A RANDOMISED CONTROLLED TRIAL OF NEOADJUVANT CHEMOTHERAPY WITH FEC vs CONCURRENT CHEMO-RADIATION WITH FEC IN LOCALLY ADVANCED BREAST CANCER

Breast cancer is the most commoncancer among urban Indian women with a CIR of 29.4/100,000 in the MMTR. Nearly50% of the patients present with locally advanced cancers to the Institute. TheInstitute has pioneered the use of concurrent chemo-radiation for Head and Neckcancers, especially oral cancer and in breast cancer (Shanta and Krishnamurthy,1976; Shanta et al., 1985; Shanta and Krsihnamurthy,1991). However, world overthe current practice is to use neo-adjuvant chemotherapy only followed bysurgery and then radiation. The use of neo-adjuvant treatment helps todownstage the tumour making it suitable for surgery ( mastectomy and at somecentres breast conservation is also done) and also assess the response of thetumours to the treatment given. In the Institute, the pathologic completeresponse (pCR) rates following concurrent chemo-radiotherapy is around 29 % ;while with the use of neo-adjuvant chemotherapy alone, the pCR rates haveranged from 13 – 25% depending on the drugs used. Since most of the patientstreated in the Institute are from the lower socio-economic group, use ofTaxanes and Trastuzumab (Herceptin), are beyond the reach of most patients.

Concurrent chemoradiation has itsadvantages- Spatial co-operation, Radiation sensitization and toxicityintdependence (Steels Principles).One of the reasons for the non-use ofneo-adjuvant concurrent chemo-radiotherapy in most centers is the fear oftoxicity, especially to the skin. While skin toxicity is known to occur, grade3 or 4 toxicity, are unusual. The ability to achieve pCR also helps inachieving a better DFS and OS, especially in node positive patients . In acountry with limited resources, optimization of the treatment modalities isessential using the proven methods of treatment.

 OBJECTIVES :

1. To compare  the clinical efficacy of neo-adjuvantchemotherapy as against neo-adjuvant concurrent chemo-radiation in Locally Advanced Breast Cancer.

2. To compare the toxicities of thetwo therapy arms

3. To compare the Pathologic CompleteResponse in the two arms

4. To compare the Disease FreeSurvival

5. To assess the Quality of life inthe randomized patients

6. To do a Proteomics profiling of the pre-treatment tumour tissue sampleand identify and validate characteristic signatures which can help predictPathologic Complete Response (pCR). Additionally, the proteins contributing tothe peaks will also be identified and validated.

LITERATURE REVIEW

INTERNATIONAL

There is limited literature availableon the use of concurrent chemoradiation in breast cancer. There are few prospectiveand retrospective comparative studies that have compared use of CMF and RT and Anthracyclinebased chemotherapy and radiation in the adjuvant setting in early stage Breastcancer(Ismaili et al., 2003). After mastectomy or BCT, the adjuvant treatmentbased on anthracycline and concurrent RT reduced breast cancer relapse rate,and significantly improved LRFS, EFS and OS in the patients receiving more than1 cycle of concurrent CT. There were more hematologic and non hematologictoxicities in the anthracycline group.

The concept of using preoperativechemotherapy in patients with operable breast cancer originated fromexperimental and clinical observations, as well as from theoretical hypotheseson tumor cell growth and dissemination. Results from nonrandomized studies withdifferent chemotherapeutic agents or combination regimens given preoperativelydemonstrated substantial clinical response rates but low pathologic tumorresponse rates. In addition, several such studies were able to show that-byreducing primary breast tumor size-preoperative chemotherapy can lead to anincrease in the rate of breast-preserving procedures The National SurgicalAdjuvant Breast and Bowel Project (NSABP) B-18 trial was the largest randomized trial that aimedto compare preoperative to postoperative chemotherapy in operable breastcancer.

The use of neo-adjuvant chemotherapyhas been increasingly used in the last decade following the results of theNSABP- 18 and other similar trials. Approximately 13% of primary breastcarcinoma cases exhibited both a clinical complete response (cCR) and apathologic complete response (absence of invasive tumor [pCR]) to preop AC. Anadditional 7% of patients exhibited a pCR in the absence of a cCR. A pCRoccurred in 38% of those patients determined to have achieved a cCR. Poornuclear grade of the tumor cells in the pre-entry FNA and/or TC specimenssignificantly predicted a pCR. Patients with the latter exhibited a better OSand DFS compared with those with a pathologic partial response (presence ofsparse invasive tumor [pPR]) or no pathologic response (pNR)

Pathological CR has been shown to bean independent predictor of prolonged DFS/OS but very few patients achieve pCRand that too is dependent upon the type of chemotherapy. Earlier trials testinganthracyclines as NACT produced pCR rates of 2-13% and there was no differencein DFS/OS.Subsequently, taxanes have been used either alone orwith anthracyclines and it has shown to improve pCR rates. Single agentdocetaxel has produced pCR rates of 16-20% and near complete responses of 18%to more than 25%. In combination with anthracyclines pCR rates of 10% to>20% are reported depending upon the stages of tumor included in thatparticular study.

Historically, NACT has not led to increased DFS/OS but achieving pCR isassociated with longer DFS/OS. In a recent update of NSABP B-18 and B-27trials, there was no survival advantage with NACT and addition of taxane topreoperative chemo did not translate into higher survivals although pCR rateswere higher.

NATIONAL

At the Institute the use ofneoadjuvant therapy has its origins dating back to the 1980 s when we usedRadiation alone or Radiation along with MeFu . The results were seen to bebetter with the concurrent use of RT and MeFU – this was not randomized. Havingfound the results with this schedule being better concurrent  chemoradiation became the protocol therapyfor the LABC patients. Initially we used CMF and RT . Later anthracycline basedchemotherapy( FAC initially and then FEC ) with RT  became more frequently used. Taxanes- aloneor with anthracycline  were usedonly  occasionally with RT

WORK PLAN:

A. CLINICAL TRIAL

B. PREDICTIVE MARKERASSESSMENT

A. Clinical Trial

Study Design: A randomized controlledclinical 2 arm study.

Sample sizeWithan expectation of 10% difference in pCR% and 10-15% difference in 5-yeardisease free survival between PCR achieved and not achieved cases, with theleast failure rate of 21%, a sample size of 250 cases in each arm isrequired at 85% power and significance level of 5% for two sided testing ofnull hypothesis that “effects of Arm I is no different from the effects of ArmII†and various power constraints. We plan to include 275 in each arm, in orderto make a provision for 10% lost to follow-up. We assume that the entire periodof the study – recruiting and completion of therapy will be about 4-5 years.

 Arms Of The Study

Arm 1: Neoadjuvant concurrentchemoradiation with FEC-60 , followed by surgery and adjuvant chemotherapy withDocetaxel  , with/without Radiation

Arm 2: Neoadjuvant chemotherapy withFEC-90 , followed by surgery, adjuvant chemotherapy with 4 cycles Docetaxel andsequential Radiation

  Primaryoutcomes to be studied

1. Pathologicalcomplete response (PCR)

 Secondary Outcomes to bestudied

1.5-year Disease freesurvival

1. 5-year Overall survival
2. Toxicity
3. Qualityof Life (QOL)

RandomizationAll consecutive cases accepted fortreatment from a pre-specified index date will be screened for eligibilitycriteria. The eligible cases will then be asked for informed consent.Patientswith operable Locally Advanced Breast Cancer will also be given an option ofinitial Mastectomy followed by adjvant therapy. Those giving consent   for the study  will be randomized to one of the arms (Arm Ior Arm II). Randomization is done by generating random number tables throughcomputer programs for the proposed total number of cases in the study. Adocument is then prepared giving the allocation of all the subjects to the twoarms in chronological order. This information is kept confidential at thestatistics department and provided only to the administering clinician onrequisition during every physical allocation of cases to the treatment arms.

INCLUSION CRITERIA

1. Patientswith Locally Advanced Breast Cancer  -stages IIB , IIIA and IIIB-operable and inoperable
2. Patients  PS I and II
3. Patientsfit for anthracycline based chemotherapy – FEC and single agent Docetaxel
4. Noprevious chemotherapy or hormone therapy

EXCLUSION CRITERIA

1. PSIII and PS IV
2. Age> 65 yrs
3. StageIII C- ipsilateral supraclavicular  nodepositive
4. InflammatoryBreast  Cancer
5. Patientswith co-morbidities that preclude the use of anthracylines such as cardiacdisease, uncontrolled diabetes
6. Patientswith liver dysfunction (s.bilirubin < 1.5 mg/dl, SGOT,  SGPT and serum  alkaline phosphatase should be less thantwice  normal)
7. Patientswith fixed axillary nodes, multiple cutaneous nodules (> 3) in the skinoverlying the breast, ipsilateral arm lymphedema.
8. Patientswith large and pendulous breasts on whom concurrent chemoradiation will have more skin toxicity

END POINTS

Primary – Pathologic Complete Response (pCR)

Secondary – Disease Free Survival, OverallSurvival, Toxicity Evaluation, QOL

Criteria for taking Patients off thestudy will include

1. Patientswho progress on Neoadjuvant chemotherapy alone
2. Patientswho continue to be inoperable after concurrent chemo-radiation
3. Lifethreatening toxicity

Statistical MethodsDescriptivestatistics will be generated in the form of two-way tables giving theproportion of cases in Arm I and Arm II pertaining to different factors relatedto the patient, disease, treatment and outcomes studied. The comparison ofthese proportions between Arm I and Arm II would be carried out usingChi-square test (for nominal scale data) and Student-t test (for interval scaledata) for statistical significance. Survival probability will be estimated byactuarial method. Log rank test would be employed to test for differences insurvival. Cox proportional hazard model would be utilized to elicit theindependent effect of the study arm on survival after adjusting for otherprognostic factors.

 STUDY PROTOCOL

Pre-Treatment Investigations

All patients will have a core needlebiopsy for histopathology and for Proteomic study. 15ml blood sample will bedrawn from the patients after their inclusion in the study. This blood samplewill be stored after separating the cells and the plasma for future studies(genomic and proteomic). Along with histopathological studies,immunohistochemistry will be done for the following markers – Estrogen receptor(ER), Progesterone Receptor (PR), HER2, EGFR, CK5/6, Ki67.

In addition,in an equal number of patients in both arms (n=50), it is proposed to drawblood samples during the chemotherapy for estimation of serum drug andmetabolite levels using HPLC and ESI MS/MS. This will be done only afterobtaining informed consent. 5 ml blood sample prior to administration ofchemotherapy, and at 30 minutes, 60 minutes, 120 minutes, 6 hours, 12 hours, 24hours, 48 hours and 72 hours. In a proportion of the women, who participate inthe study, tru-cut biopsy 24 hours after the chemotherapy is given will bedone, if they are willing and provide an informed consent. Frozen sectionconfirmation of the tumour with at least 70% tumour cells, will be done. Thesesamples will then be processed immediately for quantification of the drugs andtheir metabolites using HPLC – ESI - MS. The genotyping will be done oncefunding is obtained.

The staging evaluation includes aChest X-Ray, CT chest, Ultrasound of the liver and the pelvis, contralateralmammogram and a Bone Scan. . A MRI / PET CT scan may be requested in thosepatients who have an abnormality on the Bone Scan. A   2-D ECHO is done for all the patients prior to randomization.

All patients fulfilling the Inclusion criteriaand those who have given written Informed Consent will be included in theTrial.  Randomization is done afterobtaining the informed consent in the Dept. of Epidemiology.

Management of patients taken off thetrial

Those patients who have progressedduring chemotherapy are taken off the study and are considered for the additionof radiation.

Patients who still have InoperableLABC will have documentation of disease (FNAC) and taken off the study. Theycan be considered for alternate chemotherapy, if willing.

Patients taken off the study due tolife threatening toxicity, will be considered for CMF chemotherapy, if willing.

ARM 1 – CONCURRENT CHEMO-RADIATION ARM

Patients in this arm will receive IFEC on Day 1.

The dosage for the FEC is as follows

5-FU 600 mg/m2                                  -intravenous        Day 1

Epirubicin 60 mg/m2                            - intravenous       Day 1

Cyclosphosphamide 600mg/m2          - intravenous       Day1

The dosage of Epirubicin has been kept at 60mg/m2. Our current protocol at the Institute uses Epirubicin at60mg/m2 and at this dosage we have fairly acceptable toxicity -  Skin toxicity  is Gr III – 25 % and Gr  IV less than 5 %.

The incidence of Febrile Neutropenia is5% .

We do not wish to give Epirubicin at adosage of 75mg/m2 as we feel that the skin and haematological toxicity will behigher.

Radiation will be started within thefirst week of starting chemotherapy.

Radiation is given to the breast,axilla and supraclavicular regions to a total dose of 40 Gy. Radiation isdelivered using 6 Mev from a Linear Accelerator. The Internal Mammary nodalregion is included in the field, if feasible. During the radiation the patientwill receive the II cycle of FEC on day 22 and then continue the radiation.  On the day of chemotherapy radiationis not given. The third cycle of chemotherapy is given on day 43. Duringradiation, if the patient develops severe Grade III or Grade IV reactions thenthe chemotherapy is delayed by a few days till the reaction settles down.

ARM 2 - NEOADJUVANT CHEMOTHERAPY ARM

Patients will receive the 3 cycles FEC, once in 21 days.

5-FU 600 mg/m2                                 -intravenous   Day 1

Epirubicin 90 mg/m2                            -intravenous   Day 1

Cyclosphosphamide 600mg/m2          -intravenous   Day 1

Patients are reassessed 1 week afterthe 3 rd chemotherapy and planned for Surgery which will be Modified Radical Mastectomy.

Prophylactic Growth factors are used in allthe patients as per the NCCN guidelines

Patients are reassessed by theSurgical Oncologist and planned for the Surgery about 4 weeks from thecompletion of Radiation and about 3 weeks from the last chemotherapy.

POST-OPERATIVE TREATMENT

After the surgery the next cyclechemotherapy is given after the wound has healed well and the sutures anddrains are removed. In both the arms we plan to use single agent Docetaxelfor    4 adjuvant chemotherapy cycles.

Adjuvant Chemotherapy Schedule- samefor both arms

Inj Docetaxel    75mg/ sq m  I.V, Once in 3 weeks, 4 cycles

In patients who are not considered fitfor Docetaxel – like uncontrolled diabetes we plan to continue the samechemotherapy

In ARM II, the radiation to the chest wall and the draining regions isgiven 3 weeks after the last cycle of chemotherapy.

In the ARM I  the radiation to theInternal Mammary region is given 3 weeks after the 6th cycle – if itwas not given earlier.

All receptor positive post-menopausal patients,on completion of the chemotherapy, will be given Letrozole for 5 years. Inreceptor positive pre-menopausal women, laparoscopic BSO will be done if theycontinue to menstruate after  thecompletion  of chemotherapy.They willthen receive Letrazole for 5 years. Patients who achieve chemo inducedamenorrhoea will receive Tamoxifen for 5 years.

HISTOPATHOLOGICAL ASSESSMENT

The modified radical mastectomyspecimen will be assessed by several sections of the tumour bearing area andits surroundings (15 – 30 sections, median – 18 sections ) depending on thesize of the tumour, excluding sections taken from other quadrants, resectedmargins and axillary nodes. The presence of residual tumour, its size, gradeand the pattern of response, whether localized or diffuse/patchy as well asother morphological changes in the breast parenchyma and nodal involvement willbe recorded.

RESPONSE CRITERIA

A pathological complete response (pCR)will be defined as the absence of all microscopic evidence of tumour. Apathological partial response (pPR) will be defined as the presence of tumoureither localized/focal or diffuse and patchy surrounded by fibrous stroma.Pathological no response (pNR) will be the persistence of tumour with noresponse to treatment.

IMMUNOHISTOCHEMICAL ANALYSIS

Immunohistochemistry will be performedon formalin-fixed, paraffin embedded sections. Less than 5 micron sections onAPES coated slides will be obtained. Sections will be deparaffinized andrehydrated. The sections will then be subject to wet pressure antigen retrievalfor half an hour. After cooling, the sections will be washed in TRIS buffersaline (TBS) (pH 7.4)) and incubated with 0.03% hydrogen peroxide in TBS for 30mins at room temperature. This will be followed by incubation with primaryantibody overnight followed by incubation with secondary antibody for ½ hourthe following day. Next step will be incubation with DAB for 2-5 minutesfollowed by counterstaining with haematoxylin, dehydration, clearing andmounting. Positive and negative control slides will be processed under the sameconditions as above omitting the primary antibody in negative control. Theslides will then be viewed under a microscope and analyzed.

Quality of Life Assessment

Methodology – Cancer patients with locally advanced breast cancerreceiving chemotherapy or chemo-radiation who satisfy the selection criteriawill be assessed for QOL, Distress, Fatigue and patient satisfaction at 4different time periods. Psycho-oncologists who conduct the assessment will beblind to the study.

Assessment Schedule:

Assessment Period

Chemotherapy group

Chemo-Radiation Group

| I assessment

Baseline

Baseline

| II assessment

Half way through treatment

Halfway through treatment

| III assessment

Completion of treatment

Completion of treatment

| IV assessment

4 weeks after treatment completion

4 weeks after treatment completion

TOOLS

FOLLOW-UP

Patients will be followed up once every3 months with a thorough clinical examination. If clinically warrantedinvestigations such as Xray chest etc will be done.

Investigations are done 1 year afterthe completion of therapy. Investigations include a contralateral mammogram  and 2-D ECHO.

If a patient defaults in follow-up,the Institute’s established method of trying to get the patient back will bedone.

All patients will be followed up fortheir life time.

 B. Proteomics Study

Thecore needle biopsy will be assessed for percentage tumour cells by frozensection analysis and only samples with at least more than 60% tumour cells willbe included in the proteomics study. The flash frozen tissue samples will behomogenized in CHAPS lysis buffer with added protease and phosphataseinhibitors using a FastPrep® homogenizer at 4oC in a cold room.Following centrifugation the supernatant from the homogenized sample iscollected and will be quantitated using bicinchoninic acid (BCA) based protein assay. The quality of proteins isolatedwill be assessed by SDS PAGE analysis.

 Biomarker discovery and analysis byMALDI-TOF MS based system- CLINPROT

Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) is apowerful method for the detection of large numbers of peptides and proteins andtherefore can be used for acquisition of proteomic profile spectra from complexprotein mixtures. The work comprises of initial fractionation bysuperparamagnetic microparticles with functional surfaces designed to undergospecific interactions with peptides and proteins. This is followed byco-crystallization in a surplus of matrix molecules on a MALDI plate target.The analyte is evaporated into the gas phase using pulsed laser and thespectral data is acquired in the designated mass/charge (m/z) range. Thespectra will be anaysed using ClinProTools, a data interpretation software forbiomarker analysis.

5-10 µg of protein lysate will befractionated using magnetic beads exhibiting biochemical functionalities likehydrophobic interaction (HIC8), ionic exchange (WCX), and metal affinity(IMAC resin). The elutes from these fractionations will be co-crystallizedusing a matrix solution composed of α-cyano-4-hydroxycinnamic acid (HCCA 0.3g/l in ethanol:acetone 2:1)in a Anchor Chip MALDI plate. The ratio of sample tomatrix solution will be fixed at a ratio of 1:1. Each sample will be spotted infour replicate spots, CLINPROT standards are also spotted to calibrate the MALDImass spectrometer before data acquisition. The data will be acquired for atotal of 450-550 shots fired at 15 to 18 locations in the spot at 30 shots perlocation. Mass spectra of all the samples generated using MALDI–TOF/TOF will besmoothed and baseline-subtracted using FlexAnalysis software (version 2.2).Three workflows involved in ClinProTools ’Peak Statistic Calculation’, ’ModelGeneration’ and ’Classification’ will be performed. For Peak StatisticCalculation the spectra will be recalibrated and average spectra calculated,peak picking, peak calculation and statistic calculation will be assessed inthe Peak Statistic report. Next, Model will be generated using the spectra fromthe training data set using four different Model generating algorithms GeneticAlgorithm (GA), Support Vector Machine (SVM), Supervised Neural Network (SNN),QuickClassifier (QC). The samples used for the test set will be obtainedblinded to the clinical response details. Classification of the test data willbe performed using the models generated and presented as classification report.The spectral peaks which represent the maximum differences between the classesdenoted by their high ROC curve values will be taken up for furtheridentification.

 Identification of Biomarker byTop-Down Sequencing (TDS)

 The fractionation which yielded thecandidate biomarker will be performed again, however the reaction size will beincreased 10 fold. The eluted fraction will be separated on a reverse phase(RP) C18 capillary column and fractions of the eluent are transferred to a384-well pre- spotted MALDI target by the Bruker Proteineer FC II fractioncollector. To determine the fraction which contains the biomarker candidate allthe 384 fractions will be analysed by MALDI TOF/TOF in the linear mode. Thetarget fractions containing proteins with the expected molecular weight of thebiomarker will be taken up for MALDI-TDS. The MALDI-TDS spectra generated byin-source decay in reflector mode for the proteins are obtained and Proteinsare identified from MALDI-TDS spectra using Mascot.

 Quantitative Analysis of Proteins InCore Needle Biopsies of Breast Cancer

 Proteinexpression in the Biopsies of Breast Cancer will be assessed using Tandem MassTags (TMT) set which enables multiplexing up to six samples and NanoLC-ESI-MS/MS. Tandem Mass Tags (TMT) comprises of isobaric tagging reagentwhich within a set has the same nominal parent (precursor) mass and is composedof an amine-reactive NHS-ester group, a spacer arm and an unique MS/MS reporter.These reporter ions that are in the low mass region of the MS/MS spectrum areused to report relative protein expression levels during peptide fragmentation.The protein samples will labeled with TMT tags prior to trypsin digestion sincecombining labeled samples earlier in the sample process will reduce samplevariability. The labeled proteins are desalted and excess TMT tags are removedfrom the solution. Using strong cation exchange ion chromatography the sampleswill be fractionated prior to performing Nano LC-MS/MS with a C18 reverse phase(RP) column.

  Forthe protocol 100µg of the protein lysate will be used. 45μL of 100mM Triethylammonium bicarbonate (TEAB) is added to the sample and the final volume isadjusted to 100 μL. 5μLof the 200mM Tris(2-carboxyethyl)phosphinehydrochloride (TCEP) is added and the sample is incubated at 55oCfor 1hr. Following which 5μL 375mM iodoacetamideprepared in TEAB is added and incubation continued for another 30 minutes. Forlabeling with TMT label reagents the reduced and alkylated protein istransferred to TMT Reagent vial containing 24μL of the TMT Label Reagent foreach 100μg of protein sample. The reaction is incubated at room temperature for1 hour following which, 8μL of 5% hydroxylamine is added to the sample andincubate for 15 minutes to quench the reaction. The reaction is desalted andcleared of the unlabelled TMT tags by using 10K spin filter cartridge.  The desalted and concentrated proteins arere-suspended in 100μL of 100mM TEAB 2.5μg of trypsin per 100μg of protein isadded and the reaction incubated overnight at 37°C. The trypsin digestedproteins will be fractionated using strong cation exchange chromatography using**PolySULFOETHYLAspartamideâ„¢ (**1.0-mm i.d.)HPLC columns. Initially the peptides will be eluted using shallow gradient toabout 180 mM salt which would form most of the gradient time, this is followedby a steep gradient to ~ 0.5 M salt. Approximately over 90 fractions will becollected. The fractions will be pooled with each pool comprising of 5fractions which would result in ~18 combined fractions. These fractions will beanlysed by  Nano LC-ESI-MS/MS using aC18 RP column (15 cm) connected to a ESI ion source. The fragmentation methodused would be electron transfer dissociation (ETD) to generate the reporterions. The relative quantitation of reporter ions released fromlabeled peptides, and protein identification will be performed usingProteinScape 2.1.

  VALIDATIONOF THE PROTEINS

Oncethe differentially expressed proteins are identified between the pCR andnon-pCR tumours, an independent and blinded set of 200 tumors will be assessedby IHC.