En Dunder-rapport(studie)

3 forskare från Skottland, Dundee närmare bestämt, är på krigsstigen. Det formligen dundrar av harm i en studie de nu lägger fram. Biomarkörer för cancer är ämnet för deras upprördhet. Forskarna har hittat ett flertal missvisande data och helt utelämnande fakta i ett antal cancermarkörer som idag används och är standard för att identifiera olika typer av cancer. Dessa missade delar anser de kan ha negativ påverkan på en cancerpatient som får behandling med läkemedel biomarkörerna föreskriver. Forskarna visar ex i en bröstcancer att biomarkörerna missar vissa cancercellinjer som i ett senare skede riskerar att blomma ut i en ny bröstcancertumör. 

Hur har de listat ut detta? Och varför har de satt tänderna i detta?

Frågorna hänger ihop,men de är svåra att besvara i ett gemensamt svar/sammanhang. 

Den första frågan hur hittar vi svaret på i studien. De har använt cancerceller från en patient,studerat dessa under mikroskop,tillfört (av biomarkören) rekommenderat läkemedel och sen studerat hur cancercellerna uppför sig.
Har alla cancercellinjer (nu snackar vi avancerad mikroskopi och uthållighet i observationerna) försvunnit eller bara de mest utmärkande av biomarkören identifierade? Svaret förstår ni säkert redan.

För att kunna följa cancercellerna och tillsatta läkemedel måste dessa hållas vid liv hela vägen fram,eller så långt nu cellerna överlever utanför kroppen. Alltså gick det inte att färga in cellerna då det medför missvisande data (cellerna påverkas av infärgningen) samt att infärgningen startar en process som alltid leder till cellernas död mer förr än senare.

Vilket instrument de använde kan ni säkert lista ut vid det här laget. HoloMonitor såklart !

Cancerceller och läkemedel placerades tillsammans med HoloMonitor inne i en inkubator där de följde utvecklingen minutiöst hela vägen fram tills de hittade svaren de redovisar i denna forskningsrapport.

Andra frågan varför de satt tänderna i detta ifrågasättande av idag använda biomarkörer kan de bara svara på själva och det framgår inte i studien. Förmodligen för att cancerpatienter drabbas av "oförklarliga och svårtydda" komplikationer och/eller tumörer efter behandling. Tumörer som inte borde ha uppkommit,men lik förbannat gör det.

En rekapitulation först

Som de flesta phi,are vet har Bolaget berättat att HoloMonitor är ypperligt för att testa nya läkemedel. 
Att människoceller insjuknade av nåt elände hålls vid liv inne i en inkubator,att man sen tillför de läkemedel/substanser man hoppas ska råda bot på sjukdomstillståndet och studerar förloppet i ett HoloMonitorinstrument (Allt tillsammans inne i inkubatorn). 

99 ggr av 100 (min uppskattning) funkar det inte och man (forskare) får gå tillbaka till ritbordet och klura ut nåt annat. Under tiden vill man att cellerna i inkubatorn är vid liv så experimenterandet kan fortsätta. Det speciellt om forskaren inte har tillgång till obegränsat antal celler av specifik typ.

I denna studie har forskarna gått bakvägen kan man säga. Läkemedlen har fått visa sin effektivitet på ett gäng olika cancerceller. Bröstcancer,äggstockscancer,tjock och ändtarmscancer,blodcancer är de jag hittar som ingick i studien.Samtliga cancrar behandlade med de av biomarkörerna rekommenderade läkemedlen.

Svaren de fick redovisar de på ett hyfsat syrligt sätt. Jag klipper ur och markerar valda delar av studien.

The search for CDK4/6 inhibitor biomarkers has been hampered by inappropriate proliferation assays

Posted March 15, 2023.

SUMMARY

CDK4/6 inhibitors arrest the cell cycle in G1 and are used in combination with hormone therapy to treat advanced HR+/HER- breast cancer. To allow more effective use of these drugs in breast cancer, and to facilitate their use in other tumour types, biomarkers that can predict response are urgently needed. 

We demonstrate here that previous large-scale screens designed to identify the most sensitive tumour types and genotypes have misrepresented the responsive cell lines because of a reliance on ATP-based proliferation assays. 

When cells arrest in G1 following CDK4/6 inhibition, they continue to grow in size, producing more mitochondria and ATP. 

This cellular overgrowth masks an efficient arrest using metabolic ATP-based assays, but not if DNA-based assays are used instead. 

By comparing tumour cells using different assay types, we demonstrate that the lymphoma lines previously identified as the most responsive cell types, simply appear to respond the best because they fail to overgrow during the G1 arrest. 

Similarly, the CDK4/6 inhibitor abemaciclib appears to inhibit proliferation better than palbociclib, but this is because it also inhibits cell overgrowth through off-target effects. 

DepMap analysis of previous screening data using only the reliable assay types, demonstrates that palbociclib-sensitivity is associated with sensitivity to Cyclin D1, CDK4 and CDK6 knockout/knockdown, and resistance is associated with sensitivity to Cyclin E1, CDK2 and SKP2 knockout/knockdown. 

Furthermore, potential biomarkers of palbociclib-sensitivity are increased expression of Cyclin D1 (CCND1) and RB1, and reduced expression of Cyclin E1 (CCNE1) and CDKN2A. 

None of these associations are present when analysing DepMap using similar data from metabolic assays. 

This reinforces the importance of new screens to assess CDK4/6 inhibitors against a wide range of cancer cell types using an appropriate proliferation assay. 

This would help to better inform clinical trials and to identify much needed biomarkers of response.

INTRODUCTION

CDK4/6 Inhibitors are novel anti-cancer drugs that have revolutionised the treatment of breast cancer. They arrest the cell cycle in G1 phase and are effective at treating advanced HR+/HER2- breast cancer, when used in combination with previous standard-of-care hormone therapy. 

CDK4/6 activity is required for G1 progression in many other cell types, implying that these drugs may also benefit a wider range of cancers. 

To identify the most sensitive tumour types, previous large-scale screens have assessed the effect of CDK4/6 inhibitors on the proliferation of a wide range of cancer cell lines 3–6. 

The aim of these screens is to reveal genomic features that correlate with sensitivity, thus yielding potential biomarkers of response. 

Predictive biomarkers are urgently needed, not just to define new tumour types that may be sensitive to CDK4/6 inhibitors, but to identify the breast cancer patients most likely to respond to these drugs.

Finally, a molecular barcoding strategy known as PRISM, was recently used to characterise the response of 578 cancer lines to 4518 drugs. 

This large format was feasible because cancer lines containing DNA barcodes, which express unique mRNA transcripts, were screened together in pools. 

The pools were lysed 5 days after treatment and the relative abundance of each mRNA barcode was used to calculate the response of each cell type. 

This screen tested all three licenced CDK4/6 inhibitors, but none of these associated CDKN2A loss or mutation with sensitivity, nor identified any other potential biomarkers of response.

In summary, the GDSC1 screened appears somewhat of an outlier and most large-scale screens to date have failed to identify potential biomarkers of CDK4/6 inhibitor response. The exception is the Gong et al study, which identified genetic defects known to activate D-types cyclins, termed D-type cyclin activating features or DCAFs, which were predicted to be indicators of sensitivity to abemaciclib specifically 5. Unfortunately, these markers have yet to demonstrate predictive power in clinical studies.

We demonstrate here that a major problem with all of these screens is that they have used endpoints that do not directly measure proliferation. 

Instead, these endpoints measure the cumulative effects of cell number and cell size. 

This is particularly problematic for cells treated with CDK4/6 inhibitors, because these cells arrest in G1 but continue to grow in size 9–13. 

We show that this cell overgrowth causes scaling of mitochondria, thus cells appear to have “proliferated” using ATP-based endpoints, even though they have not. 

Cellular RNA similarly scales with growth, probably invalidating mRNA-based endpoints as well. 

We further demonstrate that the enhanced response observed in blood cancers or with abemaciclib, are due to reduce cell overgrowth under these conditions, and not due to an enhanced proliferative arrest. These misinterpretations have likely impeded the search for CDK4/6 biomarkers, because analysis of cumulative data from screens using only a reliable DNA-based assay, demonstrates expected markers of sensitivity and resistance. 

In particular CDKN2A loss is associated with sensitivity, whereas RB1 loss and Cyclin E overexpression are associated with resistance. 

This work calls for new screens to expand on this data using a reliable proliferation assay in a wide range of cancer cell lines. 

It also highlights the importance of using appropriate “proliferation” assays when assessing any anti-cancer drugs that arrests the cell cycle but permits continued cell growth.

Results

It is unclear why blood cancers fail to grow during a palbociclib arrest, but the prediction is that these cancer types will be the least sensitive to downstream toxicity and cell cycle withdrawal. 

The fact that these cells have been categorised as some of the most sensitive by current assays, further underscores the importance of using the correct endpoint in these assays. 

Similarly, abemaciclib has been proposed to inhibit proliferation better than palbociclib, when in fact, this is due to its ability to limit overgrowth through off-target effects (Figure S2). 

Whether this is beneficial in the treatment of HR+/HER2- breast cancer is currently unclear, but in cell models at least, it likely explains the rather limited cell cycle withdrawals following abemaciclib treatment, in comparison to three other licenced CDK4/6 inhibitors.

In summary, ATP-based assays are unsuitable for measuring a G1 arrest following CDK4/6 inhibition because most cell types overgrow during that arrest and produce more mitochondria.

DISCUSSION

We demonstrate here that most cells continue to grow in size when they are arrested by CDK4/6 inhibitors. 

These enlarged cells scale their mitochondria and therefore appear to still be proliferating using ATP-based assays, even though they are not. We have shown recently that total cellular protein and RNA also scales with size during the arrest 13, therefore we hypothesise that “proliferation” assays that use any of these endpoints will also misrepresent cell enlargement as cell proliferation. 

This affects every large-scale screen that has been carried out to date to assess CDK4/6 inhibitor sensitivity. The GDSC1 screen relied partially on ATP-assays 3, and the GDSC2 and Gong et all screens relied exclusively on ATP-assays 4, 5. 

The pooled PRISM assays, which screened all three licenced CDK4/6 inhibitors, relied on mRNA sequencing of lentiviral barcodes to determine the relative levels of each cell line within the pools before and after treatment 6. It is likely that these barcodes also scale with size, as do most cellular mRNAs during a CDK4/6 inhibitor arrest 13, 18. Therefore, we predict that overgrown cells will similarly be overrepresented in PRISM assays despite an effective arrest. 

There is therefore an urgent need to perform new assays that can accurately report a proliferative arrest following CDK4/6 inhibitor treatment.

The same issue may affect the assessment of many more anti-cancer drugs.

All of these confounding (förvirrande) effects of cell growth have likely hampered (förhindrat) the search for CDK4/6 biomarkers. By grouping and analysing assays using reliable endpoints, we find that the top co-dependencies are Cyclin D1, CDK4 and CDK6 for palbociclib-sensitive cells, and Cyclin E1, CDK2 and Skp2 for palbociclib-resistant cells (Figure 5a). Comparisons of gene expression, copy number and proteins levels identified decreased CDKN2A as one of the strongest a predictors of sensitivity (Figure 5D, E). This gene encodes for the CDK4/6 inhibitor p16 INK4A, and low p16 INK4A expression was previously identified as a possible marker of sensitivity to palbociclib. 

The subsequent PALOMA-1 trial showed CDKN2A copy number was not predictive of response, and the later PALOMA-2 trial showed CDKN2A mRNA or expression of p16INK4A protein were also not predictive either.

MATERIALS AND METHODS (urval)

Time Lapse Imaging

To characterise the arrest caused by palbociclib each cell line was plated at low density (15,000 per well) into an Ibidi μ-plate glass-bottomed 24 well plate. 

The following day cells were treated with drugs and then imaged using a Holomonitor M4 (Phase Holographic Imaging) at 37°C with 5% CO2. 

Images were taken every 20 minutes for a total of 4 days. 

Image analysis was performed using the Holomonitor App Suite. 

For each condition, cells were selected at random and then followed by eye to record the length of time between the first and second mitosis (or the end of the movie).

Min kommentar

Dessa forskare från Skottland, Dundee lär knappast bli inbjudna till nästa julfest hos de angivna biomarkörernas tillverkare tippar jag. Men jäklar vad strongt gjort av dem att utmana och ifrågasätta dessa biomarkörers effektivitet. Att uthålligt studera en massa cellinjer med rekommenderade läkemedel och sen hitta missvisande data som ger förklaringar till varför cancerpatienter återigen drabbas hårt fast de fått behandling. 

Tuff behandling ska sägas.Cellgifter är ingen höjdare att få in i kroppen kan undertecknad skriva under på.

Måste man sen göra om proceduren?..........NEEEEEE skriker kroppen.

Och för att komma till dessa stunning results har de använt sig av HoloMonitor !!!

Förstår ni nu varför jag är helsåld på instrumentet? 

Då kanske ni även förstår varför jag återkommande säger (skriver) att HoloMonitor är en cancerforskares bästa vän.

Fortfarande ingen som kan ringa Bill Gates och be han sponsra världens alla cancerforskare med varsitt instrument? 😎

                                          Mvh the99

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