Heavily pretreated cancer patients who have good performance status and who are willing to continue treatment are considered a challenging issue for the treating physician. The problem will be more difficult when those patients had exhausted all the recommended lines of treatment.
Polytreated patients who are ambitions to have their disease controlled motivated the clinical oncologists to search for new hope.
It is well known that cancer cells have the ability to change their biological behavior due to the presence of frequent mutations. These mutations may render the disease refractory or sensitive to a particular treatment.
Precision medicine including molecular profiling as an emerging modality can detect the new mutations in tissue samples taken from cancer patients. This modality is about to be standard of care for many cancer types. The clinical oncologists need a trusted genetic profiling that may provide reliable, high quality molecular information to guide more precise and individualized treatment decisions that will be based on these mutational changes.
Modern approaches to assess DNA, RNA and Proteins reveal the highest quality molecular blueprint to guide more precise and individualized treatment decisions that is proven to extend overall survival  .
The following panel of tests can be used alone or in combination to figure out the genetic map of tumors: Immunohistochemistry (IHC), in situ hybridization (ISH), next generation sequencing (NGS), Sanger sequencing (SS), Pyro Sequencing (PyroSeq)    .
Applying one or more of the previous tests on a new tissue biopsy from the tumor mass of refractory metastatic cancer patients who exhausted all the traditional lines of treatment may create a new chance for treatment with unexpected drug that may not be recognized in the treatment protocols of that specific cancer.
The authors of this study hypothesize that polytreated metastatic cancer patients may harbor ongoing mutations in their tumors rendering them refractory to the traditional treatment. Hence, doing molecular profiling on new tissue biopsies may be mandatory to search for new unexpected treatment that may be potentially effective.
This study has two end points. First is the identification of molecular biomarkers expression in different tumors (such as MGMT and TOPO1). Second goal was to assess the influence of using molecular profiling to guide treatment choice in patients with rare or refractory solid tumors and determine the degree of discrepancy between the elaborated drugs with potential benefit and traditional protocols used in guidelines.
This study is the first of three steps for assessing the use of genetic profiling as a new modality used in Egyptian cancer patients. The second step in a future study will assess the benefit of using this procedure in terms of median overall survival and median progression free survival. The third step in another future study will be assessment of the cost effectiveness of the procedure in Egypt.
2. Patients and Methods
A single-center, prospective observational study conducted on patients with rare or refractory solid cancer. Estimated number of patients = 25. Specific testing through molecular intelligence testing service was performed per physician request.
Patients were distributed according to tumor type, frequency of biomarkers associated with potential benefit, frequency of drugs associated with potential benefit.
All patients admitted to the oncology department in the International Medical Center. Cairo. Egypt from January 2015 to January 2017 were assessed for eligibility prior to inclusion in the study, according to the following criteria:
Inclusion criteria; male and female cancer patients with age range (>18 and ˂60). Patients with good performance status = 0 to 1 by ECOG, no co morbidities anticipated to impede the administration of systemic chemotherapy (Patients with controlled diabetes, controlled hypertension, controlled heart disease and no previous admission in any intensive care units in the last three months can join the study after meticulous clinical examination), patients must be treated with at least three systemic chemotherapy protocols relevant to the National Cancer Comprehensive Network recommendations. Naive patients with rare cancer types with no standard systemic chemotherapy recommendations.
Exclusion criteria; non-compliance to medications, non rare cancer types who did not receive any previous chemotherapy, patients with co morbidities that is extensive and that impedes the administration of systemic chemotherapy (Uncontrolled diabetes, uncontrolled hypertension, renal impairment evident by creatinine clearance less then 30 ml/min in 24 hours in urine collection test, ischaemic hear diseases, patients with previous admission in intensive care units more three or more times in the last three months, patients with bleeding disorders that preclude taking a new biopsy and patients with poor bone marrow reserve who cannot tolerate any further chemotherapy), patients with inaccessible biopsy sites and patients with double primary cancer patients with PS = 2 to 4 by ECOG, patients who refused to take a new biopsy and patients with psychological disorders who cannot sign a consent to join the study.
Molecular test was done through multiplatform approach (Caris Life Sciences® Molecular Intelligence). Analysis was done on new tissue biopsies taken from polytreated eligible metastatic cancer patients.
Tissue samples were meticulously reviewed by a certified and properly trained pathologist before being accepted for NGS testing, before enrollment, the pathologist had to document that the received tissue sample is adequate and non necrotic.
Immunohistochemistry (IHC), in situ hybridization (ISH), next generation sequencing (NGS), Sanger sequencing (SS), were the applied lab tests (alone or combined).
IHC analysis was performed on formalin-fixed paraffin-embedded tumor samples. Commercially available kits were used, automated staining techniques (Ventana, Dako, Benchmark X, AutostainerLink 48) were performed, commercially available antibodies like TUBB3 (PRB-435P, BioLegend, ERCC1 (8F1, Abcam), PGP (C494, Invitrogen) and RRM1 (10526-1-AP, Proteintech) were used.
FISH was used for evaluation EGFR [EGFR/CEP7 probe]. Direct sequence analysis was applied using the Illumina MiSeq platform on DNA genomes isolated from paraffin-embedded tumor samples. Mutation analysis by Sanger sequencing included selected regions of BRAF, KRAS, c-KIT and EGFR genes and was performed by using M13-linked PCR primers designed to amplify targeted sequences.
25 eligible patients joined the study three patients were excluded due to non compliance on treatment. The sociodemographic and clinical characteristics of the patients are demonstrated in Table 1.
Table 1 demonstrates the sociodemographic and clinical characteristics of the patients who joined the study.
• The tumor grade means the degree of differentiation (Grade I = well differentiated to grade IV = poorly differentiated) while the tumor stage means that the tumor is local or loco regional or metastatic.
• ECOG = European Cooperative Oncology Group.
• The listed co morbidities refer to controlled diseases that did not preclude joining the study according to the eligibility criteria.
TUBB3, PGP and TLE3 (indicating potentiality of paclitaxel) was detected in 22% of cases, TS (Antifolates) in 18%, TOPO1 (Irinotecan) in 14%, RRM1 (Gemcitabine) in 13%, MGMT (Temozolomide) in 7%, TOPO2 (Doxorubcin) in 7%, ERCC1 (Platinum) in 6%, BRAF (Vemurafenib) in 2%, KRAS and NRAS (anti EGFR) in 2%, C-KIT (TKIs potentiality) in 1%, hormonal receptors in 5% of cases (Antihormonal potentiality), monoSPARK and polySPARK in 3% of cases indicating nabpaclitaxel potentiality [Figure 1 and Figure 2].
Table 1. Sociodemographic characterstics of the study patients.
Figure 1. Frequency of drugs associated with potential benefit.
Figure 2. Frequency of biomarkers associated with potential benefit.
Figure 1 demonstrates the frequency of drugs suggested to be of potential benefit based on the corresponding biomarkers elaborated from the test. The frequency of drugs is arranged in descending order.
Figure 2 demonstrates the frequency of biomarkers elaborated from the test. Note that this biomarker developed due to genetic mutational changes that was detected in new tissue biopsies taken from the patients who joined the study. The detected biomarkers are arranged in descending order according to the frequency of occurrence.
Potentiality of some drugs (Based on their corresponding biomarkers) was unexpectedly detected as following; Pemetrexed, irinotecan, dacarbazine and temozolomide in breast cancer patients, platinums and taxanes in liver, Taxanes, gemcitabine, fluoropyramidines, pemetrexed, dacarbazine and temozolomide in kidney cancer, Taxanes, gemcitabine, pemetrexed, dacarbazine and temozolomide in cancer colon, irinotecan in cancer tongue, Pemetrexed and irinotecan in adrenal gland cancer [Figure 3].
Figure 3 correlates the tumor type with the drugs of suggested potential benefit. Note that some drugs were unexpectedly found to have potentiality in some cancers not in the scope of their sensitivity like using paclitaxel in adrenal gland cancer, liver cancer and cancer colon.
Anonymous bars in the figure represent novel agents under clinical trials and not stated in the guide lines.
Figure 3. Potential benefit treatment across tumor types.
The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of fluorescence-based sequencing methods with a DNA sequence, DNA sequencing like next generation sequencing (NGS) has become easier and faster   .
Behjati and his colleagues stated that NGS of DNA is the process of parallel sequencing of millions of small DNA fragments. These fragments can be pieced together by mapping the individual reads to the reference human genome using bioinformatics analyses. The human genome contains three billion bases that can be sequenced multiple times, delivering accurate data and unexpected DNA variation  .
The concept of the current study is supported with the reports of Behjati and his colleagues; the current study suggests that polytreated metastatic cancer patients may have frequent ongoing mutations in their cancer tissue. Hence, the unexpected DNA variations in those patients mandated performing a reliable gene test on new tissue samples.
NGS is a modality including many approaches to investigate human genome. The most practical approach is the targeted panel intended to detect informative genetic alterations that may impact the treatment decision of the clinician. Targeted panels are currently the most frequent used type of NGS for diagnostic molecular somatic testing in solid tumors  .
In the current study many pathological procedures were used including the targeted panel approach of NGS. IHC analysis was performed using commercially available kits, automated staining techniques (Ventana, Dako, Benchmark X, AutostainerLink 48) were performed, commercially available antibodies were used like TUBB3 (PRB-435P, BioLegend, ERCC1 (8F1, Abcam), FISH was used for evaluation EGFR [EGFR/CEP7 probe]. Direct sequence analysis was applied using the Illumina MiSeq platform on DNA genomes. Mutation analysis by Sanger sequencing included selected regions of BRAF, KRAS, c-KIT and EGFR genes and was performed using designed PCR primers M13-linked to amplify the targeted sequences.
In a retrospective study by Raphael and his colleagues NGS was applied on samples from 20 metastatic breast cancer patients. One of the study end points is detection of genomic alterations suggesting potentiality for targeted therapy. Genomic alteration was identified in 14 (70%) of patients most frequently PIK3CA (40%), TP53 (55%) and EGFR1 (20%). Raphael concluded that molecular profiling using targeted NGS panel identified genetic changes elaborating potentiality of some drugs that provided clinical benefit in 35% of cases  .
In the current study, five metastatic breast cancer cases were included, discrepancy between the drugs provided by the NGS and those stated in the traditional guidelines were 12%, 15%, 23%, 31% and 21% for cases 1 to 5 respectively (Table 2). TS, TOPO1 and MGMT were some of the unexpected biomarkers detected in the samples rendering potentiality of unexpected drugs like pemetrexed, irinotecan and temozolomide. In spite the fact that our study detected
Table 2. Degree of discrepancy between the elaborated drugs of potential benefit and the protocols stated in the international guidelines: (Except the metastasis of unknown origin).
the presence of biomarkers different from those detected in Raphael study, both data agrees with the concept of personalized treatment base on gene mapping.
Ross and his colleagues applied a comprehensive genomic profiling on epithelial ovarian cancer cases by NGS. 67 out of 141 genomic alterations were identified in 48 cases. Results stated that unexpected mutations like NF1and AKT3 predicted potentiality to mTOR inhibitors, 2% of cases had V8421 ERBB2 mutation raising potentiality for anti-ERBB2 targeted therapies and 2 cases had c-MET amplification suggesting potentiality to crizotinib  .
In the current study the two ovarian cancer cases demonstrated 1 and 2%, discrepancy between the drugs provided by the NGS and those stated in the traditional guidelines. Thymedilate synthetase was the most unexpected biomarker in the tissue samples rendering potentiality to florouracil and pamatrexed.
Gong and his colleagues studied the molecular profiling of metastatic colorectal cancer by NGS in 2017, Unexpected novel mutation KRASR68S1 was found. ERBB2 (HER2) amplified tumors were identified in 5.1% of cases that may indicate potentiality to trustuzumab  .
In the current study, specimens from cancer colon patients demonstrated activity in the following biomarkers, TS, RRM1, MGMT, TUBB3 indicating unexpected potentiality of pemetrexed, gemcitabine, temozolomide and taxanes respectively which are drugs never stated in treatment of cancer colon in any guidelines.
In a study conducted by Ross and his colleagues about the role of NGS in 29 cases of adrenocortical gland cancer (ACC), it was found that ACC is aggressive with low chances of complete surgical resection and limited chances of systemic treatment protocols  .
Treatment selection in the guidelines for ACC relied on retrospective non-randomized studies    .
Ross and colleagues identified the presence of amplified CDK4 and MDM. Although a number of drugs targeting CDK4 and MDM2 are under trials, the NGS stated their potentiality in treating ACC cases harboring those amplifications   .
In the single case of adrenal gland carcinoma who joined this study. NGS revealed the presence of TS, TOPO1, TOPO2 and ERCC1 indicating potentiality of antifolates, irinotican, anthracyclins and platinum respectively. Of note, these drugs had never been stated in any guidelines for treatment of ACC. This result clarifies the role of molecular profiling in creating new unexpected chances of treatment for some rare cancers as adrenal gland cancer. Due to the paucity of the recommended drugs stated in the guidelines for treating the adrenal carcinomas, the drugs elaborated by NGS and suggested to be of potential activity in the single case of this trial, acquired 73.2% incompatibility (Table 2).
The heterogeneity between the recognized genetic changes and biomarkers in our study and others confirms the crucial role of NGS based personalized treatment.
Based on the data of the current study, the authors are confident that polytreated metastatic cancer patients have frequent ongoing mutations. Hence, the unique genetic map of each particular case is something crucial for figuring out its personalized treatment that cannot be applied on other cases with the same disease.
The authors of the study think that including reliable tests for detection of mutational changes in new tissue samples taken from polytreated metastatic cancer patients may be an optimal treatment strategy for refractory metastatic solid tumors. Moreover, doing the test on regular basis for each patient either by taking new tissue biopsies or liquid blood sample during the treatment journey for detection of the ongoing mutations in circulating DNA may be mandatory to figure out personalized treatment. However, Cost benefit and cost effective analysis should be considered before everything.
Disclosure of Funding
This research did not receive any specific grant from funding agencies in the public commercial or not-for-profit sectors.
Some of the scientific content of this article was demonstrated as an abstract in the Cancer Genomics conference that was held in Heidelberg. Germany. November 2017.
All the authors of this work agree with the scientific content of this article and there is no conflict between them
This work follows the ethical consideration for publication and for patient’s rights. All patients signed informed consent before enrollment in the study.
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