem to have a strong predilection for the uterine cervix, they often present polyp.
Most cases have fibrosarcoma-like morphology, meaning fascicular proliferation of spindle cells, with mild to most moderate atypia.
However, one case was different, it should bland spindled to epitheliod cells set in a myxoid matrix with a prominent perivascular hyalinization.
All cases to date were pan-Trk positive and at least focally S100 positive. Slightly over half of cases expressed CD34. pan-Trk can be of help.
However, pan-Trk IHC is not entirely specific as it can be positive in a small subset of leiomyosarcomas and many BCOR- rearranged uterine sarcomas.
Most cases in the uterus harbored NTRK1 fusions only small subset with NTRK3 fusions.
About one third of reported cases showed an aggressive clinical course.
So far, one patient was successfully treated with larotrectinib.
COL1A1- PDGFB fusion-associated uterine fibrosarcoma
The COL1A1 fusion is the canonical fusion of the vast majority of soft tissue dermatofiberosarcoma protuberans (DFSPs). These tumors were known as prototypical examples of cutaneous meaning superficial soft tissue tumors. Dr Michal personally has never seen primary DFSP in deep soft tissues like retroperitoneum and medius–minimus tendon. Neither has seen a case in some parenchymal organ. Surprisingly these tumors occur in the uterus as well.
Most pathologist have been familiar with DFSP very well because it's not particularly rare in the skin. The classic DFSP, or low-grade DFSP shows this storiform arrangement of spindle cells. And a small subset of these tumors, about 1-2%, can develop areas with high-grade transformation. And these high-grade tumors are then called fibrosarcoma, ex DFSP or fibrosarcoma arising in DFSB. They differ from the low-grade counterparts. By virtue of having this fascicular arrangement of tumor cells, higher meiotic activity is present. In some cases, we can see more atypia, but these tumors are generally not very atypical. Obviously, in classic DFSPs, we can see CD34 diffusely and stromal positive. However, in the skin, we can see very often loss of the CD34 staining in the high-grade fascicular, or fibrosarcoma-like areas. CD34 stains can help detect these fascicular areas in the cutaneous DFSB. As a cutaneous tumor, DFSB can occur in the vulva.
COL1A1- PDGFB fusion-associated uterine fibrosarcoma
Only four cases have been described so far. One study Croce, et al described three cases and another one came from a case report. We put four cases together.
Two cases were in the cervix and two cases were in the uterine body. Patients were slightly older than the patients with NTRK tumors and some of them were postmenopausal patients, which have not been reported so far in NTRK fusion tumors. The size was highly variable, and the tumors were presented with a li more advanced stages than the NTRK tumors which were FIGO stage IB in 2 cases and IIIB in another two cases. 
We can basically see a cellular proliferation with storiform or herringbone architecture. The tumors were composed of uniform cells with ovoid to spindle-shaped nuclei and scant cytoplasm. Only mild to and most moderate nuclear atypia was present. Again, there was a variable miotic activity and there was no lymphocytic infiltrate, which was one of the morphological differences compared with NTRK-rearranged uterine tumors. Two cases showed necrosis. While we can see areas of classical DFSP in some of these reported cases somewhere at the periphery, all tumors had the predominant morphology of fibrosarcoma ex DFSP. 
All cases were diffusely CD34 positive. Again, learning from the cutaneous or soft tissue, we understand that the high-grade areas very often lose CD34 positivity. This also happened in one tumor where one block was completely negative. When the pathologist reviewed this block, he or she would think that it's a CD34 negative neoplasm. Although it should be positive in most cases, the negative reaction with the CD34 does not exclude possibility of fibrosarcoma DFSP area.
One study showed PDGFP positivity.  The studies that are there shows that seem to be quite sensitive for the DFSP. Dr Michal personally had no experience with this antibody though. The negative statins included S100, pan-Trk negative, ER, PR, desmin, BCOR. The negative IHC are also very helpful. 
Mostly the same as for NTRK- rearranged sarcomas except that tumors can occur in slightly older patients and also postmenopausal patients, that pan-Trk, S100 protein are negative, that CD34 should be positive in most cases.
Molecular confirmation is necessary because of the rarity. In soft tissue DFSP, we use this duel fusion probe, COL1A1/PDGFB, which works well. If we have a very high suspicion that this could be the DFSP of the uterus, we can use this FISH essay. However, we would again suggest using NGS because most NGS panels today cover also NTRK genes. Many of them also cover other fusions found in the uterine pathology. NGS is preferable.
Outcome and treatment
Only three patients of the four had the follow-up information. One patient showed no evidence of disease, yet the follow-up was very short. One patient died of disease five years after diagnosis with developing metastases to pelvis and bladder.
One patient was very interesting with a detailed description of the treatment. This lady was diagnosed with a fibroid uterus with large size. During the operation, the surgeon saw a mass emanating from the fundus and invading into the bladder and omentum. The tumor was initially a couple years ago diagnosed as a leiomyosarcoma in spite of negative muscle markers.
The patient was treated with multiple chemotherapy regimens that showed only a very limited response. They changed the protocols many times, but nothing worked. Eventually, the patient developed multiple pelvic masses from measuring from four to incredible 22 centimeters and also lung metastasis. The situation was quite desperate and fortunately the sequencing was available. Finally, they sequenced the tumor and discovered the COL1A1 fusion. They found the first paper on this fusion and figured that this was another example. The patient was started on imatinib, which was a tyrosine kinase inhibitor. The treatment resulted in good response for soft tissue DFSP with metastatic disease. After 11 months on imatinib, the patient showed a resolution of the pulmonary nodules and a significant decrease in the peritoneal deposits with shrinkage from 22 centimeters down to 6.5 centimeters. The response was clinically significant.
Summary of the chapter
Only four cases described there is there
seems to be no predilection for cervix or uterine body.
The morphology of these tumors is identical to what we know well from the soft tissue looks like a fibrosarcoma arising in the DFSB.
All were CD34 positive. Keep in mind CD34 may be lost in high-grade areas of the DFSP
S100, protein and pan-Trk are negative in these tumors,
FISH can be used to confirm the diagnosis. NGS is preferred.
These tumors can behave aggressively.
One uterine DFSP patient on imatinib showed significant treatment response,
It was noted from one review that 60% of patients with soft tissue DFSP have shown either partial or even a complete response (5%) to imatinib.
Recent advance of uterine Inflammatory myofibroblastic tumo
Inflammatory myofibroblastic tumor (IMT) was initially considered rarely affecting uterus. However recent studies from last five to six years have shown that uterine IMTs were highly under recognized. Most IMTs were misdiagnosed as a leiomyoma, soft tissue tumor with uncertain malignant potential (STUMP), or as leiomyosarcoma, a smaller subset perhaps as endometrial stromal sarcoma (ESS) of the uterus. One recent TMA tissue microarray study using ALK antibody as a screening marker found five tumors out of 1747 (0.3%) leiomyomas represented IMTs.  Two studies have shown 1/44(2.3%) and 1/23(4.3%) of leiomyosarcomas should be labelled IMTs instead. One of them was done by Dr Michal’s colleagues in Pilsen. Another paper found out ALK fusions in 14% of STUMPS. This seems to be the category which hides the highest number of IMTs. As we know, a small subset of uterine IMTs harbor other fusions than ALK. It is speculated that the number of IMTs that are hidden behind these diagnoses is just slightly higher than these numbers here.
The soft tissue IMTs showed, according to some studies, local recurrence of 25% and distant metastasis rate less than 2%. It seemed they behaved more aggressively. In 2017, there was a study has done a literature review of 46 IMTs with follow-up and found that five patients had extrauterine disease at the time of surgery and another five patients were at either one or multiple recurrences. Five patients died of disease so it seems significantly more aggressive in the uterus than in extrauterine soft tissue. Since there are such specific kinase inhibitors available to target the molecular changes that drive IMT oncogenesis. it's clinically important to recognize these tumors.
Diagnostically useful features
IMT has a strong predilection for the uterine body particularly for submucosal locations and polypoid quite often. The vast majority of IMTs contain variable prominent myxoid areas and inflammatory infiltrates consisting mainly of lymphocytes. Be noted a conspicious subset of cases has very limited myxoid area. Whenever we see the uterine tumor with myxoid areas, we should usually consider the possibility of IMTs. However, the fascicular smooth muscle-like areas that represent the other growth pattern can also predominate in IMTs. The atypia is usually mild or moderate and only very rare cases have shown severe atypia, which can be helpful clue when differentiating from leiomyosarcomas which are typically atypical. When compared to smooth muscle tumors, the nuclei of IMT seem to be fusiform or tapered, rather than blunt-ended as we can see in smooth muscle tumors. There's an intriguing and peculiar association of IMT with pregnancy which seems to be somehow related to the hormonal change of the patients. Whenever we see the mesenchymal tumor in a pregnant patient, we should think of IMT differentially. Overall, we should have a very low threshold for performing ALK immunohistochemistry, which is because it is an excellent screening marker for these tumors. It shows a good specificity. Rather, in the context of uterine pathology, it's very specific actually.
According to the studies, 80% of uterine IMTs harbor ALK gene rearrangement. ALK IHC shows a good specificity. However, if we see a tumor whose clinical pathological features are highly suggestive of IMT but ALK negative, alternative fusions may be involved, particularly in pregnant women. The recent studies have shown that extrauterine soft tissue, also the uterine IMTs can harbor alternative fusions such as NTRK3, RET, ROS1 rearrangements.  We are expecting more of these gene fusions in IMTs to be reported.
Question and Discussion
What is the relevance of CD34 expression in extrauterine cases? It is usually expressed in hematopoietic stem cells.
Yeah, that's right. CD34 expression in extrauterine soft tissue is unspecific. But I still think it's somehow useful, especially the morphology is right plus S100 protein is positive. if the CD34 is positive as well, I think it just helps to render the diagnosis. Yet it's not specific.
Why do NTRK fusions in gynecological tumors seem to be sarcoma-like?
I've never thought of thought about it this way. As far as I know, there's no gyneclogical carcinoma with NTRK fusion. As the uterine carcinomas are not my area of expertise or interest, I am not able to answer. Yes. That's great remark.
How common is NGS that includes fusion testing in gynecological tumors in Czech Republic and across Europe?
I'm not sure how it is elsewhere in Europe, I think the NGS is getting more and more available. So especially in Western Europe, you can usually get tested if you have a high suspicion. In Czech Republic, since last year we have NGSS paid by the insurance companies. We are to offer NGS much more than before in routine practice and also researches. Our department in Pilsen as well as one department in Prague serve as national-wide domain consultation centers for gynecological pathology. The Prague department has also NGS testing facilities available. I think, in our country NGS is widely available. We can speak just for our team here whenever we see a uterine sarcoma that doesn't conform to like a classical leiomyosarcoma, or some other common low-grade endometrial stromal sarcomas we just go straight to NGS testing, without even doing much immunohistochemistry because these tumors are just so rare. They won't affect the economy of our healthcare system. We try to sequence these tumors as much as we can partly because they can be interesting for our research activities.
You said NGS testing is common for you. In your experience, how long does it take to get results from NGS?
At our department, we have established department we have a great team. As I said at the beginning, we were involved in a lot of NGS testing for several pharma companies such that our molecular team has been trained very well. Typically, if we are just doing some work for pharma companies, we can get a result as soon as in 10 days, because we have enough cases to test. We need to have a certain amount of cases to turn the machines on to make it cheaper. If we don't have enough cases, all this thing gets much more expensive. It takes 10 days to two weeks at most in routine.
You mentioned there were only 16 reported cases of cervical tumors with NTRK rearrangements. Do you believe here are potentially many more that have gone unreported due to the lack of screening for fusions as far?
The fusion testing in gynecological tumors wasn't that widely available before. Only two years ago, we had the first paper on this. Since then, people became aware that these tumors exist just two years ago. I'm pretty sure we will see more and more cases reported in the literature.
Following the previous question, you also mentioned that these patients are often asymptomatic. They may have some vaginal bleeding, but due to this view, are these cervical tumors or other gynecological tumors most likely found at routine visits? If so, are they more advanced because, you know, those visits aren't very common?
Talking about NTRK tumors from what I've read in the paper, most of these tumors have been detected at early stages, like FIGO IA or IB, which is not very advanced. And I think it's very much because of the predilection for uterine cervix which can be quite well visualized during the gynecological exam. And these tumors are often set to be polyploid. The gynecologist can notice this easily. I think that's I think that's why these tumors have been mostly detected at early stages.
About the patients you mentioned, do you know the age distribution of those patients by chance? Also, does history of pregnancy impact patient?
For the NTRK tumors, the age ranges from 20 to late 40s, even 50 years of age. I think most or even all patients were pre-menopausal. And I'm not aware of a report of a pregnant woman with this tumor but I've been contacted because we reported one patient one case in the literature. I was contacted by some gynecologists from Switzerland, and they had one patient. She was pregnant and she had a NTRK-rearranged sarcoma of the uterine cervix and they were just, they were just discussing the options for treatment whether you know, hysterectomy and abortion is necessary or whether they can wait.
Would you stain all uterine sarcomas routinely with BCOR, pan-Trk, etc.?
I wouldn't stain all of them, but I would think about this diagnosis if you see a spindle cell sarcoma with mild to moderate atypia. If we see a sarcoma which is highly pleomorphic expressing desmin and smooth muscle actin, especially in a diffuse manner, we will just sign it out as leiomyosarcoma. On the other hand, for something else, we will keep our differential very wide and, and make monomorphic spindle cell tumor a candidate for NGS testing at our institution.
With NGS, most people mean DNA sequencing, would you perform RNA sequencing routinely?
We do much NGS by Archer. For me, it is referring to RNA sequencing actually. We have very large experience with Archer panels and the experience is very good. Also, our molecular biologists are very happy with this sequencing because it's just quite easy to interpret, and it also works well with archival tissues. Especially for NTRK fusions, RNA sequencing is the best option. The DNA sequencing will fail to detect many of the NTRK rearrangements. Because these genes are bulky such that they have a lot of intronic areas which cannot be covered by DNA sequencing.
 Michal M et al. Genes Chromosomes Cancer. 2019;58(9):680-685.
 Chiang S et al. Am J Surg Pathol. 2018 ;42(6):791-798.
 Rabban JT et al. Histopathology. 2020 Jan 23
 Wells AE et al. Gynecol Oncol Rep. 2019; 28:141-144
 Croce S et al. Mod Pathol. 2019;32(7):1008-1022
 Michal M et al. Am J Surg Pathol. 2019;43(8):1152-1154
 Solomon JP et al. Mod Pathol. 2020;33(1):38-46.
 Gatalica Z et al. Mod Pathol. 2019;32(1):147-153.
 Kao, YC et al. Mod Pathol. 2020 Feb 7.
 Weisman PS et al. Am J Surg Pathol. 2020;44(4):567-570
 Jahanseir K et al. Int J Gynecol Pathol. 2018;37(6):537-546
 Croce S et al. Mod Pathol. 2019;32(7):1008-1022
 Pickett JL et al. Am J Surg Pathol. 41(10):1433-1442.
 Ptiková Z et al. Virchows Arch. 2018;473(5):583-590.
 Devereaux KA et al. Am J Surg Pathol. 2019 Jan;43(1):64-74